Textbook of Aging Skin
Miranda A. Farage, Kenneth W. Miller and Howard I. Maibach Editors
Textbook of Aging Skin With 366 Figures and 156 Tables
Editors: Miranda A. Farage, Ph.D. Principal Scientist The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Kenneth W. Miller, Ph.D. Associate Director The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA Howard I. Maibach, M.D. Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 94122 USA
Library of Congress Control Number: 2009938632 ISBN 978-3-540-89655-5 This publication is available also as: Electronic publication under ISBN 978-3-540-89656-2 Print and electronic bundle under ISBN 978-3-540-89935-8 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. ß Springer-Verlag Berlin Heidelberg 2010 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Springer is part of Springer Science+Business Media www.springer.com Publishing Editor: Tobias Kemme MRW Editor: Sandra Fabiani Printed on acid‐free paper
Dedicated to those on a forgotten and sometimes lonely and scary aging journey—much more dignity and respect are deserved for all of you. – MAF, KWM and HIM
When things go wrong as they sometimes will, When the road you’re trudging seems all uphill, When the funds are low and the debts are high, And you want to smile but you have to sigh, When care is pressing you down a bit Rest if you must, but don’t you quit. Success is failure turned inside out, The silver tint on the clouds of doubt, And you can never tell how close you are, It may be near when it seems afar. So, stick to the fight when you’re hardest hit It’s when things go wrong that you mustn’t quit. —Unknown
Acknowledgments
Deep appreciation and grateful thank-yous are extended to the significant efforts of many people who contributed both knowingly and indirectly to this book, by dedicating their valuable time in preparing their chapters. This book represents the fruits of a jointly conceived and executed venture, and has also benefited from global and diverse partners. A special thank-you to Dr. Mark Dato and Mr. Ron Visscher for generously offering their time and expertise in peerreviewing the relevant chapters and extending their immense support to this book. No praise is excessive for their efforts, and our heartfelt gratitude goes to them. We would like to single out Mr. Anil Joseph Chandy (Springer Reference Editorial Office) for a special recognition of his great effort, time, discipline, and dedication in moving this book forward in a timely and organized manner. We extend our appreciation to Ms. Marion Philipp and Ms. Ellen Blasig (Springer Heidelberg) too for the same. Last but not least, we acknowledge the assistance provided by Dr. Deborah A. Hutchins, Ms. Zeinab Schwen, Ms. Wendy Wippel, Ms. Gayle Entrup, Ms. Jan Tremaine, Ms. Peggy Firth, and Dr. T. L. Nusair for this book. Their collective recommendations and input have vastly improved the texts assembled here. Above all, we extend our everlasting gratitude and love to our parents, who inspired us and to our families and children, who supported and encouraged us all the way with their incredible patience. Only their continuous care, unconditional love, and incomparable sacrifice made all this possible, and easy to achieve. Miranda A. Farage Kenneth W. Miller Howard I. Maibach
Cincinnati and San Francisco October 2009
Foreword
The population is aging rapidly. Centenarians are no longer a rarity. The fastest growing segment of the population in the United States is people over 80. In the next 25 years, half of the population in the United States will be aged over 50. These shifts will have a tremendous impact on the delivery of healthcare to the elderly and will require a new awareness of how cutaneous disorders affect the quality of life, comprising a heavy burden on health and wellbeing. Physicians and healthcare workers are woefully ignorant of the distress, discomfort, and anxieties of people afflicted by disorders of the skin. There exists a widespread misconception that skin disorders are simply cosmetic nuisances that can be self-treated by a great assortment of anti-aging creams and lotions available at the local drug store. Most of these include high-sounding ingredients such as antioxidants, vitamins, nutrients, botanicals, and ancient folkloristic remedies, the efficacy and safety of which have never been tested. They offer little more than hope in a bottle. The fact is that common skin diseases may not often be lethal but can ruin enjoyment of life. Chronic itchy rashes can be maddening, lowering one’s self-esteem, embarrassing, interfering with sleep, and often accompanied by depression, social isolation, and deterioration of appearance; they can also be uncomfortable, and, not least, costly to treat. The elderly commonly take 15–20 oral supplements daily to fight the ailments of old age. These are generally useless and may be harmful, often interacting adversely with prescription drugs. The elderly often resort to alternative medicines instead of seeing their doctor to obtain FDA-approved drugs, and also often skip their daily doses to save money. Noncompliance is common. Misdiagnosis and mistreatment of the elderly by health-care workers are common. National surveys show that skin diseases increase steadily throughout our lifespan. Old people may have as many as 5–10 coexistent cutaneous problems that are worthy of medical attention. Moreover, the clinical manifestations of skin diseases in the aged often have different appearances than in the young, confounding diagnosis. Importantly, healing of chronic lesions, especially ulcers, is impaired in the elderly. Immunity is weakened, increasing susceptibility to infections. Response to treatment is slower, leading to noncompliance. Adverse drug reactions are common and too commonly not suspected. Management of chronic conditions is difficult and frustrating. The above litany of problems makes this textbook edited by Farage, Miller, and Maibach a welcome addition to the literature. It is invaluable as a reference resource covering exhaustively an enormous number of clinical conditions. No topic is neglected including cosmetic treatments. The numerous contributions are by highly qualified experts who have a published record of expertise. This comprehensive volume is also practical and relevant to the everyday world of clinical practice. The information will be useful to physicians, manufacturers of drugs and skincare products, educators, investigators, nursing home personnel, estheticians, and federal regulators. This first edition is up-to-date, including much new material that belongs to the shelves of every library, which deals with geriatric problems. Dermatologists especially will be remiss if they do not put this volume within easy reach for consultation as they encounter a swelling clientele of aging patients. Albert M. Kligman M.D., Ph.D. Professor Emeritus University of Pennsylvania Philadelphia, PA USA
Preface
The skin is a portal of knowledge on aging. From its softness and smoothness in infancy, through its suppleness in youth, to its wrinkled texture in elders, the skin displays the most visible and accessible manifestations of aging. Due to falling birth rates and rising life expectancies in industrialized countries, the average age of the population is increasing. Research interest in the process of aging has grown and people are becoming obsessed with looking and “staying” young. Although excellent compendia exist on the subject of aging skin, the body of knowledge is burgeoning. Consequently, this handbook compiles information into one comprehensive reference. It covers a range of topics, from the basics of skin structure and function, to the cellular and molecular mechanisms of aging, to the latest bioengineering instruments used to assess age-related changes in the skin. The Nobel Prize in Physiology and Medicine awarded in 2009 to Drs. E. H. Blackburn, C. W. Greider and J. W. Szostak will stimulate research that will ameliorate the effects of aging on the organ systems of both humans and animals. This textbook will simplify approaches when the skin may be an efficient approach to aging based on Dr. Blackburn’s team research. The skin approachability and the opportunities to work on humans will provide us in the near future with rapid therapeutic and preventive applications. Contributors are internationally recognized experts from multiple disciplines germane to this topic. We gratefully acknowledge all contributors for sharing their time and expertise. We expect this handbook to be valuable to researchers and students with an interest in aging skin. Because research progress in this area is so rapid, we hope to update this compendium periodically as advances in the field dictate. The editors welcome suggestions for the second edition. Miranda A. Farage, Kenneth W. Miller, and Howard I. Maibach October 2009
Table of Contents
Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxv
Part 1
Basic Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1
Skin Aging in Animal Models: Histological Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Tapan K. Bhattacharyya
2
Histology of Microvascular Aging of Human Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Peter Helmbold
3
Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Peter Helmbold
Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4
Degenerative Changes in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
5
Skin Aging: A Brief Summary of Characteristic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Christina Raschke . Peter Elsner
6
Physiological Variations During Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Ge´rald E. Pie´rard . Philippe Paquet . Emmanuelle Xhauflaire-Uhoda . Pascale Quatresooz
7
The Stratum Corneum and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Anthony V. Rawlings
8
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Sandy Sercu . Noritaka Oyama . Joseph Merregaert
9
Pathomechanisms of Endogenously Aged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Evgenia Makrantonaki . Christos C. Zouboulis
10
Pathomechanisms of Photoaged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Jean Krutmann
xiv
Table of Contents
11
Proteoglycans in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Franc¸ois-Xavier Maquart . Ste´phane Bre´zillon . Yanusz Wegrowski
12
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases in Skin Aging Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Satoshi Amano
13
Aging and Intrinsic Aging: Pathogenesis and Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Hanan Assaf . Mohamed A. Adly . Mahmoud R. Hussein
14
Buffering Capacity Considerations in the Elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Jacquelyn Levin . Howard I. Maibach
15
Neurotrophins and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
16
Considerations for Thermal Injury: The Elderly as a Sensitive Population . . . . . . . . . . . . . . . . . 159 Donald L. Bjerke
17
Skin Reactivity of the Human Face: Functional Map and Age Related Differences . . . . . . . . . . . 173 Slaheddine Marrakchi . Howard I. Maibach
18
Cluster of Differentiation 1d (CD1d) and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
19
Aging of Epidermal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Alexandra Charruyer . Ruby Ghadially
20
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging . . . . . . . . . . . . . . . . . . . 201 Byung-Soon Park . Won-Serk Kim
21
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Stacy S. Hawkins . William Shingleton . Jean Adamus . Helen Meldrum
22
Hyaluronan and the Process of Aging in Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Robert Stern
23
Changes in Nail in the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Nelly Rubeiz . Ossama Abbas . Abdul Ghani Kibbi
Specialized Skin: Genital . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 24
Vaginal Secretions with Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Paul R. Summers
25
Unique Skin Immunology of the Lower Female Genital Tract with Age . . . . . . . . . . . . . . . . . . . 253 Paul R. Summers
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26
Aging Genital Skin and Hormone Replacement Therapy Benefits . . . . . . . . . . . . . . . . . . . . . . . . 257 William J. Ledger
Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 27
Facial Skin Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Ge´rald E. Pie´rard . Fre´de´rique Henry . Pascale Quatresooz
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 28
Pathology of Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Qunshan Jia . J. Frank Nash
Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 29
Alterations of Energy Metabolism in Cutaneous Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Thomas Blatt . Horst Wenck . Klaus-Peter Wittern
30
Cellular Energy Metabolism and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Regina Hourigan
Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 31
DNA Damage and Repair in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Daniel B. Yarosh
32
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Satoshi Amano
Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 33
Sebum Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Claudine Pie´rard-Franchimont . Pascale Quatresooz . Ge´rald E. Pie´rard
34
Climacteric Aging and Oral Hormone Replacement Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Pascale Quatresooz . Claudine Pie´rard-Franchimont . Ge´rald E. Pie´rard
35
Biological Effects of Estrogen on Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Zack Thompson . Howard I. Maibach
Stratum Corneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 36
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones . . . . . . . . . . . . . . . . . . . 371 Enzo Berardesca . Joachim Fluhr
37
Stratum Corneum Cell Layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 Hachiro Tagami
xv
xvi
Table of Contents
38
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Mutsumi Okazaki
39
Cyanoacrylate Skin Surface Strippings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Claudine Pie´rard-Franchimont . Jorge Arrese-Estrada . Pascale Quatresooz . Ge´rald E. Pie´rard
40
Biology of Stratum Corneum: Tape Stripping and Protein Quantification . . . . . . . . . . . . . . . . . . 401 Ali Alikhan . Howard I. Maibach
Endogenous and Exogenous Factors in Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . 409 41
Effect of Ozone on Cutaneous Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Giuseppe Valacchi
42
Infrared A-induced Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Peter Schroeder . Jean Krutmann
43
Global Warming and its Dermatologic Impact on Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Young Hui . Haw-Yueh Thong . Howard I. Maibach
44
Skin Photodamage Prevention: State of the Art and New Prospects . . . . . . . . . . . . . . . . . . . . . 429 Denize Ainbinder . Elka Touitou
45
Environmental and Genetic Factors in Facial Aging in Twins . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 David J. Rowe . Bahman Guyuron
46
Tobacco Smoke and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 Akimichi Morita
Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 47
DNA Biomarkers in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Kimberly G. Norman . Alex Eshaghian . James E. Sligh
In vitro Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 48
The Use of Reconstructed Skin to Create New In Vitro Models of Skin Aging with Special Emphasis on the Flexibility of Reconstructed Skin . . . . . . . . . . . . . . . . . . . . . . . . . 461 Daniel Asselineau . Sylvie Ricois . Herve´ Pageon . He´le`ne Zucchi . Sole`ne Mine . Sarah Girardeau . Flore Nallet . Se´verine Teluob . Gae¨lle Claviez-Homberg
49
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 Akira Date . Tomohiro Hakozaki
50
Aging of Skin Cells in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 Suresh I. S. Rattan
Table of Contents
Pigmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 51
Hyperpigmentation in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Tomohiro Hakozaki . Cheri L. Swanson . Donald L. Bissett
52
Pigmentation in Ethnic Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503 Rupa Pugashetti . Howard I. Maibach
53
The New Face of Pigmentation and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 John Nip . S. Brian Potterf . Sheila Rocha . Shilpa Vora . Carol Bosko
Part 2
Disease State/Conditions with Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
Diseases Associated with Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 54
Non-neoplastic Disorders of the Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Malignant Skin Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543 55
Neoplastic Skin Lesions in the Elderly Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach . Isaac M. Neuhaus
56
Carcinogenesis: UV Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 Douglas E. Brash . Timothy P. Heffernan . Paul Nghiem
57
Melanoma and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Salina M. Torres . Marianne Berwick
58
Aging-associated Non-melanoma Skin Cancer: A Role for the Dermis . . . . . . . . . . . . . . . . . . . . 587 Davina A. Lewis . Jeffrey B. Travers . Dan F. Spandau
59
Non-surgical Modalities of Treatment for Primary Cutaneous Cancers . . . . . . . . . . . . . . . . . . . . 601 Ossama Abbas . Salah Salman
60
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 Panthea Heydari . Andia Heydari . Howard I. Maibach
Non-Malignant Skin Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617 61
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation . . . . . . . . . . . . . . . . . . . . 619 Jerrold Scott Petrofsky . Gurinder Singh Bains
62
Atopic Dermatitis in the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639 Alexandra Katsarou . Melina C. Armenaka
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63
Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes . . . . . . . . . . . . . . . . . . . 653 Shingo Sakai . Hachiro Tagami
64
Cutaneous Effects and Sensitive Skin with Incontinence in the Aged . . . . . . . . . . . . . . . . . . . . . 663 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Part 3
Techniques and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673
Bioengineering Methods and Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675 65
Bioengineering Methods and Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677 Francesca Giusti . Stefania Seidenari
66
Hydration of the Skin Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687 Hachiro Tagami
67
Transepidermal Water Loss and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695 Ali Alikhan . Klaus-Peter Wilhelm . Fatima S. Alikhan . Howard I. Maibach
68
Corneocyte Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 Tetsuji Hirao
69
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715 Georgios Stamatas
70
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance . . . . . . . . . . 725 Kouichi Nakagawa
71
Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy . . . . . . . . . . . . 735 Jonathan M. Crowther . Paul J. Matts
72
The Measurement and Perception of Uneven Coloration in Aging Skin . . . . . . . . . . . . . . . . . . . 749 Paul J. Matts
Quality of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755 73
Assessing Quality of Life in Older Adult Patients with Skin Disorders . . . . . . . . . . . . . . . . . . . . 757 Miranda A. Farage . Kenneth W. Miller . Susan N. Sherman . Joel Tsevat
Percutaneous Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769 74
Percutaneous Penetration of Chemicals and Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 Michael F. Hughes
75
Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Sara Flores . Farzam Gorouhi . Howard I. Maibach
Table of Contents
Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787 76
Skin Aging: A Generalization of the Micro-inflammatory Hypothesis . . . . . . . . . . . . . . . . . . . . . 789 Paolo U. Giacomoni . Glen Rein
Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797 77
The Potential of Probiotics and Prebiotics for Skin Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799 Arthur C. Ouwehand . Kirsti Tiihonen . Sampo Lahtinen
78
Probiotics in Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811 Benedetta Cinque . Paola Palumbo . Cristina La Torre . Esterina Melchiorre . Daniele Corridoni . Gianfranca Miconi . Luisa Di Marzio . Maria Grazia Cifone . Maurizio Giuliani
Part 4
Toxicological/Safety and General Considerations . . . . . . . . . . . . . . . . . . . . 821
Safety Evaluation for the Elderly Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823 79
Irritant Contact Dermatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825 Florian Seyfarth . Peter Elsner
80
Susceptibility to Irritation in the Elderly: New Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833 Miranda A. Farage . Kenneth W. Miller . G. Frank Gerberick . Cindy A. Ryan . Howard I. Maibach
81
Safety Evaluation in the Elderly via Dermatological Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . 845 Mario Bramante
82
Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly . . . . . . . . . . . . . . . 857 Prashant Rai . Daniel S. Marsman . Susan P. Felter
Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 83
Aging Skin Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871 Duane L. Charbonneau . Yen L. Song . Cheng Xu Liu
84
The Vaginal Microbiota in Menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883 Miranda A. Farage . Kenneth W. Miller . Jack D. Sobel
Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 85
Impaired Wound Repair and Delayed Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 Matthew J. Ranzer . Luisa A. DiPietro
Wrinkles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909 86
Facial Wrinkling: The Marquee Clinical Sign of Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911 Greg G. Hillebrand
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Table of Contents
Scales and Typing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919 87
Assessing Quality of Ordinal Scales Depicting Skin Aging Severity . . . . . . . . . . . . . . . . . . . . . . 921 Fabien Valet . Khaled Ezzedine . Denis Malvy . Jean-Yves Mary . Christiane Guinot
88
The Baumann Skin Typing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929 Leslie S. Baumann
Part 5
Global Skin Aging and its Management: Perception, Needs, Differences and Responses to Skin Aging . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Psychosocial Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947 89
Psychological and Social Implications of Aging Skin: Normal Aging and the Effects of Cutaneous Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949 Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
90
Aging Skin: Some Psychosomatic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959 Madhulika A. Gupta
Aging Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971 91
Facial Skin Attributes and Age Perception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973 Alex Nkengne . Georgios Stamatas . Christiane Bertin
Gender, Ethnicity and Lifestyle Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981 92
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983 Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
93
Gender Differences in Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999 Sarah Fitzmaurice . Howard I. Maibach
94
Aging in Asian Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019 Low Chai Ling
Sensitive Skin and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025 95
Perceptions of Sensitive Skin with Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027 Miranda A. Farage
96
Aging and Skin Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047 Michael K. Robinson
Ingredients and Products for Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053 97
Aging and Anti-aging Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055 Giuseppina Candore . Giovanni Scapagnini . Calogero Caruso
Table of Contents
98
Cosmetics and Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063 Robert L. Bronaugh . Linda M. Katz
99
Cosmetic Anti-aging Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069 Donald L. Bissett . Mary B. Johnson
100
Topical Growth Factors for Skin Rejuvenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079 Vijayeta Rangarajan . Frank Dreher
101
Topical Peptides and Proteins for Aging Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1089 Farzam Gorouhi . Howard I. Maibach
Fem Care Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1119 102
Solutions and Products for Managing Female Urinary Incontinence . . . . . . . . . . . . . . . . . . . . . 1121 David J. Caracci
103
Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine Wipes in Postmenopausal Women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1127 Miranda A. Farage . Kenneth W. Miller . William J. Ledger
Cosmetic Surgeries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137 104
A New Paradigm for the Aging Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1139 Samuel M. Lam
105
Cosmetic Surgery in the Elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147 Dwight Scarborough . Kimberly M. Eickhorst . Emil Bisaccia
106
Facial Rejuvenation: A Chronology of Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175 Alexander S. Donath
Part 6 107
Global Market Place for the Aged . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
Marketing and Product Design of Anti-aging Skin Care Products . . . . . . . . . . . . . . . . . . . . . . . 1187 Nancy C. Dawes
108
Key Trends Driving Anti-aging Skin Care in 2009 and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . 1197 Mary Carmen Gasco-Buisson
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1207
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Editors
Miranda A. Farage, Ph.D. Principal Scientist The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA
[email protected] Kenneth W. Miller, Ph.D. Associate Director The Procter & Gamble Company 6110 Center Hill Avenue Cincinnati, OH 45224 USA
[email protected] Howard I. Maibach, M.D. Professor Department of Dermatology University of California, School of Medicine San Francisco, CA 94122 USA
[email protected] Contributors
Ossama Abbas Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Jean Adamus Unilever R&D Trumbull, CT USA Mohamed A. Adly Department of Zoology Faculty of Science Sohag University Sohag Egypt Denize Ainbinder School of Pharmacy Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel Ali Alikhan Department of Dermatology School of Medicine University of California, Davis Sacramento, CA USA Fatima S. Alikhan University of California, Berkeley Berkeley, CA USA Satoshi Amano Shiseido Research Center Tsuzuki-ku, Yokohama Japan
Melina C. Armenaka Department of Dermatology University of Athens Kesariani, Athens Greece Jorge Arrese-Estrada Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Hanan Assaf Department of Dermatology and Venereology Faculty of Science Sohag University Sohag Egypt Daniel Asselineau L’Ore´al Life Sciences Research Clichy France Gurinder Singh Bains Department of Physical Therapy School of Allied Health Professions Loma Linda University Loma Linda, CA USA Leslie S. Baumann Department of Dermatology and Baumann Cosmetic Medicine and Research Institute University of Miami Miami, FL USA Thomas Blatt Skin Research Centre Beiersdorf AG Hamburg Germany
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Contributors
Enzo Berardesca San Gallicano Dermatological Institute IRRCS Roma Rome Italy Christiane Bertin Johnson & Johnson Consumer France Issy-les-Moulineaux France Marianne Berwick Division of Epidemiology and Biostatistics Department of Internal Medicine University of New Mexico Albuquerque, NM USA Tapan K. Bhattacharyya Department of Otolaryngology-Head & Neck Surgery University of Illinois, Chicago Chicago, IL USA Emil Bisaccia College of Physicians and Surgeons Columbia University New York, NY USA Donald L. Bissett The Procter & Gamble Company Cincinnati, OH USA Donald L. Bjerke The Procter & Gamble Company Cincinnati, OH USA Carol Bosko Unilever R&D Trumbull, CT USA Mario Bramante Procter & Gamble Service GmbH Schwalbach am Taunus, Hesse Germany
Douglas E. Brash Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA Robert L. Bronaugh CFSAN Food and Drug Administration College Park, MD USA Ste´phane Bre´zillon Laboratory of Biochemistry and Molecular Biology Faculty of Medicine University of Reims Champagne-Ardenne Reims France Giuseppina Candore Department of Pathobiology and Biomedical Methodologies University of Palermo Palermo Italy David J. Caracci The Procter & Gamble Company Cincinnati, OH USA Calogero Caruso Department of Pathobiology and Biomedical Methodologies University of Palermo Palermo Italy Duane L. Charbonneau The Procter and Gamble Company Cincinnati, OH USA Alexandra Charruyer Department of Dermatology and Institute for Regeneration Medicine University of California, San Francisco San Francisco, CA USA
Contributors
Maria Grazia Cifone Department of General Pathology Faculty of Medicine and Surgery University of L’Aquila Coppito Italy Benedetta Cinque Department of Health Sciences University of L’Aquila Coppito Italy Gae¨lle Claviez-Homberg L’Ore´al Life Sciences Research Clichy France Daniele Corridoni Department of Health Sciences University of L’Aquila Coppito Italy Jonathan M. Crowther Procter & Gamble Technical Centres Ltd Egham, Surrey UK Akira Date Procter & Gamble Japan K.K Kobe, Hyogo Japan Nancy C. Dawes The Procter and Gamble Company Cincinnati, OH USA Luisa A. DiPietro Center for Wound Healing and Tissue Regeneration College of Dentistry University of Illinois, Chicago Chicago, IL USA Alexander S. Donath Cincinnati Facial Plastic Surgery Cincinnati, OH USA
Frank Dreher NEOCUTIS Inc. San Francisco, CA USA Kimberly M. Eickhorst Procedural Dermatology Fellow Morristown, NJ USA Peter Elsner Department of Dermatology and Allergology University of Jena Jena Germany Alex Eshaghian Department of Internal Medicine University of New Mexico Albuquerque, NM USA Khaled Ezzedine Department of Dermatology CHU Saint-Andre´ Bordeaux France Miranda A. Farage The Procter & Gamble Company Cincinnati, OH USA Susan P. Felter The Procter & Gamble Company Cincinnati, OH USA Sarah Fitzmaurice School of Medicine University of California, Davis Sacramento, CA USA Sara Flores Department of Dermatology School of Medicine University of California, San Francisco San Francisco, CA USA
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Contributors
Joachim Fluhr Bioskin, GmBH Hamburg Germany G. Frank Gerberick The Procter & Gamble Company Cincinnati, OH USA Mary Carmen Gasco-Buisson The Procter and Gamble Company Cincinnati, OH USA Paolo U. Giacomoni Clinique Laboratories Melville, NY USA Ruby Ghadially Department of Dermatology University of California, San Francisco and Veteran’s Affairs Medical Center San Francisco, CA USA Sarah Girardeau L’Ore´al Life Sciences Research Clichy France Maurizio Giuliani Department of Health Sciences University of L’Aquila Coppito Italy Francesca Giusti Department of Dermatology University of Modena and Reggio Emilia Modena Italy Farzam Gorouhi Department of Dermatology University of California, San Francisco San Francisco, CA USA
Christiane Guinot Biometrics and Epidemiology Unit CE.R.I.E.S. Neuilly-sur-Seine France Madhulika A. Gupta Department of Psychiatry Schulich School of Medicine and Dentistry University of Western Ontario London, ON Canada Bahman Guyuron Department of Plastic Surgery University Hospitals of Cleveland and Case Western Reserve University Cleveland, OH USA Tomohiro Hakozaki The Procter & Gamble Company Cincinnati, OH USA Stacy S. Hawkins Unilever R&D Trumbull, CT USA Timothy P. Heffernan Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA Peter Helmbold Department of Dermatology University of Heidelberg Heidelberg Germany Fre´de´rique Henry Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium
Contributors
Andia Heydari University of California, Los Angeles Los Angeles, CA USA Panthea Heydari University of California, Los Angeles Los Angeles, CA USA Greg G. Hillebrand The Procter & Gamble Company Cincinnati, OH USA Tetsuji Hirao Shiseido Research Center Tsuzuki-ku, Yokohama Japan Regina Hourigan Colgate-Palmolive Company Piscataway, NJ USA Michael F. Hughes Office of Research and Development US Environmental Protection Agency National Health and Environmental Effects Research Laboratory Research Triangle Park, NC USA Young Hui University of California, San Francisco San Francisco, CA USA
Mary B. Johnson The Procter & Gamble Company Cincinnati, OH USA Alexandra Katsarou Department of Dermatology University of Athens Kesariani, Athens Greece Linda M. Katz Office of Cosmetics and Colors CFSAN Food and Drug Administration College Park, MD USA Abdul Ghani Kibbi Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Won-Serk Kim Department of Dermatology Kangbuk Samsung Hospital Sungkyunkwan University School of Medicine Seoul Korea Jean Krutmann Environmental Health Research Institute (IUF) Heinrich-Heine-University Duesseldorf Germany
Mahmoud R. Hussein Department of Pathology Faculty of Medicine Assuit University Abha Saudi Arabia
Cristina La Torre Department of Health Sciences University of L’Aquila Coppito Italy
Qunshan Jia The Procter & Gamble Company Cincinnati, OH USA
Sampo Lahtinen Danisco Health and Nutrition Kantvik Finland
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Contributors
Samuel M. Lam Willow Bend Wellness Center and Lam Facial Plastic Surgery Center & Hair Restoration Institute Plano, TX USA
Denis Malvy Department of Internal Medicine and Infectious Diseases CHU St.Andre´ Bordeaux France
William J. Ledger Department of Obstetrics and Gynecology Cornell University-Weill Medical College New York, NY USA
Franc¸ois-Xavier Maquart Laboratoire Central de Biochimie Hoˆpital Robert Debre´ Reims France
Jacquelyn Levin Arizona College of Osteopathic Medicine Midwestern University Glendale, AZ USA
Slaheddine Marrakchi Department of Dermatology Hedi Chaker Hospital Sfax Tunisia
Davina A. Lewis Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA
Daniel S. Marsman The Procter & Gamble Company Cincinnati, OH USA
Low Chai Ling The Sloane Clinic Singapore Singapore Cheng Xu Liu The Procter and Gamble Company Cincinnati, OH USA Howard I. Maibach Department of Dermatology School of Medicine University of California, San Francisco San Francisco, CA USA Evgenia Makrantonaki Departments of Dermatology, Venereology Allergology and Immunology Dessau Medical Center Dessau Germany
Jean-Yves Mary INSERM Department of Biostatistics and Clinical Epidemiology Saint-Louis Hospital Paris France Luisa Di Marzio Department of Health Sciences University of L’Aquila Coppito Italy Paul J. Matts Procter & Gamble Technical Centres Ltd Egham, Surrey UK Esterina Melchiorre Department of Health Sciences University of L’Aquila Coppito Italy
Contributors
Helen Meldrum Unilever R&D Trumbull, CT USA Joseph Merregaert Laboratory of Molecular Biotechnology Department of Biomedical Sciences University of Antwerp Antwerp Belgium Gianfranca Miconi Department of Health Sciences University of L’Aquila Coppito Italy Kenneth W. Miller The Procter & Gamble Company Cincinnati, OH USA Sole`ne Mine L’Ore´al Life Sciences Research Clichy France Akimichi Morita Department of Geriatric and Environmental Dermatology Nagoya City University Nagoya Japan Kouichi Nakagawa RI Research center Fukushima Medical University Fukushima Japan Flore Nallet L’Ore´al Life Sciences Research Clichy France J. Frank Nash The Procter & Gamble Company Cincinnati, OH USA
Isaac M. Neuhaus Department of Dermatology University of California, San Francisco San Francisco, CA USA Paul Nghiem Departments of Therapeutic Radiology, Genetics and Dermatology School of Medicine Yale University New Haven, CT USA
John Nip Unilever R&D Trumbull, CT USA Alex Nkengne Johnson & Johnson Consumer France Issy-les-Moulineaux France Kimberly G. Norman Vanderbilt University Medical Center Nashville, TN USA Mutsumi Okazaki Department of Plastic and Reconstructive Surgery Tokyo Medical and Dental University Bunkyo-ku, Tokyo Japan
Arthur C. Ouwehand Danisco Health and Nutrition Kantvik Finland
Noritaka Oyama Department of Dermatology School of Medicine Fukushima Medical University Fukushima Japan
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Contributors
Herve´ Pageon L’Ore´al Life Sciences Research Clichy France Paola Palumbo Department of Health Sciences University of L’Aquila Coppito Italy Philippe Paquet Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Byung-Soon Park Department of Dermatology College of Medicine Seoul National University Seoul Korea Jerrold Scott Petrofsky School of Allied Health Loma Linda University Loma Linda, CA USA Ge´rald E. Pie´rard Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Claudine Pie´rard-Franchimont Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium S. Brian Potterf Unilever R&D Trumbull, CT USA
Rupa Pugashetti UCSF Psoriasis Treatment Center School of Medicine University of California, Irvine Irvine, CA USA Pascale Quatresooz Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium Prashant Rai Procter and Gamble Japan KK Higashinada-ku Japan Vijayeta Rangarajan NEOCUTIS Inc. San Francisco, CA USA Matthew J. Ranzer Center for Wound Healing and Tissue Regeneration College of Dentistry University of Illinois, Chicago Chicago, IL USA Christina Raschke Department of Dermatology and Allergology University of Jena Jena Germany Suresh I. S. Rattan Laboratory of Cellular Ageing Department of Molecular Biology Aarhus University Aarhus Denmark Anthony V. Rawlings AVR Consulting Ltd Northwich, Cheshire England UK
Contributors
Glen Rein Estee Lauder Companies Melville, NY USA Sylvie Ricois L’Ore´al Life Sciences Research Clichy France Michael K. Robinson The Procter & Gamble Company Cincinnati, OH USA Sheila Rocha Unilever R&D Trumbull, CT USA David J. Rowe University Hospitals Case Western Reserve University Cleveland, OH USA Nelly Rubeiz Department of Dermatology American University of Beirut Medical Center Beirut Lebanon Cindy A. Ryan The Procter & Gamble Company Cincinnati, OH USA Shingo Sakai Basic Research Laboratory Kanebo Cosmetics Inc. Kanagawa Japan Salah Salman Department of Dermatology American University of Beirut Medical Center Beirut Lebanon
Giovanni Scapagnini Department of Health Sciences University of Molise Campobasso Italy Dwight Scarborough Division of Dermatology Ohio State University Hospital Columbus, OH USA Peter Schroeder Environmental Health Research Institute (IUF) Heinrich-Heine-University Duesseldorf Germany Stefania Seidenari Department of Dermatology University of Modena and Reggio Emilia Modena Italy Sandy Sercu Laboratory of Molecular Biotechnology Department of Biomedical Sciences University of Antwerp Antwerp Belgium Florian Seyfarth Department of Dermatology and Allergology University of Jena Jena Germany Susan N. Sherman SNS Research Cincinnati, OH USA William Shingleton Unilever R&D Colworth UK James E. Sligh Skin Diseases Research Center Vanderbilt University Medical Center Nashville, TN USA
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Contributors
Jack D. Sobel Division of Infectious Diseases Department of Internal Medicine Wayne State University Detroit, MI USA Yen L. Song The Procter and Gamble Company Cincinnati, OH USA Dan F. Spandau Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA Georgios Stamatas Johnson & Johnson Consumer France Issy-les-Moulineaux France Robert Stern Department of Pathology Touro College of Osteopathic Medicine New York, NY USA Paul R. Summers University Health Care University of Utah Salt Lake City, UT USA Cheri L. Swanson The Procter & Gamble Company Cincinnati, OH USA Hachiro Tagami Department of Dermatology Tohoku University School of Medicine Sendai Japan Se´verine Teluob L’Ore´al Life Sciences Research Clichy ce´dex France
Zack Thompson University of California, San Francisco San Francisco, CA USA Haw-Yueh Thong Department of Dermatology University of California, San Francisco San Francisco, CA USA Kirsti Tiihonen Danisco Health and Nutrition Kantvik Finland Salina M. Torres Division of Epidemiology and Biostatistics Department of Internal Medicine University of New Mexico Albuquerque, NM USA Elka Touitou School of Pharmacy Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel Jeffrey B. Travers Department of Dermatology School of Medicine Indiana University Indianapolis, IN USA Joel Tsevat Department of Internal Medicine College of Medicine University of Cincinnati Cincinnati, OH USA Giuseppe Valacchi Department of Biomedical Sciences University of Siena Siena Italy
Contributors
Fabien Valet INSERM, Biostatistics and Clinical Epidemiology DBIM, Saint-Louis Hospital University Paris 7 France
Klaus-Peter Wittern Skin Research Centre Beiersdorf AG Hamburg Germany
Shilpa Vora Unilever R&D Bangalore India
Emmanuelle Xhauflaire-Uhoda Laboratory of Skin Bioengineering and Imaging Department of Dermatopathology University Hospital of Lie`ge Lie`ge Belgium
Yanusz Wegrowski Laboratory of Biochemistry and Molecular Biology Faculty of Medicie University of Reims Champagne-Ardenne Reims France Horst Wenck Skin Research Centre Beiersdorf AG Hamburg Germany Klaus-Peter Wilhelm proDERM Institut fuer Angewandte Dermatologische Forschung GmbH Hamburg Germany
Daniel B. Yarosh Estee Lauder Companies Melville, NY USA Christos C. Zouboulis Institute of Clinical Pharmacology & Department of Toxicology Charite´ Universitaetsmedizin Berlin Campus Benjamin Franklin Berlin Germany He´le`ne Zucchi L’Ore´al Life Sciences Research Clichy France
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20 Adipose-derived Stem Cells and their Secretory Factors for Skin Aging Byung-Soon Park . Won-Serk Kim
Introduction The term ‘‘stem cell’’ has attracted increasing attention of the scientific community as well as of the general public. In many aspects, however, it is still confusing and difficult to understand and interpret information about stem cells. They are vital to humans for numerous reasons. Groups of stem cells in some adult tissues give rise to replacement cells for the tissues that are destroyed through injury, disease, or aging [1]. Knowledge relating to how healthy cells replace diseased or otherwise damaged cells, would allow development of medical therapies focusing on creation of compatible cell lines to replace aged or diseased cells in the body. The concept of regenerative medicine using the body’s own stem cells and growth factors to repair tissue may be realizable as science and clinical experience converge to develop alternative therapeutic strategies to treat the damaged or diseased tissue. Stem cell-based therapies are also being tried in tissue engineering: The aim of tissue engineering is to repair and regenerate damaged organs or tissues using a combination of cells, biomaterials, and cytokines [1–4]. This chapter addresses the human subcutaneous adipose tissue as a promising source of adult stem cells. Adipose-derived stem cells (ADSCs) may offer a solution for the problem of limited availability of human cells that are capable of self-renewal and differentiation. ADSCs can be easily obtained from liposuction of human adipose tissue, cultured in a large scale, and display multi-lineage developmental plasticity. In addition, ADSCs secrete various cytokines and growth factors, which control and manage the damaged neighboring cells, and this has been identified as essential functions of ADSCs [5–7]. As reviewed elsewhere in this book, aging and photoaging are complex processes involving the wound-healing cascade and/or repetitive oxidative stress. Conventional anti-aging skin treatments such as light-based or radiofrequency devices and/or peelings have been less than satisfactory because their primary mechanism is mainly inducing new collagen synthesis via activation of dermal fibroblasts. On the basis of previous studies that
demonstrated wound healing, antioxidant, antiwrinkle, and antimelanogenic effects of ADSCs and their secretory factors, they may be good candidates for the treatment of aging [5–9]. Therefore, this chapter describes the authors’ recent research and clinical developments on the anti-aging effects of ADSCs and their secretory factors.
Stem Cells and ADSCs Stem cells are a population of immature tissue precursor cells capable of self-renewal and provision of multi-lineage differentiable cells for tissues. Although embryonic stem cell has multi-potency, there are many limitations such as difficulties in control of differentiation and issues relating to ethics. As a result, use of adult stem cells with fewer implicating issues is becoming an area of increased interest in stem cell medicine. Given the vast potential of treatments utilizing stem cells, validation and evaluation regarding safety and efficacy will result in greater benefits.
ADSCs and Regeneration Due to the lack of a specific and universal molecular marker for adult stem cells, functional assays for multiple differentiations must be used to identify stem cells in a tissue. Mesenchymal stem cells (MSCs) were first characterized in bone marrow, but many studies have reported the existence of MSCs in the connective tissue of several organs [10, 11]. The role of these cells is not entirely clear, but they are generally believed to constitute a reserve for tissue maintenance and repair. It was recently demonstrated that the most abundant and accessible source of adult stem cells is adipose tissue. The yield of MSCs from adipose tissue is approximately 40-fold greater than that from bone marrow [12–14]. The following are the highly consistent, although not identical, expression profiles of cell-surface proteins on ADSCs [2, 15]: adhesion molecules, receptor molecules, surface enzymes, extracellular matrix (ECM) proteins,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_20, # Springer-Verlag Berlin Heidelberg 2010
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and glycoproteins. However, hematopoietic cell markers such as CD14, CD31, and CD45 are not expressed. Interestingly, the immunophenotype of ADSCs resembles that reported for other adult stem cells prepared from human bone marrow (bone marrow stromal cell [BMSC]) and skeletal muscle [2]. Differentiation of ADSCs is not restricted to the adipocyte lineage, but they can be differentiated into chondrocyte, osteocyte, cardiomyocyte, neuron, etc. [16, 17]. In addition, activity comparison with BMSC revealed a similar regenerative capacity. Therefore, this abundant and accessible cell population has potential clinical utility for regenerating damaged or aged tissue and tissue engineering. As with many rapidly developing fields, diverse names have been proposed to describe the plastic-adherent cell population isolated from collagenase digests of adipose tissue: adipose-derived stem/stromal cells, adiposederived adult stem cells, adipose-derived adult stromal cells, adipose stromal cells (ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblast, pericyte, preadipocyte, and processed lipoaspirate (PLA) cells. To address the confusion due to diverse nomenclature, the International Fat Applied Technology Society reached a consensus to adopt the term ‘‘adipose-derived stem cells’’ to identify the isolated, plastic-adherent, multipotent cell population. Questioning the validity of the term ‘‘stem cell’’, led to the use of the acronym to mean ‘‘adipose-derived stromal cells’’ [18]. Although studies are limited, the quality and quantity of the ADSCs varies according to interperson differences, the harvest site, harvesting method, and culture conditions. Age and sex are the most obvious of the interperson differences. Stem cell recovery varies between subcutaneous white adipose tissue depots [19, 20]. Yield and growth characteristics of ADSC (> Fig. 20.1) are also affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction [21].
Mechanism of Action for Regeneration Stem cell therapy is a safe, practical, and effective source for repair of damaged tissue [22, 23]. Despite rapid translation to the bedside, the mechanism of action for regeneration is not well characterized. It was initially hypothesized that immature stem cells migrate to the injured area, differentiate into the phenotype of injured
. Figure 20.1 ADSCs display adherent and fibroblastic morphology. They show abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume (Reprinted with permission from Elsevier, Kim WS et al. [6])
tissue, repopulate the diseased organ with healthy cells, and subsequently repair the tissue (building-block function). However, this theory has some drawbacks because the levels of engraftment and survival of engrafted cells are too low to be therapeutically relevant [24]. In addition, acute stem cell-mediated improvement within days or even hours makes it difficult to fully explain the mechanisms by which regeneration occurs [25, 26]. Instead, much of the functional improvement and attenuation of injury afforded by stem cells can be repeated by treatment with cell-free conditioned media derived from ADSCs (ADSCCM) [27]. Thus, it can be deduced that ADSCs may exert their beneficial effects via complex paracrine actions (manager function) in addition to building-block function.
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
Proteomic Analysis of ADSCs and Their Secretomes Proteomics, large-scale studies of proteins, can be used to analyze the intracellular and secretory proteins of ADSCs. For example, Roche et al., conducted a 2-DE gel analysis of BMSCs and ADSCs, and confirmed the similarity [28]. Zvonic et al., also analyzed the ADSC-CM by 2-DE gel electrophoresis, detected approximately 300 features from ADSC-CM, and found that secretomes are up-/downregulated by induction of adipogenesis [29]. Although the intracellular and secretory proteins of ADSCs have been analyzed through 2-DE-coupled mass spectrometry or non-gel-based mass spectrometry, the active proteins of ADSCs responsible for the tissue regeneration are not fully identified. This may be due to the fact that studies using proteomics has limitations as this approach is capable of analyzing highly abundant proteins only. Therefore, new mass spectrometry-based proteomic analysis techniques for stem cell proteins in correlation with other state-of-the-art analytical tools and functional study by neutralizing the candidate proteins are needed to clearly characterize the active proteins of regeneration.
Diverse Pharmacologic Actions of ADSCs and Their Secretory Factors Wound-Healing Effect of ADSCs Several studies of the pathophysiology of photoaging have detected similarities with certain aspects of acute and/or chronic wounds. Histologically, photoaged skin shows marked alterations in ECM composition. Skin wound repair by adult stem cells was originally demonstrated using BMSC. Wu et al. showed that BMSC injection around the wound significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts [30]. Sasaki et al. demonstrated that BMSCs can differentiate into multiple skin cell types including keratinocytes, pericytes, and endothelial cells, which contribute to wound repair [31]. Notably, analyses of proteins in conditioned medium of BMSC (BMSC-CM) indicated that BMSCs secret distinctively different cytokines and chemokines compared to dermal fibroblasts [32]. ADSCs have surface markers and gene profiling similar to BMSCs and their soluble factors are not significantly different [6, 10]. Given their convenient isolation compared with BMSCs and extensive proliferative capacities ex vivo, ADSCs hold great promise
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for use in wound repair and regeneration. However, there is little evidence demonstrating the wound-healing effects of ADSCs. It was also demonstrated that ADSCs accelerate wound healing, especially with regard to fibroblast activation [6]. They promote proliferation of dermal fibroblasts, not only by direct cell-to-cell contact, but also by paracrine activation through secretory factors. This fibroblast-stimulating effect of ADSCs was superior to that of the fibroblasts. Furthermore, ADSC-CM enhanced secretion of type I collagen from dermal fibroblasts and stimulated fibroblast migration in in vitro wound-healing models. ADSCs secreted a variety of growth factors such as basic fibroblast growth factor (bFGF), KGF, TGF-b, hepatocyte growth factor (HGF), and VEGF into the conditioned medium, which might mediate the woundhealing effect of ADSCs. In addition to the in vitro evidence, the wound-healing effect of ADSCs was also verified in an animal study, which showed that topical administration of ADSCs significantly reduced the wound size (34% reduction) and accelerated the re-epithelialization at the wound edge (> Fig. 20.2). Similar to ADSC treatment, ADSC-CM treatment also accelerated wound healing in laser-induced burn mouse models (authors’ unpublished data). In this experiment, burn wounds were made by laser surgery in the epidermis and they were significantly reduced by single and multiple administration of ADSC-CM. As ADSCs are physiologically located beneath dermal fibroblasts, they may interact with dermal fibroblasts. However, ADSCs and secretomes of ADSCs may reach the epidermis in wounded area and may affect the recovery of this layer. As such, ADSC-CM was treated in cultured primary human keratinocytes and shown to increase the proliferation and migration of keratinocytes (authors’ unpublished data). This result suggests that secretomes of ADSC also accelerate the healing of epidermal layer.
Antioxidant and Antimelanogenic Effects of ADSC Reactive oxygen species (ROS) produced in the catalytic reactions by many environmental stimuli may be involved in the pathogenesis of a number of skin disorders including photoaging, photosensitivity diseases, and some types of cutaneous malignancy. Antioxidants, as a popular term in drug and cosmetics, take the form of enzymes, hormones, vitamins, and minerals. In biological systems, the normal processes of oxidation produce highly reactive free radicals, which may continue to damage even the
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. Figure 20.2 Wound healing effect of ADSCs in nude mice. Artificial wounds were made using a 6-mm punch biopsy and ADSCs were topically applied. The wound size was reduced significantly in the ADSC-treated side (right side of the back) 7 days after surgery (Reprinted with permission from Elsevier, Kim WS et al. [6])
body’s own cells. Antioxidants scavenge free radicals before they get a chance to harm the body. As of now, there are few reports on the antioxidant action of stem cells. However, some evidences support the protective role of secretomes of ADSCs against the skin damage induced by reactive oxygen species. For example, IGF reportedly protects fibroblasts and intestinal epithelial cells from free radicals [33, 34]. HGF protects the retinal pigment epithelium against oxidative stress induced by glutathione depletion [35]. Pigment epithelium-derived factor (PEDF) is an anti-angiogenic/neurotropic factor and has been shown to have antioxidant effects [36]. Interleukin-6 (IL-6) reduces the epithelial cell death induced by hydrogen peroxide [37]. In addition, subtypes of superoxide dismutase (SOD) are expressed and secreted from ADSC [38]. Therefore, antioxidant function of ADSC was investigated in dermal fibroblasts after inducing chemical oxidative stress by the tert-butyl hydroperoxide (tbOOH). Morphological change and cell survival assay revealed that incubation with ADSC-CM aided dermal fibroblasts to resist free radicals induced by tbOOH. In addition, activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were enhanced in the dermal fibroblasts treated with ADSC-CM. In a cell cycle analysis, ADSC-CM treatment reversed the apoptotic cell death induced by ROS, which was demonstrated by a significant decrease of subG1 phase of dermal fibroblasts [8]. Photoaging is believed to be responsible for up to almost 80% of the skin changes commonly attributed to the aging process. The study further investigated the antioxidant and protective effects of ADSCs in the photodamage of the primarily cultured dermal fibroblasts (> Fig. 20.3). In this experiment, ADSC-CM pretreatment significantly
. Figure 20.3 Antioxidant effect of ADSCs in UVB-irradiated fibroblasts as shown by cell cycle analysis of DNA contents. Untreated fibroblasts showed little or no sub-G1 phases (a). However, UVB irradiation significantly increased sub-G1 (apoptotic) cells (b), which were reversed by ADSC-CM pretreatment (c) (Reprinted with permission from Elsevier, Kim WS et al. [5])
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
reduced the apoptosis of dermal fibroblasts from UVBinduced damage, which was demonstrated by a significant decrease of sub-G1 phase of dermal fibroblasts after ADSC-CM pretreatment. In addition, ADSC-CM treatment increased the production of collagen and reduced the expression of matrix metalloproteinase-1 in the dermal fibroblasts. These results indicated that ADSCs can play a key role in protecting dermal fibroblast from UVBinduced oxidative stress [5]. As antioxidants inhibit the chemical reactions leading to melanin formation, change the type of melanin formed,
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and interfere with the distribution of pigment and melanosome transfer, they are good candidates for skin whitening resources. As ADSC-CM is a free radical scavenger and has potent antioxidant activity, antimelanogenic effect of ADSC was investigated. ADSC-CM treatment inhibited the synthesis of melanin and the activity of tyrosinase in melanoma B16 cells. In addition, expressions of tyrosinase and tyrosinase-relating protein 1 were down-regulated by ADSC-CM treatment, which indicated the mechanism of action for antimelanogenic effect of ADSCs and their soluble factors (> Fig. 20.4) [9].
. Figure 20.4 (a) Antimelanogenic effect of ADSC-CM. Expression of MITF and TRP2 remained unchanged, but expressions of tyrosinase and TRP1 were down-regulated by ADSC-CM treatment in B16 melanoma cells. (b) The inhibitory effect of ADSC on melanin synthesis is schematically represented (Reproduced with permission from Pharmacological Society of Japan, Kim WS et al. [9])
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Animal Studies for Skin Aging To study the effects on in vivo skin, ADSCs (1 106 cells) and ADSCs-CM were intradermally injected on the back of a micropig, twice in a 14-day interval (n = 3). One month after the second injection, skin samples were obtained at the treatment and the control sites of adjacent normal skin. Although the increase in the dermal thickness was not significant, increased collagen expression was noted by western blot in the ADSCs- and ADSCs-CMtreated skin samples (> Fig. 20.5) [7]. In another experiment, photodamage was induced by an 8-week UVB irradiation in hairless mice. The irradiation dose was one MED (minimal erythema dose; 60 mJ/cm2) in the first 2 weeks, two MED in the third week, three MED in the forth week, and four MED in the fifth through eighth weeks. After wrinkle induction, varying numbers of ADSCs (A group: control; B group: 1 103 cells; C group: 1 104 cells; and D group: 1 105 cells) were subcutaneously injected into the mice (n = 8 for each group). In a replica analysis, parameters involving skin roughness were improved with mid-level and higher dose groups of ADSCs (C and D group) (> Fig. 20.6). Dermal thickness was increased in the ADSC-injected groups (16% and 28% in C and D groups, respectively) and ECM contents in the dermis were also increased by Massson’s trichrome staining results of collagen (blue) in the ADSCtreated groups (> Fig. 20.7). As cell transplantation between species mediates immune rejection, the survival of
ADSC from humans was investigated after injection of ADSCs labeled with PKH26 (red color, > Fig. 20.8 inset). As shown in > Fig. 20.8, survival of the ADSCs was clearly demonstrated [5].
Clinical Application of ADSCs and the ADSC Protein Extract ADSCs and the ADSC Protein Extract for Skin Aging As a pilot study, intradermal injections of purified autologous PLA cells (1 106 cells), which contain approximately 20–30% ADSCs, were tried with photoaged skin of one patient [7] after informed conset. The female patient had two successive injections at 2-week intervals. Two months after the second injection, the patient showed improvements in general skin texture and wrinkling as evidenced by medical photographs of periorbital wrinkles. Measurements of dermal thickness by a 20 MHz high-frequency ultrasonographs (Dermascan-C, Cortex, Hadsund, Denmark) also indicated increased thickness (2.054 vs. 2.317 mm) (> Fig. 20.9). In a large-scale pilot study, the effects of the ADSC protein extract applied transdermally in the treatment of the various signs of skin aging were evaluated: (1) wrinkles, (2) acquired pigmentary lesions, and (3) dilated pores [39]. Korean patients visiting for the treatment of
. Figure 20.5 Micropig experiment shows the change of dermal thickness without (a) and with (b) intradermal injections of ADSCs. Increased collagen expression was noted by western blot in the ADSCs- and ADSCs-CM-treated skin (c) (Reproduced with permission from Wiley-Blackwell, Park BS et al. [7])
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. Figure 20.6 Antiwrinkle effects of ADSCs. Photodamage was induced by 8-week UVB irradiation in hairless mice, and ADSCs were intradermally and subcutaneously injected three times. Wrinkles were evaluated by replica analysis. (a) Control; (b) 1 ¥ 103 cells; (c) 1 ¥ 104 cells; (d) 1 ¥ 105 cells (Reprined with permission from Elsevier, Kim WS et al. [5])
. Figure 20.7 Massson’s trichrome staining shows that collagen contents (blue) are significantly increased in the mid-level and higher dose groups of ADSCs (c and d groups in photodamaged hairless mice experiment) (Reprined with permission from Elsevier, Kim WS et al. [5])
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. Figure 20.8 Survival of ADSCs labeled with PKH26 (insert) injected in the skin of hairless mice. Two weeks after injection, mouse skin block was cryosectioned and counterstained with green-fluorescent nucleic acid stain. ADSCs are stained red (Reprined with permission from Elsevier, Kim WS et al. [5])
skin aging were recruited during September 2006–August 2007. The population (n = 235) aged 28–71 years (mean 41 years) had skin phototypes III and IV with mild to moderate photodamage. The advanced ADSC Protein Extract(AAPE; Prostemics Inc., Seoul, Korea) was applied three to twelve times at two-week intervals. The changes were evaluated objectively by photographic documentation and Robo Skin Aanlyzer CS100/VA100 (Inforward Inc., Tokyo, Japan), and subjectively by patient questionnaire. The evaluation score was based upon the following scales: 0 = poor/worsend; 1 = no change/no change; 2 = fair/mild improvement; 3 = good/moderate improvement; and 4 = excellent/marked improvement. As compared to 47.4% showing good to excellent improvement in wrinkle, 63% of the patients were judged to have good to excellent improvement in acquired pigmentary lesions and dilated pores (> Fig. 20.10). Melasma is a multifactorial disorder caused by sun exposure, hormonal imbalance, and genetic predisposition. In many countries including Asia, melasma ranks among the top ten most common skin conditions. Ethnic differences between Asian and other skin types may influence the efficacy and tolerability of melasma treatments.
. Figure 20.9 Clinical study using intradermal injections of purified autologous PLA cells. Medical photographs of periorbital wrinkles were taken before (a) and after (b) treatment, and dermal thickness was measured by ultrasonographs before (c) and after (d) treatment. Improved general skin texture and increase thickness (2.054 vs. 2.317 mm) were evident 2 months after two injections (b and d) (Reprined with permission from Wiley-Blackwell, Park BS et al. [7])
Adipose-derived Stem Cells and their Secretory Factors for Skin Aging
. Figure 20.10 Objective and subjective evaluation of the protein extracts from ADSC-CM in a large-scale (n = 235) pilot study in terms of: (a) wrinkles, (b) acquired pigmentary lesions, and (c) dilated pores. The evaluation score is based upon the following scales: 0 = poor/worsend; 1 = no change/no change; 2 = fair/mild improvement; 3 = good/moderate improvement; and 4 = excellent/marked improvement. As compared to 47.4% showing good to excellent improvement in wrinkle (a), 63% of the patients were judged to have good to excellent improvement in acquired pigmentary lesions (b) and dilated pores (c)
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Recent clinicopathologic studies on melasma show that lesional skin showed more prominent solar elastosis when compared to the normal skin [40]. Moreover, it has been suggested that interactions between the cutaneous vasculature and melanocytes might have an influence on the development of pigmentation [41]. The coexistence of telangiectasia and/or solar elastosis with melasma points out that photodamage is closely linked to the pathogenesis of melasma. The actions of ADSCs in wound healing, antioxidation, antimelanogenic effects and the reversal of photodamage in vitro, and in animal models prompted the clinicians to bring these biologic actions to bedside. The representative cases with marked response in photoaging and melasma were shown in > Fig. 20.11a–d. These clinical results for the past 4 years suggest that the ADSCs and the protein extract are promising rational strategies for melasma and photodamage.
Combination with Other Procedures and Active Transdermal Delivery Various light source and radiofrequency devices have been used for the treatment of skin aging by selectively heating up the collagen in the dermis to stimulate collagen remodeling. In general, both ablative and nonablative techniques lead to new collagen formation. As ADSCs and their secretory factors promote the wound healing by activating dermal fibroblasts, it can be speculated that when combined, ADSCs and the protein extracts might augment the clinical effects beyond the intrinsic fibroblast-stimulatory effect of the various devices. Based upon the previous documentation of woundhealing and antimelanogenic effects of ADSCs, the efficacy of the protein extract of ADSCs in reducing healing time and PIH or erythema was investigated after fractional CO2 laser treatment (MiXto SX1, Lasering, Italy) in a pilot study as prospective, randomized, placebocontrolled, double-blinded, and split-face setting [42]. CO2 fractional treatments have emerged as one of the new technologies in skin rejuvenation. However, comparatively increased incidence of PIH is problematic especially in dark-skinned patients. In this study, Korean patients of Fitzpatrick skin types III and IV (mean age 45.7 years) with facial wrinkles were treated with full-face fractional CO2 laser (Parameter: 8 W, index level 8). All subjects were randomly allocated to split-face application of either the protein extracts of ADSCs or emollient only. Serial photographs were taken at each visit during the treatment and three-month follow-up period. Marked difference in
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. Figure 20.11 The representative cases with marked response before (a, c) and after (b, d) treatment with the protein extracts of ADSCs
the duration of erythema and healing was observed (> Fig. 20.12). The quality of wound healing was noted to be improved. This therapy was well-tolerated by majority of patients with minimal adverse effects. It was concluded that the protein extracts of ADSCs can be safely and effectively used to prevent PIH and to accelerate wound healing after fractional CO2 laser treatment in dark skin. As the secretory factors of ADSC generally contain ingredients of large molecular weights, various new ‘‘active’’ enhancement technologies designed to transiently circumvent the barrier function of the stratum corneum would be required for transdermal delivery: e.g., iontophoresis, sonophoresis, electroporation, or microneedle arrays or skinstamp.
Conclusion The current topics of increasing interest in the dermatological field are anatomical–functional damage to the skin
and every possible means to counteract the injurious effects. In the beginning, ADSCs were shown to increase the survival rate in fat transplantation [43]. This chapter explains that ADSCs and their secretory factors have diverse pharmacologic effects for skin aging. However, clinical application of cultured ADSCs for human skin is in the early stage and might be related to issues/concerns: the threat of passing on viruses, passing on diseases from other animal source nutrients to cultured stem cells in the laboratory, uncontrolled growth, and misdirected growth, especially of embryonic stem cells. In addition, ADSCs have to overcome the obstacles in that they are difficult both to handle and to commercialize in an industrial point of view: how to store the ADSCs, the containers to store them, how to transport them to the point, and the shelf life in various environments. Therefore, new methods and materials to overcome these limitations are needed. Secretomes of ADSC have some advantages over cell-based therapies and might have greater potential in skin regeneration, because they can
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. Figure 20.12 A split-face comparison shows that the application of the protein extract of ADSCs results in less intense erythema and microcrusting 2 days after fractional CO2 laser resurfacing
be manufactured in a large scale with long-term stability and they are relatively devoid of safety issues. As such, the study demonstrated that photodamage can be reversed by utilizing the ADSCs/their secretory factors alone [5, 7, 39], or in combination with other devices minimizing unwanted effects [42]. Identification of active proteins will be the next goal, and drug development using these proteins will suggest better strategies for skin aging in the future.
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32. Chen L, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE. 2008;3:e1886. 33. Baregamian N, et al. IGF-1 protects intestinal epithelial cells from oxidative stressinduced apoptosis. Surg Res. 2006;136:31–37. 34. Rahman ZA, et al. Antioxidant effects of glutathione and IGF in a hyperglycaemic cell culture model of fibroblasts: some actions of advanced glycaemic end products (AGE) and nicotine. Endocr Metab Immune Disord Drug Targets. 2006;6:279–286. 35. Shibuki H, et al. Expression and neuroprotective effect of hepatocyte growth factor in retinal ischemia-reperfusion injury. Invest Ophthalmol Visual Sci. 2002;43:528–536. 36. Tsao YP, et al. Pigment epithelium derived factor inhibits oxidative stress-induced cell death by activation of extracellular signalregulated kinases in cultured retinal pigment epithelial cells. Life Sci. 2006;79:545–550. 37. Kida H, et al. Protective effect of IL-6 on alveolar epithelial cell death induced by hydrogen peroxide. Am J Physiol. 2005;288:342–349. 38. Liochev SI, et al. How does superoxide dismutase protect against tumor necrosis factor: a hypothesis informed by effect of superoxide on ‘‘free’’ iron. Free Radic Biol Med. 1997;23:668–671. 39. Kang SH, et al. Improvement of melasma and scars with the secretory factors from ADSCs. Korean J Dermatol. 2007;45(Suppl. 2):136. 40. Kang WH, et al. Melasma: histopathological characteristics in 56 Korean patients. Br J Dermatol. 2002;146:228–237. 41. Kim EH, et al. The vascular charateristics of melasma. J Dermatol Sci. 2007;46:111–116. 42. Park BS, et al. Rejuvenation of aging skin using fractional CO2 laser resurfacing followed by topical application of ADSC protein extract. Kor J Dermatol. 2008;46(Suppl 1):266–267. 43. Matsumoto D, et al. Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12:3375–3382.
13 Aging and Intrinsic Aging: Pathogenesis and Manifestations Hanan Assaf . Mohamed A. Adly . Mahmoud R. Hussein
Introduction Cutaneous aging is a complex biological phenomenon consisting of two components: intrinsic aging and extrinsic aging. Intrinsic aging is also termed true aging which is an inevitable change attributable to the passage of time alone and is manifested primarily by physiologic alterations with subtle but undoubtedly important consequences for both healthy and diseased skin and is largely genetically determined [1]. Extrinsic aging is caused by environmental exposure, primarily to UV light, and more commonly termed photoaging. In sun-exposed areas, photoaging involves changes in cellular biosynthetic activity that lead to gross disorganisation of the dermal matrix [2]. The intrinsic rate of skin aging in any individual can be dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light. Photodamage, which considerably accelerates the visible aging of skin, also greatly increases the risk of cutaneous neoplasms. So, the processes of intrinsic and extrinsic aging are superimposed. As the population ages, dermatological focus must shift from ameliorating the cosmetic consequences of skin aging to decreasing the genuine morbidity associated with problems of the aging skin. Therefore, a better understanding of both the intrinsic and extrinsic influences on the aging of the skin, as well as distinguishing the retractable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the irretractable cutaneous aging (primarily intrinsic aging), is very important to solve the problem of aging [2].
Pathogenesis of Intrinsic Aging Logic dictates that one or more molecular events must underlie the aging process. These changes are now beginning to be unraveled and are discussed. As these mechanisms are identified, further insights into the underlying processes of skin aging should emerge and better strategies to prevent the undesirable effects of age on skin appearance should follow. The process of intrinsic
skin aging resembles that seen in most internal organs and an explanation is thought to involve decreased proliferative capacity leading to cellular senescence, and altered biosynthetic activity of skin derived cells [2]. The molecular mechanisms partly underlying skin aging comprise a multifaceted process influenced by various factors affecting different body sites at variable degrees. A stochastic process that implies random cell damage as a result of mutations during metabolic processes due to the production of free radicals is also implicated [2–4]. As the molecular mechanisms leading to human senescence are complex processes, different research approaches are used to study aging including studies of monogenic segmental progeroid syndromes. Two progeria syndromes, Werner’s syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS), which are characterized by clinical features mimicking physiological aging at an early age, provide insights into the mechanisms of natural aging. They suggest a model of human aging. Based on recent findings on WS and HGPS, human aging can be triggered by two main mechanisms: telomere shortening and DNA damage. In telomere-dependent aging, telomere shortening and dysfunction may lead to DNA damage responses which induce cellular senescence. In DNA damage-initiated aging, DNA damage accumulates, along with DNA repair deficiencies, resulting in genomic instability and accelerated cellular senescence. In addition, aging due to both mechanisms (DNA damage and telomere shortening) is strongly dependent on p53 status. These two mechanisms can also act cooperatively to increase the overall level of genomic instability, triggering the onset of human aging phenotypes [3, 5]. Data from another trial revealing the molecular changes of intrinsic skin aging were analyzed by applying ‘‘Serial Analysis of Gene Expression’’ (SAGE(TM)) to skin biopsies of young and aged donors. The analysis resulted in several hundred differentially expressed genes with varying statistical significance. Of these, several genes were identified that either have never been described in skin aging before (e.g., APP) or have no identified function (e.g., EST sequences). This is the first time that
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_13, # Springer-Verlag Berlin Heidelberg 2010
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intrinsic skin aging has been analyzed in such a comprehensive manner, offering a new and partially unexpected set of target genes that have to be analyzed in more detail in terms of their contribution to the skin aging process [4, 6]. Moreover, normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging [5, 7].
Manifestation of Skin Aging Various expressions of intrinsic aging include smooth, thinning skin with exaggerated expression lines. Extrinsically aged skin is characterized by photodamage as wrinkles, pigmented lesions, patchy hypopigmentations, and actinic keratoses [2].
The Wrinkle and Its Measurement There is a new method for the measurement of the size and function of the wrinkle, called ‘‘Profilometric’’ method. Facial wrinkles are not a single groove, but comprise an anatomical and functional unit (the ‘‘Wrinkle Unit’’) along with the surrounding skin. This Wrinkle Unit participates in the functions of a central neuromuscular system of the face responsible for protection, expression, and communication. Thus, the Wrinkle Unit, the superficial musculoaponeurotic system (superficial fascia of the face), the underlying muscles controlled by the CNS and Psyche, are considered to be a ‘‘Functional Psycho-Neuro-Muscular System of the Face for Protection, Expression and Communication’’. The three major functions of this system exerted in the central part of the face and around the eyes are: (1) to open and close the orifices (eyes, nose, and mouth), contributing to their functions; (2) to protect the eyes from sun, foreign bodies, etc.; and (3) to contribute to facial expression, reflecting emotions (real, pretended, or theatrical) during social communication. These functions are exercised immediately and easily, without any opposition (‘‘Wrinkling Ability’’) because of the presence of the Wrinkle Unit that gives (a) the site of refolding (the wrinkle is a waiting fold, ready to respond quickly at any moment for any skin
mobility need) and (b) the appropriate skin tissue for extension or compression (this reservoir of tissue is measured by the parameter of WTRV). The ‘‘Wrinkling Ability’’ of a skin area is linked to the wrinkle’s functions and can be measured by the parameter of ‘‘Skin Tissue Volume Compressed around the Wrinkle’’ in cubic millimetre per 30 mm wrinkle during maximum wrinkling. The presence of wrinkles is a sign that the skin’s ‘‘Recovery Ability’’ has declined progressively with age. The skin’s ‘‘Recovery Ability’’ is linked to undesirable cosmetic effects of aging and wrinkling. This new Profilometric method can be applied in studies where the effectiveness of anti-wrinkle preparations or the cosmetic results of surgery modalities are tested, as well as in studies focused on the functional physiology of the Wrinkle Unit [6]. Nevertheless, the gradual physiologic decline of aging skin is well documented [7, 8].
Aging of the Epidermis The most striking and consistent histologic change is flattening of the dermoepidermal junction, a considerably smaller surface between the two compartments and presumably less communication and nutrient transfer [9]. Dermal–epidermal separation has been demonstrated to occur more readily in old skin under experimental conditions. Inter-rete epidermal thickness probably remains constant with advancing age, but variability in epidermal thickness and in individual keratinocyte size increases [9]. Average thickness and degree of compaction of the stratum corneum appears constant with increasing age, although individual corneocytes become larger. The skin surface pattern examination reveals slight age-associated loss of regularity. There is also an age-related decrease in the barrier function of intact stratum corneum as measured by percutaneous absorption of at least some substances. Subsequent work suggests that age effects on percutaneous absorption depend, in part, on drug structure, with hydrophilic substances being less well absorbed through the skin of old subjects, but hydrophobic substances being equally well absorbed [1]. More recent work indicates that there is an age-associated decrease in percutaneous absorption for hydrophilic substances like hydrocortisone and benzoic acid, but no change for hydrophobic substances like testosterone and estradiol [10]. An age-associated decrease in epidermal turnover rate of approximately 30–50% between the third and eighth decades has been determined by a study of desquamation rates for corneocytes at selected body sites. The thymidine-labeling index of the epidermis in vivo has been reported to decline nearly 50% with age [1].
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There is also a corresponding 100% prolongation in stratum corneum replacement rate and a decrease in the linear growth rates for hair and nails. A study of epidermal wound healing showed that the repair rate in the skin linkwise declines with age, as restoration of normal skin surface markings in deroofed subcorneal blister sites required a median of approximately 3 weeks in subjects aged 18–25 years, but 5 weeks in subjects 65–75 years. Healing was essentially complete in all young subjects who were healed by 7 weeks, and the last by 8 weeks [11]. Clinical observations suggest that the development of chronic wounds frequently associate with persistent low tissue oxygen supply (hypoxia). The prolonged tissue hypoxia exposes wounds to bacterial infection, a prolonged inflammatory response, and eventually tissue necrosis. The elderly population accounts for a large portion of this morbidity [12]. Consistent with clinical observations, compelling evidence from laboratory studies has shown that age affects wound healing in several aspects: sprouting of aged microvessels was significantly less than the sprouting of young microvessels [13], increased gelatinase and collaginase levels in skin of aged donors and in wound fluid from chronic leg ulcers, and decreased TIMP (tissue inhibitor of matrix metalloproteinase) levels in the skin of aged donors as well as reduced deposition of matrix components and re-epithelialization [14–16]. A novel study about wound healing with increasing age done by Yu-Ping Xia et al. (2001) [17] revealed that keratinocytes isolated from elderly donors, in contrast to those from young individuals, had depressed migratory activity when they were exposed to hypoxia [17]. Analysis of underlying biochemical changes demonstrated a differential activation of matrix metalloproteinases by hypoxia in keratinocytes isolated from young and old ages. Matrix metalloproteinases-1 and Matrix metalloproteinases-9 and tissue inhibitor of matrix metaloproteinases-1 were strongly upregulated by hypoxia in young cells, whereas no induction was observed in aged cells. Furthermore, transforming growth factor-b1 signaling appears to be involved in keratinocyte differential response to hypoxia, as transforming growth factor-b type 1 receptor was upregulated by hypoxia in young cells, while there was no induction in aged cells. Transforming growth factor-b neutralizing reagents blocked hypoxia-induced matrix metalloproteinase-1, matrix metalloproteinase-9 expression, and hypoxia-induced cell migration as well. These results introduced by Yu-Ping Xia et al. (2001) [17] suggest that an age-related decrease in response to hypoxia plays a crucial part in the pathogenesis of related re-epithelialization in wounds [17]. A decrease in the number of the enzymatically active melanocytes per unit surface area of the skin,
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approximately 10 20% of the remaining cell population each decade, has been repeatedly documented [8]. It is not known whether the cells truly disappear or simply become undetectable by ceasing to produce pigment, but in either case the protective barrier of the body against ultraviolet radiation presumably is reduced. The number of melanocytic nevi (moles) also progressively decreases with age from a peak of 15 40 in the third and fourth decade to an average of four per person after age 50; moles are rarely observed in persons above age 80 [18]. The contribution of extracellular matrix components to intrinsic skin aging has been investigated thoroughly; however, there is little information as to the role of the cytoskeletal proteins in this process. Therefore, the new studies highlight the importance of the cellular compartment in this process and demonstrate that special attention has to be given to RNA as well as protein normalization in aging studies. OeNder et al. (2008) demonstrated that the mRNA levels of the genes for K1, K3, K4, K9, K13, K15, K18, K19 and K20 are downregulated in aged skin, K5 and K14 are unchanged, and K2, K16 and K17 are upregulated in aged skin. The mRNA data were confirmed on the protein level. This diverse picture is in contrast to other cytoskeletal proteins including components of the desmosome (JUP), microtubuli (TUBA) and microfilaments (ACTB) – often regarded as house-keeping genes – that were all reduced in aged skin [19]. The incidence of cancers, infectious diseases, and autoimmune disorders increases with advancing age [20]. In addition, aging is accompanied by a number of changes in immune function such as decreased lymphocyte proliferative responses to both mitogens and antigens, reduced delayed type hypersensitivity reactions, and decreased antibody responses to vaccination and infection [19]. Murine models of aging have demonstrated that there is an age-associated dysregulation in cytokine production, as evidenced by consistently decreased production of interleukin-2 (IL-2) and generally increased production of interleukin-4 [21]. These data, coupled with the increased incidence of cancer and the recurrence of latent viral infection in aged humans, have led to the hypothesis that the process of aging per se induces a switch from a predominantly type 1 cytokine (IL-2, INF-g, IL-12) profile supporting a dominant cellmediated immune response, to a predominantly type 2 cytokine (IL-4, IL-5, IL-6, IL-10) profile, promoting a dominant humoral response. A 20 50% reduction in the number of morphologically identifiable epidermal Langerhans cells occurs between early and late adulthood and may account in part for the age-associated decrease in immune responsiveness observed in the skin [7, 8].
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In principle, inhibition of aging should delay cancer. But the question which arises is whether it is possible to slow aging. As recently proposed, the nutrient-sensing TOR (target of rapamycin) pathway is involved in cellular and organismal aging. In rodents, certain conditions that interfere with the TOR pathway slow aging and prevent cancer. Retrospective analysis of clinical data reveals that in animals, from worms to mammals, caloric restrictions, life-extending agents, and numerous mutations that increase longevity all converge on the TOR pathway. And, in humans, cell hypertrophy, hyper-function and hyperplasia, typically associated with activation of TOR, contribute to diseases of aging. Theoretical and clinical considerations suggest that rapamycin may be effective against atherosclerosis, hypertension and hyper-coagulation (thus, preventing myocardial infarction and stroke), osteoporosis, cancer, autoimmune diseases and arthritis, obesity, diabetes, macula-degeneration, Alzheimer’s and Parkinson’s diseases. Finally, the extended life span will reveal new causes for aging (e.g., ROS, ‘‘wear and tear’’, Hayflick limit, stem cell exhaustion) that play a limited role now, in relation to TOR. So, there is a potential clinical use of TOR inhibitors in order to slow aging and delay cancer [22]. Richardson et al. (2004) also emphasized that regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals [23]. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and TOR signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. Moreover, one of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced the understanding of the molecular mechanism of TOR activation. So, the regulation of TOR signaling is very important to control cell growth during normal development and tumorigenesis [24]. Vitamin D production, which is an important endocrine function of human epidermis, is suspected to decline with age. With advancing age, bone mass decreases markedly, especially in postmenopausal women, predisposing to trabicular bone fractures. Osteoporosis, or lack of cortical and trabicular bone, is a prominent factor, but some elderly individuals also have osteomalacia, since the decreased mineralization of bone is classically associated
with vitamin D deficiency. Although avoidance of dairy products, the principal dietary source of vitamin D, insufficient sun exposure, and sun screen use, undoubtedly contribute to vitamin D deficiency in the elderly [25]. The level of epidermal 7-dehydrocholesterol per unit skin surface area also appears to decrease linearly by approximately 75% between early and late adulthood suggesting that lack of its immediate biosynthetic precursor may also limit vitamin D production. In one study, old adult volunteers exposed to total body UV irradiation produced far less vitamin D3, over the ensuing week, than did complexion-matched young adult volunteers exposed to the same UV dose [2]. Neoplasia is associated with aging in virtually all organ systems, but is especially characteristic for aged skin. Acrochorrdon, cherry angioma, seborrheic keratosis, lentigo, sebaceous hyperplasia, one or more of these benign epidermal tumors is present in nearly every adult beyond age 65 years, and most individuals have dozens of lesions [20]. Actinically induced basal cell carcinoma and squamous cell carcinoma are by far the most common human malignancies. These benign and malignant neoplasms almost certainly reflect in part the loss of proliferative homeostasis with age.
Epidermal Proteins and Skin Aging Recent studies revealed by gel electrophoresis of healthy human skin (sun protected skin) from donors of different ages that there are age-associated changes in five proteins, which were identified as Keratin 10, Involucrin, Prealbumin, Hsp 27, and Rho B. More recently, it was found by the tool of immunohistochemistry that these epidermal proteins are expressed in the human skin epidermis and that their expression patterns undergo age-associated changes [26]. Examination of cryosections from healthy sun-protected skin derived from individuals ranging from the first decade of life until the ninth decade revealed that Keratin 10, Involucrin, Prealbumin, Hsp 27, and Rho B proteins are present in high density nearly in all layers of young epidermis (> Fig. 13.1). In contrast, the expression of the investigated epidermal proteins is reduced as a function of age. In some skin specimens from very old individuals, the proteins were not detectable [26]. All five proteins are known to be closely related to differentiation and proliferation of keratinocytes. Moreover, prealbumin, Hsp27 and Rho B have been demonstrated to play a crucial role in tumor cell biology. Thus, decreased expression of these proteins could be
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. Figure 13.1 Expression of Hsp27 protein in normal human skin. A moderate to strong expression is found in most epidermal layers, including the stratum corneum. a: Tyramid signal amplification (TSA) technique (200x), b: Avidin-Biotin complex (ABC) technique (200x). (Reprinted with permission from Adly et al. [47].) ß 2006, Elsevier
one reason for the increased prevalence of skin cancer in old individuals. Additionally, these proteins are possible marker proteins for intrinsic aging of the epidermis [26]. Additionally, Hsp27 and other epidermal proteins were found to be expressed in the human skin hair follicles and involved in hair follicle cycle control [26]. (> Fig. 13.2).
Aging of the Dermis Loss of dermal thickness approaches 20% in elderly individuals, although in sun-protected sites significant thinning occurs only after the eighth decade [27]; the remaining tissue is relatively acellular and avascular [1, 9]. Precise histologic concomitants of wrinkling, if any, are unknown, although the age-related loss of normal elastin fibers may be contributory [9]. Deep expression lines seem to result from contractions of connective tissue septae within the subcutaneous fat. In one study, an approximately 50% reduction in mast cells and a 30% reduction in venular cross-sections was noted in the papillary dermis of buttock skin from elderly adults compared to that from young adult controls, associated with a corresponding reduction in histamine released and other manifestations of the inflammatory response following UV radiation exposure [7]. The striking age-associated loss of vascular bed, especially of the vertical capillary loops that occupy the dermal papillae in young
skin, is felt to underlie many of the physiologic alterations in old skin. Reduction in the vascular network surrounding hair bulbs and eccrine, apocrine, and sebaceous glands may contribute to their gradual atrophy and fibrosis with age [1]. An age-associated decrease in dermal clearance of trans-epidermally absorbed materials has been reported and is probably due to alterations in both the vascular and extracellular matrix. A previous study showed that wheal resorption after intradermal saline injection required almost twice as long on average in elderly versus young adult subjects [1]. Controversely, the time required for development of a tense blister after topical application of 50% ammonium hydroxide is nearly twice as long in older subjects, suggesting a decreased transduction rate with age in injured skin. Impaired transfer of cells as well as solutes between the extravascular and intravascular dermal compartments is suggested by several studies to occur by age, but it is difficult to isolate these components in a complex inflammatory reaction. Decreased vascular responsiveness in the skin of older individuals has been documented clinically assessing vasodilation and transudation after application of standardized irritants, histamine, and the mast cell degranulating agent 48/80 [11]. Intensity of erythema following a standardized UV exposure is also decreased with age in normal skin, although factors other than decreased vascular responsiveness may also contribute [7]. A previous study that assessed cutaneous vascular response to the vasodilator
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. Figure 13.2 Expression of Hsp27 protein in normal human scalp skin anagen VI hair follicle, immunostained with (ABC) and (TSA) techniques. a and d show the distal region (200x), b and e show the central region (200x), c and f show the proximal bulb region (200x). D: shows schematic representation of anagen VI hair follicle. (Reprinted with permission from Adly et al. [47].) ß 2006, Elsevier
methyl nicotinate concluded that there was no difference between young adult and old adult subjects after correction for rate of drug absorption [28]. Compromised thermoregulation, which predisposes the elderly to sometimes fatal heat stroke, or hypothermia may be due in part to reduced vascularity of dermal arterioles, and in the latter instance, to loss of subcutaneous fat as well. The skin of healthy older subjects is less sensitive to dinitrochlorobenzene (DNCB) and to standard recall antigens, compared to the skin of young adult controls [29]. This decrease undoubtedly reflects the well documented decrease in total number of circulating thymus derived lymphocytes and in their responsiveness to standard mitogens. The elastic fibers in the skin are less well studied, but have been reported to show progressive cross linkage and
calcification with age in adult skin. On average, older skin has thicker elastic fibers than young skin, and elastic fiber alterations extend deeper into the dermis with advancing age [30, 31]. Small cysts and lacuna are common in aging elastic fibers, sometimes progressing to complete fragmentation. Similar changes can be produced experimentally by incubation of dermal slices with elastase or chymotrypsin (but not collagenase) in vitro, suggesting that enzymatic degradation of elastin may be a mechanism of normal dermal aging [30, 31]. The dermal microvasculature in middle-aged or elderly subjects may show mild vascular wall thickening; vascular wall thinning to less than half of the normal young adult measurement, associated with absent or reduced perivascular veil cells that has been reported in skin of very old subjects and probably contributes to vascular fragility [30, 31].
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Significant decrease in thrombomodulin-positive cells and vascularity were evidenced in the aged group. Specific subsets of the dermal dendrocyte populations and the blood microvasculature appear affected by aging. Capsaicin may limit these aging effects. [32]. Biochemical changes in collagen, elastin and dermal ground substance that have been described during foetal and early postnatal development are far greater than those that have been described with advancing age during adulthood. With advancing adult age, rat tail collagen does manifest a slight increase in the force of contraction (isometric tension), when heated above its shrinkage temperature, consistent with increasing cross-linkage of the collagen molecule [33]. Both rat tail tendon and human skin display a progressive decrease in the ratio of soluble to insoluble collagen [34]. The predominant cross-links in skin have been reported to decrease and virtually disappear with age in mature animals, however, using techniques that measure borohydride reducible cross-links, despite evidence of increasing mechanical stability. This suggests that some collagen cross-links in vivo may be progressively reduced or oxidized and are, therefore, no longer measurable. Certain non enzymatic cross-links in connective tissue, such as histidinoalanine and the Millard reaction product, do show a strong positive correlation with adult age, and have been suggested to contribute to age-associated changes in the dermis [1]. The proportion of recently synthesized dermal collagen, as determined by neutral salt extraction, is small and does not vary with age in adult [35, 36]. However, there is a significant decrease with age in the percent of total collagen that is released by pepsin digestion, and hence incompletely cross-linked, from approximately 25% at age 30 years to approximately 10% at age 75 years with a proportionate increase in the percent of insoluble collagen from approximately 70 88%. The amount of ketoaminelinked glycosylation of insoluble dermal collagen also increases with age, possibly related to slower collagen turnover or higher average glucose levels in the tissue [35, 36]. Prolyl- and lysyl-hydroxylase, enzymes necessary for intercellular stabilization of the collagen triple helix and for its intermolecular cross-linking, show an ageassociated decline in activity in human skin, although these coenzyme activities in cultured dermal fibroblasts from donors ranging in age from a few months to 94 years do not. This apparent contradiction could be explained by an age-associated decrease in either dermal fibroblast number in vivo or fibroblast responsiveness to a serumderived enzyme stimulating factor in vitro. There is some data concerning the possible postmaturational age-associated changes in mucopolysaccharides
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(glycosaminoglycans and proteoglycans) or other molecules of the ground substance in which collagen and elastic fibers are embedded. There appears to be a slight decrease with age in mucopolysaccharide content relative to dry weight or collagen content of the skin, especially for hyaluronic acid. Although mucopolysaccharides constitute only 0.1 0.3% of dry weight for whole skin, their decrease may adversely influence skin turgor, as proteoglycans bind a high volume of water in the dermis [37, 38]. Mechanical properties of the skin also change with age. Uniaxial and biaxial tension tests performed on excised abdominal skin stripes demonstrate progressive loss of elastic recovery, consistent with gradual destruction of the dermal elastin network, and the time required for excised skin to return to its original thickness after 50% compression is markedly prolonged [39]. This early work, which has been confirmed and extended by in vivo studies of ventral forearm skin of 133 volunteers in each decade of life, showed linear declines of approximately 25% in both men and women for elasticity and extensibility. Loss of elasticity began in childhood and continued through the ninth decade, while extensibility was constant through the sixth decade and then declines more rapidly thereafter. Overall, a picture emerges of aging dermis as an increasingly rigid, inelastic and unresponsive tissue, less capable of undergoing modification in response to stress. IGF-I is a key regulator of human skin aging and declining IGF-I levels with age may play a significant role in the reduction of skin surface lipids and thickness [40].
Nerves and Appendages By the end of the fifth decade, approximately half the population has at least 50% grey (white) body hair with an even higher proportion of depigmented scalp hair, and virtually everyone has some degree of greying due to progressive and eventually total loss of melanocytes from the hair bulb. Loss of melanocytes is believed to occur more rapidly in hair than in skin because the cells proliferate and manufacture melanin at maximal rates during the anagen phase of the hair cycle, while epidermal melanocytes are comparatively inactive throughout their lifespan. Scalp hair may gray more rapidly than other body hair because its anagen to telogen ratio is considerably greater than that of other body hair. Advancing age is also accompanied by a modest decrease in number of hair follicles. Remaining hairs may be smaller in diameter and grow more slowly. The process called balding results primarily from the androgen-dependent conversion of the relatively dark thick scalp hairs to lightly pigmented short
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fine hairs similar to those on the ventral forearm. Bitemporal hair line recession begins during late adolescence in most women and virtually all men. Assessment of baldness is hampered by lack of a precise definition, but by certain criteria advanced bitemporal and occipital hair loss in men increases in prevalence, respectively from 20% and 3% at the end of the third decade to more than 60% by the seventh decade. Eccrine glands decrease approximately 15% in average number during childhood in most body sites. Spontaneous sweating in response to dry heat is further reduced by more than 70% in healthy old subjects as compared to young controls, attributable primarily to a decreased output per gland. Maximal sweat production has not been quantified in the elderly, but is almost certainly reduced and probably predisposes to heat stroke in this age group [41]. Similar studies have not been performed for apocrine glands, although the apparently decreased requirement for under-arm deodorants in the elderly suggests decreased function. Lipofuscin gradually accumulates with age in the secretory cells of both eccrine and apocrine glands. Sebaceous gland size and number appear not to change with age [9]. The exponential decrease in sebum production of approximately 23% per decade beginning in the second decade in both men and women, approximately 60% over the adult lifespan, is attributed to the concomitant decrease in production of gonadal or adrenal androgen to which sebaceous glands are exquisitely sensitive. The clinical effects of decreased sebum production, if any, are unknown. There is no direct relationship to xerosis or seborrheic dermatitis [42]. Pacinian and Meissner’s corpuscles, the cutaneous end organs responsible for pressure perception, light and touch, progressively decrease to approximately one third of their initial average density between the second and ninth decades of life and display greater size variation. There are very few histologically demonstrable aging related changes in Merkel corpuscles or in free nerve endings. Decreased sensory perception was documented in old skin more than 3 decades ago by several techniques. Cutaneous pain threshold has been reported to increase up to 20% with advancing age [43]. The available data do not permit differentiation among an age-associated increase in the prevalence of peripheral neuropathy, a true aging change in healthy subjects, increased rate of heat dispersion in old skin due to age-associated dermal alterations, an increased peripheral nerve threshold to painful stimuli, and an increased central threshold to pain perception [43]. The many psychological and social factors influencing an individual reaction to pain may also be presumed to vary with age. In any case, either decreased awareness of, or reaction to, noxious stimuli
would facilitate wounding and irritation of old skin. Sympathetic nervous system activity is altered in aging.
Aging and Skin Diseases Disorders of the skin are known to be common and bothersome in the elderly, but existing incidence and morbidity figures are suspect [44]. Few dermatologic disorders occur predominantly in the elderly, and none is restricted to this age group. Perhaps the prototypic disease of old skin is bullous pemphigoid, characterized by subepidermal blister formation with fixation of complement and immunoglobulins along the basement membrane. Its predilection for the elderly may partially explained by the age-associated increase in circulating autoantibodies and ease of dermal epidermal separation, although other autoimmune and blistering dermatoses are not more common in old age. Possibly age-associated changes in the basement membrane itself render it specifically vulnerable to the disease process. More than two thirds of herpes zoster cases occur after the fifth decade, with an age adjusted annual incidence rate of approximately 0.25% at 20 50 years vs. more than 1% at age 80 years [45]. Post-herpetic neuralgia, uncommon in patients less than 40 years old, occurs frequently in older patients; more than half of those beyond age 60 years. In one large series this altered response to varicella virus has been established, however no mechanism explaining this altered response to varicella virus has been found. Recurrent herpes simplex infection also involves reactivation of latent virus in regional ganglia and T cell-mediated host defenses, but is more common in young adults and indeed rare among immunocompetent elders. The general phenomenon of impaired wound healing in the elderly may account for slower resolution of the acute eruption, but its relevance, if any, to post-herpetic neuralgia is unclear [45]. Age-associated muting of the inflammatory response might indeed be expected to reduce the risk of neuralgia, since prophylactic use of anti-inflammatory corticosteriods is often successful. Xerosis, the dry rough quality of old skin, may be attributable to a subtle disorder of epidermal maturation, although histologic studies reveal little alteration of either the viable epidermis or the stratum corneum with age. Available data fails to support water loss [1], decreased stratum corneum lipids [42] or altered amino acid composition as etiologic factors [42]. The surface irregularity may also be attributed simply to slower transit of corneocytes through the stratum corneum, allowing accumulation of damage in situ. Similarly, there is no explanation
Aging and Intrinsic Aging: Pathogenesis and Manifestations
for the pruritus that often accompanies xerosis. Unsupported hypotheses include frequent penetration of irritants through an abnormal stratum corneum and an altered sensory threshold due to subtle neuropathy. Many dermatoses more commonly observed in the elderly reflect the higher prevalence of systemic diseases such as diabetes, vascular insufficiency, and various neurologic syndromes in this population. In the case of chronic leg ulcers, for example, healing of previously recalcitrant lesions can sometimes be achieved by use of neonatal epidermal allograft, postulated to elaborate needed growth factors and/or matrix materials that the surrounding senescent host epithelium is incapable of producing. The allegedly increased incidence of other disorders such as tinea pedis or seborrheic dermatitis may reflect reduced local skin care with subsequent exacerbation of previously unapparent problems, rather than an age-associated change in the skin itself. Alternatively, subtle changes in the immune status may be responsible, in analogy to the increased prevalence and severity of those disorders in patients with acquired immunodeficiency syndrome [1]. Reduced tolerance to systematically administrated drugs is well documented in the elderly due to the decrements in lean body mass and metabolism and renal excretion of the active ingredients [46]. Comparable data for topically applied medication do not exist, but it is tempting to postulate that retarded dermal clearance of absorbed material reduced dermal mass and cellularity, and possibly altered metabolic capacity may render old skin more susceptible to both beneficial and adverse effects of topical medications, or at least alter the optimal dosage frequency [1]. In the case of corticosteriod preparations, relative vascular unresponsiveness may render blanching of erythema as an unreliable indicator of other effects in old skin.
Conclusion A better understanding of both the intrinsic and extrinsic influences on the aging of the skin, as well as distinguishing the retractable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the irretractable cutaneous aging (primarily intrinsic aging), is very important to solve the problem of aging.
Cross-references > Degenerative
Changes in Aging Skin
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References 1. Balin AK, Pratt LA. Physiological consequences of human skin aging. Cutis. 1989;43(5):431–436. 2. Puizina-Ivic N. Skin aging. Acta Dermatovenerol Alp Panonica Adriat. 2008;17(2):47–54. 3. Ding SL, Shen CY. Model of human aging: recent findings on Werner’s and Hutchinson-Gilford progeria syndromes. Clin Interv Aging. 2008;3(3):431–444. 4. Holtkotter O, Schlotmann K, Hofheinz H, Olbrisch RR, Petersohn D. Unveiling the molecular basis of intrinsic skin aging(1). Int J Cosmet Sci. 2005;27(5):263–269. 5. Chondrogianni N, de CMSD, Franceschi C, Gonos ES. Cloning of differentially expressed genes in skin fibroblasts from centenarians. Biogerontology. 2004;5(6):401–409. 6. Hatzis J. The wrinkle and its measurement – a skin surface Profilometric method. Micron. 2004;35(3):201–219. 7. Gilchrest BA, Stoff JS, Soter NA. Chronologic aging alters the response to ultraviolet-induced inflammation in human skin. J Invest Dermatol. 1982;79(1):11–15. 8. Gilchrest BA. Age-associated changes in the skin. J Am Geriatr Soc. 1982;30(2):139–143. 9. Montagna W, Carlisle K. Structural changes in aging human skin. J Invest Dermatol. 1979;73(1):47–53. 10. Roskos KV, Maibach HI, Guy RH. The effect of aging on percutaneous absorption in man. J Pharmacokinet Biopharm. 1989;17(6): 617–630. 11. Grove GL. Age-related differences in healing of superficial skin wounds in humans. Arch Dermatol Res. 1982;272(3–4):381–385. 12. Van de Kerkhof PC, Van Bergen B, Spruijt K, Kuiper JP. Agerelated changes in wound healing. Clin Exp Dermatol. 1994;19(5): 369–374. 13. Arthur WT, Vernon RB, Sage EH, Reed MJ. Growth factors reverse the impaired sprouting of microvessels from aged mice. Microvasc Res. 1998;55(3):260–270. 14. Ashcroft GS, Horan MA, Ferguson MW. Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol. 1997;108(4):430–437. 15. Ashcroft GS, Herrick SE, Tarnuzzer RW, Horan MA, Schultz GS, Ferguson MW. Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA. J Pathol. 1997;183(2):169–176. 16. Ashcroft GS, Kielty CM, Horan MA, Ferguson MW. Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans. J Pathol. 1997;183(1):80–89. 17. Xia YP, Zhao Y, Tyrone JW, Chen A, Mustoe TA. Differential activation of migration by hypoxia in keratinocytes isolated from donors of increasing age: implication for chronic wounds in the elderly. J Invest Dermatol. 2001;116(1):50–56. 18. Maize JC, Foster G. Age-related changes in melanocytic naevi. Clin Exp Dermatol. 1979;4(1):49–58. 19. Oender K, Trost A, Lanschuetzer C, Laimer M, Emberger M, Breitenbach M, Richter K, Hintner H, Bauer JW. Cytokeratin-related loss of cellular integrity is not a major driving force of human intrinsic skin aging. Mech Ageing Dev. 2008;129(10):563–571. 20. Blagosklonny MV. Prevention of cancer by inhibiting aging. Cancer Biol Ther. 2008;7(10):1520–1524.
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21. Albright JW, Mease RC, Lambert C, Albright JF. Trypanosoma musculi: tracking parasites and circulating lymphoid cells in host mice. Exp Parasitol. 1999;91(2):185–195. 22. Blagosklonny MV. Aging: ROS or TOR. Cell Cycle. 2008;7(21): 3344–3354. 23. Richardson CJ, Schalm SS, Blenis J. PI3-kinase and TOR: PIKTORing cell growth. Semin Cell Dev Biol. 2004;15(2):147–159. 24. Inoki K, Ouyang H, Li Y, Guan KL. Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev. 2005; 69(1):79–100. 25. Matsuoka LY, Wortsman J, Hanifan N, Holick MF. Chronic sunscreen use decreases circulating concentrations of 25-hydroxyvitamin D. A preliminary study. Arch Dermatol. 1988;124(12): 1802–1804. 26. Hussein MR. Analysis of p53, BCL-2 and epidermal growth factor receptor protein expression in the partial and complete hydatidiform moles. Exp Mol Pathol. 2009. 27. de Rigal J, Escoffier C, Querleux B, Faivre B, Agache P, Leveque JL. Assessment of aging of the human skin by in vivo ultrasonic imaging. J Invest Dermatol. 1989;93(5):621–625. 28. Roskos KV, Bircher AJ, Maibach HI, Guy RH. Pharmacodynamic measurements of methyl nicotinate percutaneous absorption: the effect of aging on microcirculation. Br J Dermatol. 1990;122(2): 165–171. 29. Wayne SJ, Rhyne RL, Garry PJ, Goodwin JS. Cell-mediated immunity as a predictor of morbidity and mortality in subjects over 60. J Gerontol. 1990;45(2):M45–48. 30. Braverman IM, Fonferko E. Studies in cutaneous aging: II. The microvasculature. J Invest Dermatol. 1982;78(5):444–448. 31. Braverman IM, Fonferko E. Studies in cutaneous aging: I. The elastic fiber network. J Invest Dermatol. 1982;78(5):434–443. 32. Quatresooz P, Pierard GE. Immunohistochemical clues at aging of the skin microvascular unit. J Cutan Pathol. 2009;36(1):39–43. 33. Escoffier C, de Rigal J, Rochefort A, Vasselet R, Leveque JL, Agache PG. Age-related mechanical properties of human skin: an in vivo study. J Invest Dermatol. 1989;93(3):353–357.
34. Miyahara T, Murai A, Tanaka T, Shiozawa S, Kameyama M. Agerelated differences in human skin collagen: solubility in solvent, susceptibility to pepsin digestion, and the spectrum of the solubilized polymeric collagen molecules. J Gerontol. 1982;37(6):651–655. 35. Schnider SL, Kohn RR. Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen. J Clin Invest. 1981;67(6):1630–1635. 36. Schnider SL, Kohn RR. Effects of age and diabetes mellitus on the solubility of collagen from human skin, tracheal cartilage and dura mater. Exp Gerontol. 1982;17(3):185–194. 37. Lipson MJ, Silbert JE. Acid mucopolysaccharides of tadpole tail fin and back skin. Biochim Biophys Acta. 1965;101(3):279–284. 38. Kondo K, Seno N, Anno K. Mucopolysaccharides from chicken skin of three age groups. Biochim Biophys Acta. 1971;244(3):513–522. 39. Daly CH, Odland GF. Age-related changes in the mechanical properties of human skin. J Invest Dermatol. 1979;73(1):84–87. 40. Makrantonaki E, Vogel K, Fimmel S, Oeff M, Seltmann H, Zouboulis CC. Interplay of IGF-I and 17beta-estradiol at age-specific levels in human sebocytes and fibroblasts in vitro. Exp Gerontol. 2008;43 (10):939–946. 41. Silver AF, Chase HB. An in vivo method for studying the hair cycle. Nature. 1966;210(5040):1051. 42. Downing DT, Stewart ME, Strauss JS. Changes in sebum secretion and the sebaceous gland. Dermatol Clin. 1986;4(3):419–423. 43. Procacci P, Zoppi M, Maresca M. Experimental pain in man. Pain. 1979;6(2):123–140. 44. Beauregard S, Gilchrest BA. A survey of skin problems and skin care regimens in the elderly. Arch Dermatol. 1987;123(12):1638–1643. 45. Hope-Simpson RE. The Nature of Herpes Zoster: A Long-Term Study and a New Hypothesis. Proc R Soc Med. 1965;58:9–20. 46. Vestal RE. Aging and pharmacology. Cancer. 1997;80(7):1302–1310. 47. Adly MA, Assaf HA, Hussein MR. Expression of the heat shock protein-27 in the adult human scalp skin and hair follicle: hair cycle-dependent changes. J Am Acad Dermatol. 2006;54(5):811–817.
38 Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast Mutsumi Okazaki
Introduction
Fibroblast
In the skin pigmentation, the actinic damage plays a major role [1], but the effect of chronologic cellular aging is also an important factor. The chief cellular components of the skin other than melanocytes are keratinocytes and fibroblasts, whose paracrine effects on melanocytes (rather than melanocyte itself) play an important role in the epidermal pigmentation [2–11]. Human keratinocytes express several melanogenic cytokines, such as endothelin-1 (ET-1) [2–4], granulocyte macrophage colony stimulating factor (GM-CSF) [5], stem cell factor (SCF), and basic fibroblast growth factor (bFGF) [7–9]. Human fibroblasts, on the other hand, secrete several melanogenic cytokines such as bFGF, HGF, and SCF [6, 10, 11]. Further, interleukin-1a (IL-1a), a pro-inflammatory cytokine, stimulates the production of ET-1 by keratinocytes and of HGF by fibroblasts [3, 12–14]. It has been reported that the overexpression of these melanogenic cytokines is responsible for the age-related pigment ary cutaneous disorders [15–17]. The age-associated change was studied in cytokine secretion by keratinocytes and fibroblasts based upon this paracrine cytokine network within the skin for epidermal pigmentation mechanisms.
Normal skin specimens were taken from Japanese patients (disused skin during plastic surgery, i.e., after the dog-ear correction). Informed consent was obtained from all patients. Fibroblasts were cultured from 19 specimens (age = 26.7 15.6, from 7 to 65 year old, 8 males, 11 females). The methods of isolation and culture of fibroblasts were reported previously [18]. Fibroblasts were grown in the fibroblast growth medium (FGM), which consists of Dulbecco’s modified Eagle’s medium (DMEM), 0.6 mg/mL glutamine, and 10% fetal calf serum (FCS). Third cultures of fibroblasts were used for the experiments. Fibroblasts were seeded in a 60 mm culture dish at a density of 5 105 cells/5 mL and cultured in FGM. After human fibroblasts had been cultured for 96 h at 37 C under a 5% CO2 atmosphere, the medium was collected to quantify HGF, SCF, and bFGF, respectively, by ELISA.
Correlation Between Age and Secretion of Melanogenic Cytokine Studies were planned to elucidate whether the aging of keratinocytes and fibroblasts was related to the potential to secrete several melanogenic cytokines. In the first experiment, the keratinocytes and fibroblasts derived from the skin of different chronological ages were cultured, and the secretions of melanogenic cytokines were evaluated by ELISA (enzyme-linked immuno-sorbent assay). The series of study were carried out with the informed consent of the person whose skin samples were used.
Keratinocyte Normal skin specimens were obtained from Japanese patients, and keratinocytes were cultured from 16 specimens (age = 28.0 17.1, from 7 to 64 years, 6 males, 10 females). The methods of isolation and culture of keratinocytes were reported previously [18]. Keratinocytes were grown in the serum-free keratinocyte growth medium (KGM; Kyokuto Seiyaku, Tokyo) which consists of MCDB153 with high concentrations of amino acids, transferrin (final concentration 10 g/mL), insulin (5 g/mL), hydrocortisone (0.5 g/mL), phosphorylethanolamine (14.1 g/mL), and bovine pituitary extract (40 g/mL). The final concentration of Ca2+ in the medium was 0.03 mM. Second cultures of keratinocytes were used for the experiment. Keratinocytes were seeded in a 60 mm culture dish at a density of 1.5 105 cells/5 mL, and cultured in KGM supplemented with 0.5% FCS. After human
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Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast
keratinocytes had been cultured at 37 C under a 5% CO2 atmosphere for 72 h, the keratinocyte-conditioned medium was collected to quantify IL-1a, ET-1, and GM-CSF, respectively, by ELISA. The comparison of the cytokine concentration between male and female was carried out using unpaired t-test. And the scatter diagrams showing the relationship between age and cytokine concentration were drawn, and simple linear regression equations were calculated, and simple linear regression test was used to determine whether there was any correlation between age and
concentration of cytokine. A value of P Fig. 38.1a–c).
. Figure 38.1 Scatter diagram showing the relationship between donor age and value of cytokine concentration in fibroblasts. The lines represent the linear regression equation (n = 19; R, coefficient of determination). (a) HGF y = 159 0.034x, R = 0.0054, P = 0.98; (b) SCF y = 191 0.84x, R = 0.19, P = 0.44; (c) bFGF y = 2.62 0.00079x, R = 0.0064, P = 0.98
Aging and Melanocytes Stimulating Cytokine Expressed by Keratinocyte and Fibroblast
2. Cytokine secretion of keratinocytes No gender differences in the donor age and cytokine concentration were found between male and female. There was a significant correlation between age and IL-1a concentration (R = 0.71, P = 0.002). There was a relatively weak correlation between age and ET-1 concentration, but the correlation was not significant (R = 0.41, P = 0.051). No correlation existed between age and GM-CSF concentration (R = 0.32, P = 0.23) (> Fig. 38.2a–c).
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In the second experiment, the secretions of IL-1a and ET-1 by keratinocytes were compared between the second and fifth cultures. In this study, the fifth cultures of keratinocytes derived from nine donors who were less than 30 years old were used. Cell cultures and ELISA assay were performed in the same way as the first experiments. A comparison of the cytokine concentrations between the second and fifth culture keratinocytes was carried out using paired t-test. A value of P Fig. 26.1) These microscopic changes in the bacterial flora can be confirmed by culture. One report shows a marked diminishment in the number of probiotic lactobacilli, with lactobacilli the dominant vaginal species in only 13% of the menopausal subjects [2]. Anaerobes become the dominant species. The numbers of these organisms were previously held in check by the dominance of acid-producing bacteria like the lactobacilli. There are clinical consequences of these changes in the vaginal bacterial flora that can result in lifestyle alterations for these women. Perimenopausal and particularly post-menopausal women are colonized by Escherichiae coli at an increased incidence, and this is inversely related to the presence of lactobacilli [3]. These vaginal bacterial changes make the woman much more susceptible to lower urinary tract infection due to E. coli [4]. The protective effect of the lactobacilli, keeping the numbers of these gram-negative aerobes in check, has been lost, and the relatively short female urethra becomes an easier transit site for these pathogens. The perimenopausal woman is also much more prone to suffer from a grossly purulent and persistent vaginitis, named Desquamative Inflammatory Vaginitis (DIV) [5]. The hallmarks of this troublesome syndrome,
Estrogen The lack of the female hormone estrogen brings down hormonal activity that helps maintain the health of lower genital tract tissue. This loss results in the many changes that confront post-menopausal women. This absence is not an abrupt shift, the curtain does not suddenly fall one evening as the human drama of reproductive life ends, with the new playbill, postmenopausal existence beginning the next day. Instead, perimenopause, the transition period’s new name, describes the very gradual changes in the years or decades before the cessation of menses. This is a slow and inexorable process in which minute bodily alterations occur over months and years that are gradually perceived by the woman involved. Although early on, menstruation continues unabated, there is a drastic falloff in reproductive success. Assisted reproductive physicians note a stunning drop after the age of 42 in the ability of women to become pregnant, and those few who succeed have a high number of spontaneous first-trimester pregnancy losses [1]. Subsequently, these perimenopausal women notice changes in their menstrual cycle, which can vary from too frequent and heavier periods, to less frequent and lighter periods. Along with these menstrual changes, these women become aware of ‘‘hot flashes,’’ i.e., vasomotor instability, sleeping trouble, a dry and less lubricated vagina that makes intercourse much less pleasurable.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_26, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 26.1 Microscopic examination of vaginal secretions of an asymptomatic post-menopausal woman
. Figure 26.2 Superficial tissue changes of post-menopausal woman not on hormone replacement therapy
seen most commonly in women over the age of 40, are an alkaline pH, a negative whiff test when a drop of vaginal secretion is placed in a 10% potassium hydroxide mixture, the absence of lactobacilli, an increase in other bacterial forms, many immature vaginal squamous cells, and most significantly, an outpouring of inflammatory white cells. These vaginal changes have made older women more susceptible to urinary tract and vaginal infections.
Some women of this age group who are susceptible will develop a chronic inflammatory skin disease, lichen sclerosis, which predominantly affects the skin and mucous membranes of the vulvar and rectal area. This thinned vulvar skin also loses much of its natural surface defense mechanisms and is more likely to become and stay inflamed. This is an expected response. Post-menopausal women, not taking estrogen, have higher induced levels of the pro-inflammatory cytokine interleukin-1, interleukin-6, and tumor necrosis factor a when compared to women in their reproductive years [7]. The body’s response to this new inflammation is a repair process with new tissue formation. This new tissue regeneration in an inflammatory site is almost always accompanied by vulvar and perineal itching. This initiates a vicious cycle. The itching leads to scratching, often when the patient is asleep and unaware of her response. The scratching increases the tissue inflammation, and the cycle begins anew. This becomes a constant source of symptomatology leading to patient frustration. The thinning of the keratin layer of the labia majora and labia minora also diminishes the protection normally afforded against bacterial and fungal adhesion to this tissue site. The epithelial cells of reproductive-age women also produce many substances, such as peptides [8], that can kill potential bacterial or fungal pathogens. Lessened production of these substances in menopausal women increases the chances of infection. The resulting skin infections add to the inflammation. Vulvar fungal infections occur commonly in this aging population (> Fig. 26.3), and if not eradicated by treatment contribute to the cycle of inflammation,
Vulvar Changes There are visually apparent alterations in the vulva of postmenopausal women. The tremendous reduction in the levels of estrogen results in a loss of tissue elasticity and an obvious thinning of the vulvar tissue (> Fig. 26.2). This thinning, apparent to the naked eye, is accompanied by other significant subcutaneous changes. These become more noticeable when the V-600 Syris imaging system is used, which allows the observer to view the tissue two cell layers beneath the surface [6]. The thinning of the skin is highlighted. In addition, using a system of cross-polarized light visualization, there is decreased vascularity, more dryness, and more subdermal inflammation. On the surface, the vulvar skin becomes retracted with the tissue demarcations between the labia majora and labia minora becoming blunted. These changes are particularly apparent using the magnification of the colposcope. This thinner tissue area is much more fragile and more prone to splitting and cracking, at times leaving the distressed patient with a new symptom: a painful, slow-healing vulvar cut.
Aging Genital Skin and Hormone Replacement Therapy Benefits
. Figure 26.3 Candida Albicans infection of vulva of post-menopausal woman
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this response by using commercially available, over-thecounter lubricants can have unintended results. These products contain propylene glycol, a chemical preservative to which some women with inflamed mucous membranes can develop sensitivities. This contact dermatitis increases even more the local inflammation and pain. To avoid this possibility, these couples should be advised to use mineral oil or olive oil as a pre-coital lubricant. They are unquestionably messy, but do not dry out over time with firm residual kernels of commercial products that can be an additional source of irritation, and they contain no chemicals that could cause a local tissue reaction.
Outside Influences: Hormone Replacement Therapy (HRT)
itching, scratching, and more inflammation. Bacterial infection of the vulva can be a life-threatening problem in post-menopausal women, particularly in diabetics in whom synergistic bacterial vulvar infections can occur, with tissue death requiring operative intervention for survival [9].
Outside Influences: Male The television advertisements of the American pharmaceutical industry are dominated by concerns about both male urinary function and sexual prowess. The theme of male erectile dysfunction vies with difficulty in urinary voiding as a lead-in to capture the attention of the male drug consumer. The models in the ads blend the knowing smiles of the older, but still physically rugged and vigorous male with the ‘‘come hither’’ looks of their very attractive female partners. For the viewer, the obvious result will be the nirvana of continued sexual satisfaction. For the post-menopausal sexual partner of these newly invigorated older males, there can be a tremendous downside for vaginal and vulvar skin health. The resulting increase in heterosexual activity can cause vaginal and/or vulvar lacerations because of the dryness and decreased elasticity of the lower genital tract skin and mucous membranes. This genital tract pain sets in place a cascade of continuing problems. Pain will reduce vaginal secretion and the memory of prior discomfort can result in heightened pelvic floor muscle contraction when insertion is attempted, making a prior unpleasant sexual experience even more uncomfortable. Reflex attempts to modify
Since most of the changes in the health of the vagina and vulva in perimenopausal and menopausal women are related to a lessened production of estrogen, it would be logical to assume that estrogen supplementation would be an aid for these women. This is true, within limits. Estrogen therapy is most helpful as a preventive measure. It is usually beneficial if given before the aging vaginal and vulvar tissue changes of thinning, loss of turgor and elasticity have become grossly apparent. It either prevents or markedly slows these genital skin and mucous membrane alterations. In the vagina of women receiving estrogen supplementation, the pH is as acidic as it was in the reproductive years, and the dominant bacterial flora remains the acid-producing lactobacilli. If the progressive tissue changes of thinning, inflammation, or lacerations occur, the effects of estrogen are much less dramatic. They may accelerate the healing of these cuts and decrease inflammation, but they do not return the tissue to a premenopausal state. These are not formulae for the ‘‘fountain of youth,’’ but they do halt further deterioration. Other pharmaceutical agents, such as topical adrenocortical steroids, are much better in combating local tissue inflammation and are frequently prescribed as an alternative therapy in women with inflammatory vulvar changes. Despite these shortcomings, the lifestyle of women taking supplemental estrogen is usually much improved. They experience less discomfort with intercourse and have fewer lower urinary tract infections. All of these are positive observations. The reader should reflexively wonder why there has not been a deluge of television advertisements for the improvement in female well-being and pleasure with estrogen replacement therapy to parallel the onslaught of ads trumpeting male well-being. This would give equal time to the current focus upon male
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sexual satisfaction. Instead, there is an absence of these ads and market evidence of a decrease in the current use of hormone replacement therapy (HRT) in American women. Why? This phenomenon of the current less frequent use of HRT in the United States has been largely driven by the publicity surrounding the results of Women’s Health Initiative (WHI) studies. These studies, sponsored by the National Institutes of Health (NIH), have highlighted the potential dangers for women taking systemic HRT. There is a small but increased risk of developing breast cancer in women taking HRT for 5 years or more [10]. This had been previously reported in earlier studies [11]. Other unexpected adverse outcomes were noted in asymptomatic menopausal women taking HRT. They had a greater chance of having either a heart attack or a stroke as compared to the population of women taking a placebo [10]. This was a surprise, for prior studies of HRT had shown beneficial effects, with a lowering of blood lipids [12]. This had been the basis for the medical dogma of the 1980s that HRT had cardiovascular and cerebrovascular benefits for all post-menopausal women. These WHI studies raised doubts about that hypothesis and led to a precipitous drop in the numbers American women using HRT. There were some major problems in the WHI postmenopausal women study design that have been too often overlooked. The focus of the WHI study was upon asympotomatic menopausal women. This in itself was appropriate, for if symptomatic women had been studied, there would have been an inordinate loss of women recruited for the study who were given the placebo. Their symptoms would have continued, and they would have been more likely to drop out of the study. Using asymptomatic women avoided this, but to recruit this population, one third of the women had been menopausal for over 5 years and another third had been menopausal for over 10 years. This fact should raise some concerns about the interpretation of the study. This is not the usual target group for HRT. The focus should be upon women who become symptomatic with perimenopause, with symptoms becoming more pronounced as they become menopausal. For the skin, the greatest benefits from estrogen treatment result from the prevention or slowing of the skinaging process and not from the treatment of established aging skin changes. This goal of HRT therapy – prevention, not treatment – was not reached in two thirds of the WHI study population. An interesting aside is that the minority one-third of these WHI study patients who received HRT within the first 5 years of menopause did not have the
increased cardiovascular and cerebrovascular risks noted in the other two-thirds. There are dangers of the use of systemic HRT for women, particularly an increased risk of breast cancer [13], but these have been over-emphasized to include local estrogren products while downplaying the benefits. Many women with vaginal or vulvar symptoms due to skin aging will benefit from a wide variety of local estrogen products, including creams, vaginal tablets, or a vaginal ring, none of which to date have been associated with any of the systemic risks that have been attributed to systemic HRT. The concerns about HRTs have been too widely publicized. All too often, many women will avoid these potentially beneficial local medications for they have been led to believe that the risks associated with systemic HRT apply to all estrogen products. This is unfortunate, because for many women, these agents improve their sense of worth for they help to modify at least some of the pitfalls of aging. Most of the older women using estrogen look and feel better. They are a selected population, for they were symptomatic prior to the use of these hormones. For them, the hormones improve their daily lives and the quality of their social interactions. These benefits for women should be cited as they have been for men. Males, taking drugs for erectile dysfunction, have an increased risk of heart attacks and blindness, both serious medical problems. These have not been the primary focus in discussion as have been the concerns about the risks for HRT for women.
Conclusion There is a gender gap here. For women, downplay benefits and stress the risks; for men, emphasize pleasure and gloss over the risks. This is a curious commentary upon the current American scene.
Cross-references > Biological
Effects of Estrogen on Skin Aging and Oral Hormone Replacement
> Climacteric
Therapy
References 1. American Society for Reproductive Medicine. Age and Fertility: A Guide for Patients (Patient Information Series). Birmingham: American Society for Reproductive Medicine, 2003.
Aging Genital Skin and Hormone Replacement Therapy Benefits 2. Hillier SL, Lau RJ. Vaginal microflora in post-menopausal women who have not received estrogen replacement therapy. Clin Infect Dis. 1997;25(Suppl 2):S123–126. 3. Pabich WL, Fihn SD, Stamm WE, et al. Prevalence and determinants of vaginal flora alternations in post-menopausal women. J Infect Dis. 2003;188:1054–1058. 4. Raz R, Stamm WE. A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infection. N Engl J Med. 1993;329:753–756. 5. Sobel JD. Desquamative inflammatory vaginitis: a new subgroup of purulent vaginitis responsive to topical 2% clindamycin therapy. Am J Obstet Gynecol. 1994;171:1215–1220. 6. Farage M, Singh M, Ledger WJ. Investigation of the sensitivity of a cross-polarized light visualization system to detect subclinical erythema and dryness in women with vulvovagintis. Am J Obstet Gynecol. 2009;201:1e1–1e6. 7. Pfeilschifter J, Kodtiz R, Pfohl M, et al. Changes in proinflammatory cytokine activity after menopause. Endocr Rev. 2002;23:90–119.
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8. Hancock REW. Lationic peptides: effectors in innate immunity and novel antimocrobials. Lancet Infect Dis. 2001;1:156–164. 9. Addison WA, Livengood CH III, Hill GB, et al. Necrotizing fasciitis of vulvar origin in diabetes patients. Obstet Gynecol. 1984;63: 473–479. 10. Rossouw TE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomize controlled trial. JAMA. 2002;288:321–333. 11. Hulley S, Grady B, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in post-menopausal women. JAMA. 1998;280:605–613. 12. Rossouw JE, Prentice RL, Manson JE, et al. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA. 2007;297:1465–1477. 13. Chlebowski RT, Kuller LH, Prentice RL, et al. Breast cancer after use of estrogen plus progestin in post-menopausal women. N Engl J Med. 2009;360:573–582.
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19 Aging of Epidermal Stem Cells Alexandra Charruyer . Ruby Ghadially
Introduction Advances in aging biology indicate that stem cells have a crucial role in organ maturation and aging. Studies have demonstrated molecular and biochemical changes in tissue-resident progenitor cells and their microenvironments during chronological aging of tissues such as the heart [1], brain [2], and hematopoietic system [3]. In this chapter knowledge in the field of aging and stem cells derived from tissues other than the epidermis is reviewed, and the challenges of studying aging stem cells discussed. Subsequently, epidermal stem cells are reviewed and changes in progenitor populations of the epidermis that occur with age discussed. Finally, the body of knowledge specifically related to the aging of epidermal stem cells and the implications of stem cell aging for carcinogenesis are examined.
Aging and Stem Cells Changes in Stem Cell Frequency with Aging Information about the impact of aging on stem cells has been obtained from the hematopoietic system and it remains an ideal system for this type of study as stem and progenitor cells are most well defined in the hematopoietic system. The loss of immune function and the increased incidence of myeloid leukemia associated with aging were thought to be due to a decrease in hematopoietic stem cell frequency. Research has now contradicted this assumption. Several studies indicate that murine hematopoietic stem cell numbers increase substantially with age [4, 5]. Limiting dilution assays have shown that hematopoietic stem cells from aged mice were more efficient at myeloid reconstitution than hematopoietic stem cells from young mice. Aged hematopoietic stem cells were found to be five times as numerous, but one quarter as efficient at engrafting, as young hematopoietic stem cells, suggesting a small increase in reconstitution ability [5]. In another study, the relative number of the most primitive stem cell was found to be three- to fourfold higher in aged over young mice but there was a decrease in the proliferative activity
of aged hematopoietic stem cells [4]. These studies show that there is an increase in primitive precursors and a decrease in proliferative ability with aging. In skeletal muscle, estimates of stem cell (satellite cell) number have produced varying results. One study looking at DNA content and nuclei count by electron microscopy demonstrated an increase in the satellite cell number in aging rats [6]. Using flow cytometry and CD34 expression there was no difference in satellite cell frequency in aged and young murine muscles [7]. Finally, a microscopic study of skeletal muscle reported a decrease in satellite cells during aging [8]. Thus, a consensus has yet to be reached regarding the effect of aging on skeletal stem cell frequency.
Intrinsic Cellular Modifications in Stem Cells with Aging While changes in stem cell frequency play a role in aging, there is also evidence for intrinsic cellular modifications in stem cells with aging. It is challenging to distinguish intrinsic cellular aging from the effects of the cellular milieu when stem cells are studied in their natural environment, and the isolation of a pure stem cell population is needed (for review see [9]). Epidermal stem cells isolated from young and old mice based on their Hoechst dye exclusion were analyzed for gene expression profile by cDNA arrays. There was similar gene expression of 422 genes assayed in young and old epidermal stem cells [10]. Expression profiles of highly purified long-term repopulating hematopoietic stem cells showed that aging was associated with a down-regulation of genes mediating lymphoid function and up-regulation of genes involved in myeloid fate, indicating that the loss of immune function and the increase in leukemia in the elderly is due to intrinsic alterations in hematopoietic stem cells [11]. These latter studies provide evidence that the intrinsic properties of stem cells change with age.
Changes in the Stem Cell Niche with Aging Stem cell homeostasis is maintained not only by intrinsic factors, but also by extrinsic factors, such as the local
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_19, # Springer-Verlag Berlin Heidelberg 2010
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environment of the stem cell niche, the surrounding tissue, the systemic milieu of the organism and the external environment (for review see [9, 12]). Age-related modifications of extrinsic factors may include alterations in the composition of the extracellular matrix, in membrane proteins and lipids, and in factors that constitute the systemic milieu. Modifications in the stem cell niche during the aging process have been addressed in Drosophila. The Drosophila germline stem cells together with the somatic cells present in the niche is one of the most well-defined stem cell niches [13]. In somatic niche cells from older Drosophila testes there is a decrease in expression of a cell adhesion molecule (DE-cadherin) and a self-renewal ligand (unpaired). This was correlated with an overall decrease in stem cell numbers inside the niche [14]. Furthermore, restoring self-renewal (unpaired) resulted in an increase of the number of germline stem cells in older males compared with age-matched controls. Thus modifications of the somatic cells that constitute the niche in aged testes, can affect the frequency of stem cells inside the niche. Furthermore, murine spermatogonial stem cells could be serially transplanted in young mice recipients without showing any decline in stem cell number or colony forming ability for more than 3 years, indicating that a young environment can influence stem cell self renewal capacity, and that the failure of niche integrity plays a key role in the reproductive deficit in aged mice [15]. The effect of environment on skeletal muscle satellite cells has been studied [16]. No significant differences were found in mass or maximum force between old muscles grafted into young hosts and young muscle grafted into those same young hosts. Conversely, young muscles grafted into old recipients did not regenerate better than old muscles grafted into the same old hosts, indicating an important role for the environment in muscle regeneration after transplantation. Conditioned medium from differentiated myotubes of young mice exhibited a strong mitogenic action on aged satellite cells in vitro, whereas no mitogenic action was observed from conditioned media of myotubes from aged mice, either on young or on aged satellite cells [17] (for review see [18]). More recently, systemic influences on aged satellite cells were investigated using parabiotic pairings in which regenerating tissues in aged animals were exposed either to their own serum or that of young mice (isochronic or heterochronic parabioses respectively) [19]. In this study, exposing injured muscles from old mice to heterochronic parabioses greatly improved muscle regeneration and myotubule formation was similar to that observed in young mice. The authors concluded that a young systemic environment
could improve the impaired regenerative ability of aged skeletal stem cells. These studies demonstrate the important influence of both the local and systemic environment within which stem cells reside.
Symmetric and Asymmetric Stem Cell Division and Aging Stem cells in the niche undergo two types of division, symmetric self-renewal divisions leading to two identical daughter cells and asymmetric divisions leading to one daughter cell identical to the original stem cell and another non-stem daughter cell that leaves the niche and undergoes differentiation [13, 20]. It is predicted that aged stem cells would have preserved or increased self-renewal potential (symmetrical divisions) and decreased asymmetrical divisions, and in order to maintain a constant rate of cellular production the aged will have more proliferation in the transit amplifying cells [21, 22]. Studies agree with this prediction; in different stem cell compartments including the hematopoietic system and the intestinal crypt there is an increase in transit amplifying cells with age [23] (for review see [21]).
Challenges of Studying Aging and Stem Cells Issues surrounding the study of aging include the study of aging versus development, the study of animals of an appropriate age, the heterogeneity of aged animals, and the lack of a pure population of stem cells from most tissues. Many studies are confounded by the use of neonatal versus adult or neonatal versus aged tissue. Such studies may not reflect changes of aging, but rather changes occurring during development from birth to adulthood. It is important to keep these studies distinct, and the focus here is on aging of epidermal stem cells from the adult to aged individual. In aging studies, the age of the aged animals to be studied is of great importance, and guidelines for such studies are limited. The definition of aging varies depending on the physiological system under question. For example, many age-related changes in the immune system are evident by the 70% survival point and even earlier, while kidney changes start later. The most common age used to model old age is somewhere around the 50% survival point, although it may vary from the 70% survival point to the 30% survival point. It has been stated that ‘‘without epidemic disease or exaggerated or lopsided
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tumor incidence, the 50% survival point can be considered as an indicator of the onset of the senescent period’’ [24]. It is important to note that results from extremely aged animals are not reliable because they may be the consequence of advanced disease rather than aging. The best approach, although often difficult in practice, is to study multiple time points during the senescent period [25]. Aging is also difficult to study because of the heterogeneity that is associated with the aging process. Indeed, the variance in vital characteristics in the elderly is substantially higher compared with other groups of the population [26]. One universal characteristic of aging is the accumulation of molecular damage, which induces alterations in gene expression, genomic instability, mutations, tissue disorganization and organ dysfunction. Because of the low probability that two molecules will be damaged in the same way and with the same intensity substantial molecular heterogeneity results, and this leads to clinical heterogeneity in the elderly population [27]. While hematopoietic stem cells can be isolated at a single cell level [28], epidermal stem cell markers that allow isolation of epidermal stem cells at a single cell level are yet to be found. Studying populations of stem cells that are of unknown purity poses a challenge. Furthermore, the lack of a good understanding of the stem cell hierarchy in epidermis complicates studies further. In summary, studies of aging and in particular aged epidermal stem cells are profoundly affected by the choice of age for the young cohort, the choice of age for the aged cohort, the heterogeneity of the aged cohort, and the lack of a pure epidermal stem cell population for study.
Characterization of Epidermal Stem Cells The integrity of the epidermis is maintained by division of cells in the proliferative basal layer that replace differentiated cells in the outermost stratum corneum layer. Not all proliferative cells in the basal layer are stem cells. Transit-amplifying cells amplify the basal cell population but are limited to a finite number of divisions before they differentiate and are lost from the epidermis [29]. Stem cells of the basal layer are responsible for maintaining and generating the adult epidermis and its appendages, including hair follicles and sebaceous glands. Different stem cell niches have been described: (1) the follicular stem cell that resides in the hair follicle bulge [30, 31] (for review see [32]), (2) the interfollicular stem cell [33] (for review see [34]), and (3) the melanocyte stem cell localized in the hair follicle [35].
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Phenotypic analysis of hematopoietic stem cells has provided the ability to separate the long-term proliferating cell from the less primitive cells detected in colonyforming assays [36]. These types of studies have allowed a hierarchy of hematopoietic stem cell differentiation to be determined. Different methods to isolate epidermal stem cells have been proposed, although very little has been achieved in defining a hierarchy of progenitors in the epidermis. Several of the most prevalent methods for isolating putative epidermal stem cells that have been used to study aging of epidermal stem cells are discussed here (> Table 19.1), including (1) side population (SP) cells that efflux Hoechst 33342 fluorescent dye [37, 38], (2) integrin a6 bright/CD71 dim human keratinocytes [39, 40], (3) collagen adhesion, and (4) a quantitative epidermal regeneration assay [33, 41]. Other methods/ markers such as label retaining cells [42], p63 [43], keratin 19 [44], keratin 15 [45] and elevated levels of b catenin [46] have also been reported as putative stem cell markers. Hoechst dye exclusion is an example of a method borrowed from other tissues that may be useful for isolating epidermal stem cells. Hematopoietic and muscle stem cells are identified by the ability of stem cells to exclude Hoechst 33342 dye [47, 48]. A multidrug resistance P-glycoprotein pump present in stem cells mediates this exclusion. This multidrug resistance pump is also associated with resistance to anti-cancer drugs and is over expressed in some cancer cell lines [49]. Basal epidermal cells also express this P-glycoprotein [50]. Cells showing Hoechst exclusion were defined as stem cells by their clonal ability, high proliferative potential, and the ability to recapitulate an epidermis in vitro [51]. Transit amplifying cells were identified as cells showing a medium forward and orthogonal scatter without regard to Hoechst exclusion. These cells could not maintain an epidermis in vitro [51]. A recent study of mouse epidermis, characterizing side population and non-side population cells showed that a6 integrin, b1 integrin, Sca-1, keratin 14, and keratin 19 were all highly expressed by side population cells, while CD34, CD71, and E-cadherin were more weakly expressed by side population cells than by non-side population cells. This demonstrates that side population cells express previously established stem cell associated proteins [52]. In contrast, it was found that side population keratinocytes are distinct from the label-retaining cell population, since side population cells and label retaining cells showed distinctly different and non-overlapping expression profiles of b1 and a6 integrins [37]. Thus although the frequency of epidermal stem cells in the Hoechst dye excluding population is unknown, this population
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. Table 19.1 Prevalent methods for isolating putative epidermal stem cells Method Side population
Description
References
Cells that efflux Hoechst 33342 dye are highly clonogenic and recapitulate an epidermis in vitro.
Terunuma et al. [37], Triel et al. [38]
Integrin Quiescent cells. Li A, Simmons PJ, a6brightCD71dim High long-term Kaur P. [39], proliferative Terunuma et al. [40] capacity. Produce human skin equivalents with a stratified epidermis. Collagen adhesion
Cells that adhere rapidly to type IV collagen are highly proliferative in vitro. Can form a robust stratified epidermis in vitro.
Watt FM. [55], Jones PH, Harper S, Watt FM. [56], Kaur et al. [57]
In vivo epidermal repopulating unit
In vivo Schneider et al. [33], transplantation Strachan et al. [41], model (in chambers, Charruyer et al. [78] or by subcutaneous injection). Individual keratinocytes regenerate a cornified, stratified epithelium (epidermal repopulating unit/ ERU) for the long term.
Others
Label retaining cells, p63, keratin 19, keratin 15, b catenin.
Bickenbach JR. [42], Yang et al. [43], Stasiak et al. [44], Lyle et al. [45], Zhu AJ, Watt FM. [46]
contains cells with phenotypic attributes of stem cells and high proliferative potential in vitro. High a6 integrin and low transferrin receptor (CD71) expression are perhaps the most accepted epidermal stem cell markers to date [39, 53]. The a6 integrin bright, CD71 dull cells are relatively quiescent and populations of these cells have very high long-term proliferative capacity [39]. Tani et al observed that a6briCD71dim murine dorsal keratinocytes were a quiescent population of small cells, with
a high nuclear to cytoplasmic ratio, consistent with primitive cells. In addition, 1.4% of total isolated keratinocytes were both a6briCD71dim and label retaining cells [53]. In human skin, the a6briCD71dim population contained smaller cells with a high nuclear to cytoplasmic ratio, was capable of producing a high number of large colonies after 10 days of culture, and produced skin equivalents with a stratified and thick epidermis [54]. In summary, although the stem cell purity is unknown, in both human and mouse epidermis the a6briCD71dim population contains undifferentiated cells with colony forming ability in vitro. Determining how in vitro assays relate to stem cell behavior in vivo is complex and much of the evidence is inconsistent with the concept that a colony forming cell is a stem cell [33, 41]. Nonetheless the analysis of colony formation in vitro is an attractive and prevalent procedure used to define stem cell behavior. b1 integrin is expressed in all basal keratinocytes. As keratinocytes leave the basal layer they down-regulate the expression of b1 integrin [55]. Cells that adhere rapidly to a b1 integrin ligand, type IV collagen, were found to have a high proliferative potential in vitro, whereas cells that adhere slowly divide only a few times before all of their progeny undergo terminal differentiation [56]. Rapidly adherent cells also form a robust stratified epidermis in vitro [54]. 20–40% of basal cells have high b1 integrin expression, which is in great excess of the proportion of basal cells that are estimated to be stem cells in vivo and thus it is likely that high b1 integrin expression does not uniquely select for epidermal stem cells [56]. It is believed that stem cells cannot be defined based solely on their proliferative behavior in culture [57]. Stem cell populations lack the appropriate stimuli for growth in vitro and surrogate in vitro analyses to assess putative stem cell behavior may not accurately distinguish between epidermal stem cells and progenitors cells [41]. A quantitative epidermal regeneration assay was developed that involved the use of dissociated keratinocytes allowed to regenerate a cornified, stratified epithelium on top of dermal fibroblasts seeded onto the subcutaneous fascia of immunodeficient mice. By seeding progressively lower numbers of GFP-positive keratinocytes in this repopulation assay, limiting dilution analysis quantifies the frequency of cells with long-term repopulating ability in a given population. Chambers are kept for 9 weeks [33]. The value of an in vivo assay for epidermal stem cells is well recognized [41, 58]. Using the quantitative in vivo regeneration assay described above it is possible to quantify epidermal stem cells from different keratinocyte populations. The study of epidermal stem cells from different sources such as aged versus young or diseased versus
Aging of Epidermal Stem Cells
healthy would be greatly enhanced by the availability of pure populations of epidermal stem cells. To date techniques have been developed for enriching populations of keratinocytes for early progenitors, but not at the single cell level. Given the difficulty in finding specific stem cell markers, investigation is needed to determine combinations of markers that can enrich for epidermal stem cells at a single cell level. Markers found in stem cells from other tissues, embryonic stem cell markers, or even cancer stem cell markers found in tumorigenic tissues may provide useful strategies for the isolation of normal epidermal stem cells at a single cell level.
Aging and the Keratinocyte Proliferative Compartment Abundant historical studies in both human and animal models demonstrate that aging of the epidermis is accompanied by decreased proliferation, both basally and in response to proliferative stimuli [59–66]. These studies have led to the concept that epidermal stem cells, which are responsible for the maintenance of the epidermis, are involved in the aging process. The effect of aging on the keratinocyte proliferative compartment (stem cells and transit amplifying cells) has been studied in vivo. After skin injury, the regeneration of tissue requires stem cell mobilization [30, 67], which has made wound healing a valuable model for the study of the impact of aging on epidermal stem cells. A battlefield surgeon performed an early study of wound healing and age during World War I [68]. Using a cicatrisation index, the biological age of an injured soldier could be determined by measuring the rate of closure of war wounds. Soldiers in their thirties healed more slowly than those in their twenties. Standardized superficial skin wounds were created in young adults (18–25 years) and aged adults (65–75 years) and healing was monitored. By day 28, the younger group had completely restored their original skin markings, while the older cohort took double the time (56 days) [62]. A nonradioactive method, using Dansyl chloride that binds only to nonviable corneocytes, was used to measure stratum corneum transit time. Transit time was increased in aged persons and there was no difference in the number of horny layers, indicating that the increase in stratum corneum transit time in the aged was a reflection of decreased epidermal proliferation [63]. These studies of stem cells and transit amplifying cells provide some information on how the proliferative compartment of epidermis changes with aging in vivo, but not specifically on how the stem cell changes.
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The effect of aging on keratinocyte proliferation has also been studied using in vitro models of aging. Rheinwald and Green compared the proliferative behavior of seven human newborn-derived keratinocyte cultures with 3-, 12- and 34-year-derived cultures [69]. Newborn-derived cultures were able to undergo 25– 51 cell generations versus 20–27 for older person-derived cultures and could be maintained through three to six passages versus two to three passages. In addition, plating efficiency (colony forming ability) was up to 15.7% for the newborn versus 0.7% for the older person-derived cultures. Another study using newborn human keratinocytes compared with adult human keratinocytes reported that while attachment rate is independent of donor age, plating efficiency was strongly dependent on donor age. Plating efficiency was 2–10% in newborn cultures and below 0.01% in adult cultures [61, 70]. It should be noted that the above in vitro studies may reflect development, aging, or a combination of development and aging since they used newborn human keratinocytes.
Aging and Epidermal Stem Cells The above in vivo and in vitro observations of aging and proliferation were made on the entire keratinocyte population and not on different proliferative subpopulations. To address the differences in behavior of proliferative keratinocyte subpopulations (stem cells versus transit amplifying cells), the growth potential of individual proliferative clones from different donors (two neonatal, one 64-year-old, and one 78-year-old) was studied [71]. After plating individual cells, resultant clones were passaged into a second dish. The original clone was classified by the appearance of cells in the secondary dish into holoclones (cells that form large rapidly growing colonies), paraclones (that form uniformly small, terminal colonies) and meroclones (that form both types of colonies). With age, the number of holoclones decreased and the number of paraclones increased when compared with newborns. The authors concluded that the culture lifetime of a keratinocyte population declines with the age of the donor, as demonstrated by the change in the proportion of the three clone types. After studying the effects of aging on keratinocyte proliferation in vitro, and with the study of stem cell frequency in the hematopoietic system, and the discovery of epidermal stem cell markers, several studies have now addressed the influence of aging on epidermal stem cell number. Such studies have produced varying results. There were twice as many stem cells in neonatal mouse
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epidermis (1–2 days) as in adult (8–14 weeks) (8.4% versus 3.8%, respectively), using small size and Hoechst fluorescence to define a stem cell [72]. In a study of four human foreskins (from 1, 4, 35 and 61 year old donors), there was a decrease in the number of putative epidermal stem cells in adult versus neonatal human epidermis, as defined by a6 integrinhi CD71lo expression [73]. Such comparisons of adult and neonatal samples may reveal changes of development and/or aging. However, while neonatal murine epidermis had three times as many Hoechst dye-excluding cells as adult epidermis, the number of cells that could exclude Hoechst dye was unchanged in aged adult versus young adult murine epidermis [10]. The epidermal stem cells from young adults and aged adults had similar characteristics in culture, similar gene expression, and did not show extensive loss of telomeres with age. In the same study, epidermal stem cells isolated from 22-month-old transgenic mice that expressed GFP were injected into mouse blastocysts. Six months later various tissues of the resultant mice contained GFP positive cells, demonstrating that the developmental potency of murine epidermal stem cells is not altered with aging. It was concluded from this study that epidermal stem cells are resistant to cellular aging [10]. Furthermore, human and mouse keratinocytes that exclude Hoechst dye showed little variability in protein expression profiles in aged versus neonatal epidermis, suggesting that as epidermal stem cells age they do not substantially change their cellular characteristics [74]. The differing findings presented above could result from the study of keratinocytes of varying ages, their human or murine derivation, the possibility that different putative epidermal stem cells markers are not isolating the same population of progenitor cells, or that different methods have different efficacy in isolating epidermal stem cells. While the previous in vitro studies are very informative, skin aging has been difficult to study in vivo due to the lack of relevant models. A recent study attempted to address this issue by studying the effect of aging on keratin 15 positive progenitors from young (2–6 months) and aged (22–26 months) mice [75]. Using whole-mount immunostaining, they observed a similar number of keratin 15 positive bulge stem cells in follicles of both young and aged mice, suggesting that epidermal stem cell frequency is not affected by aging. Surprisingly, there was only a modest, and not statistically significant, decrease in proliferation as measured by Ki67. This is different from previous in vitro studies, and suggests that in vivo assays may produce different results in the study of skin aging and epidermal stem cells.
In order to examine in vivo, whether the decreased proliferative ability of aged epidermis could be explained by either quantitative and/or qualitative alterations in the stem and/or transit amplifying cell proliferative compartments, a quantitative in vivo transplantation assay was used similar to the hematopoietic assays that have been informative about changes in hematopoietic progenitors with aging [5, 76, 77]. In vivo transplantation assays of aged and young adult keratinocytes showed that while no significant difference in epidermal stem cell frequency could be detected, transit amplifying cell frequency was greater in the aged. With aging there was both an increased growth fraction (proportion of actively cycling cells) and longer cell cycle duration, resulting in prolonged existence of the short term repopulating cells in vivo. Finally, there was decreased cellular output from both individual epidermal stem cells and transit amplifying cells with aging (> Fig. 19.1). This suggests that increased cell cycle
. Figure 19.1 Epidermal repopulating units from aged progenitors are smaller and contain less cells than those from young progenitors at 1 and 11 weeks (bar = 10 mm)
Aging of Epidermal Stem Cells
duration contributes to the decreased cellular output from epidermal progenitors, while the larger growth fraction may be a compensatory mechanism [78].
5. 6.
Aging, Carcinogenesis, and Stem Cells Given the ability of stem cells to self-renew, proliferate and maintain homeostasis and the increase in incidence of cancer with aging, it is assumed that stem cells are involved in carcinogenesis. Recently a mechanistic link has been made between aging and carcinogenesis. It has been shown that senescent cells are more resistant to apoptosis [79] and have impaired DNA repair mechanisms [80]. Furthermore, senescent cells can survive for long periods of time, leading to the accumulation of damaged cells [81]. The resulting genetic instability leads to an increase in carcinogenesis (for review see [82]). The cancer stem cell hypothesis is being verified in different tissues including skin (for review see [83]). Because of similarities in the behavior of aging stem cells, senescent cells and cancer stem cells, it is thought that aged stem cells have a major role in the increased carcinogenesis associated with aging (for review see [84]).
7. 8.
9. 10. 11. 12. 13. 14.
15. 16.
17.
Conclusion This review discusses the changes in progenitor populations that occur with aging, and more specifically changes of the epidermis that occur with aging. The consensus of opinion appears to be that changes responsible for aging of tissues occur not only in the stem cell pool itself, but also in the transit amplifying cell compartment and in the stem cell environment. In order to study aging of epidermal stem cells it is essential to isolate epidermal stem cells at the single cell level to better define them at a molecular level. It will also be important to study the intrinsic and extrinsic changes that occur in the environment/niche of the epidermal stem cell with aging.
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30. Taylor G, et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell. 2000;102:451–461. 31. Oshima H, et al. Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell. 2001;104:233–245. 32. Ghadially R. In search of the elusive epidermal stem cell. In: The Promises and Challenges of Regenerative Medicine. Heidelberg: Springer, 2005. p. 45–62. 33. Schneider TE, et al. Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells. Proc Natl Acad Sci USA. 2003;100:11412–11417. 34. Kaur P. Interfollicular epidermal stem cells: identification, challenges, potential. J Invest Dermatol. 2006;126:1450–1458. 35. Nishimura EK, et al. Dominant role of the niche in melanocyte stemcell fate determination. Nature. 2002;416:854–860. 36. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994;1:661–673. 37. Terunuma A, et al. Side population keratinocytes resembling bone marrow side population stem cells are distinct from labelretaining keratinocyte stem cells. J Invest Dermatol. 2003;121: 1095–1103. 38. Triel C, et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res. 2004;295:79–90. 39. Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA. 1998;95:3902–3907. 40. Terunuma A, et al. Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay. Stem Cells. 2007;25:664–669. 41. Strachan LR, et al. Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro. Stem Cells. 2008;26:235–243. 42. Bickenbach JR. Identification and behavior of label-retaining cells in oral mucosa and skin. J Dent Res. 1981;60 (Spec No C):1611–1620. 43. Yang A, et al. P63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999;398:714–718. 44. Stasiak PC, et al. Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J Invest Dermatol. 1989;92:707–716. 45. Lyle S, et al. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci. 1998;111(Pt 21):3179–3188. 46. Zhu AJ, Watt FM. Beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development. 1999;126:2285–2298. 47. Lin KK, Goodell MA. Purification of hematopoietic stem cells using the side population. Methods Enzymol. 2006;420:255–264. 48. Uezumi A, et al. Functional heterogeneity of side population cells in skeletal muscle. Biochem Biophys Res Commun. 2006;341:864–873. 49. Kohno K, et al. The direct activation of human multidrug resistance gene (MDR1) by anticancer agents. Biochem Biophys Res Commun. 1989;165:1415–1421. 50. Sleeman MA, Watson JD, Murison JG. Neonatal murine epidermal cells express a functional multidrug-resistant pump. J Invest Dermatol. 2000;115:19–23. 51. Dunnwald M, et al. Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol. 2001;10:45–54. 52. Yano S, et al. Characterization and localization of side population cells in mouse skin. Stem Cells. 2005;23:834–841.
53. Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA. 2000; 97:10960–10965. 54. Kim DS, et al. Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents. Cell Mol Life Sci. 2004; 61:2774–2781. 55. Watt FM. Studies with cultured human epidermal keratinocytes: potential relevance to corneal wound healing. Eye. 1994;8(Pt 2): 161–162. 56. Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell. 1995;80:83–93. 57. Kaur P, et al. Keratinocyte stem cell assays: an evolving science. J Investig Dermatol Symp Proc. 2004;9:238–247. 58. Kolodka TM, Garlick JA, Taichman LB. Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes. Proc Natl Acad Sci USA. 1998;95:4356–4361. 59. Cerimele D, Celleno L, Serri F. Physiological changes in ageing skin. Br J Dermatol. 1990;122(Suppl 35):13–20. 60. Gerstein AD, et al. Wound healing and aging. Dermatol Clin. 1993;11:749–757. 61. Gilchrest BA. In vitro assessment of keratinocyte aging. J Invest Dermatol. 1983;81:184s–189s. 62. Grove GL. Age-related differences in healing of superficial skin wounds in humans. Arch Dermatol Res. 1982;272:381–385. 63. Grove GL, Kligman AM. Age-associated changes in human epidermal cell renewal. J Gerontol. 1983;38:137–142. 64. Haratake A, et al. Intrinsically aged epidermis displays diminished UVB-induced alterations in barrier function associated with decreased proliferation. J Invest Dermatol. 1997;108:319–323. 65. Leyden JJ, et al. Age-related differences in the rate of desquamation of skin surface cells [proceedings]. Adv Exp Med Biol. 1978;97: 297–298. 66. Roberts D, Marks R. The determination of regional and age variations in the rate of desquamation: a comparison of four techniques. J Invest Dermatol. 1980;74:13–16. 67. Ito M, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med. 2005;11: 1351–1354. 69. Nouy PLD. Biological Time. New York: The Macmillan Company, 1937. 69. Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature. 1977;265:421–424. 70. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975;6:331–343. 71. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA. 1987;84:2302–2306. 72. Dunnwald M, et al. Mouse epidermal stem cells proceed through the cell cycle. J Cell Physiol. 2003;195:194–201. 73. Youn SW, et al. Cellular senescence induced loss of stem cell proportion in the skin in vitro. J Dermatol Sci. 2004;35:113–123. 74. Liang L, et al. As epidermal stem cells age they do not substantially change their characteristics. J Investig Dermatol Symp Proc. 2004;9:229–237. 75. Giangreco A, et al. Epidermal stem cells are retained in vivo throughout skin aging. Aging Cell. 2008;7:250–259.
Aging of Epidermal Stem Cells 76. Harrison DE, Astle CM, Stone M. Numbers and functions of transplantable primitive immunohematopoietic stem cells. Effects of age. J Immunol. 1989;142:3833–3840. 77. Sudo K, et al. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. 2000;192:1273–1280. 78. Charruyer A, et al. Transit-amplifying cell frequency and cell cycle kinetics are altered in aged epidermis. J Invest Dermatol. 2009; 129(11):2574–2583. 79. Gniadecki R, Hansen M, Wulf HC. Resistance of senescent keratinocytes to UV-induced apoptosis. Cell Mol Biol (Noisy-le-grand). 2000;46:121–127. 80. Matta JL, et al. DNA repair and nonmelanoma skin cancer in Puerto Rican populations. J Am Acad Dermatol. 2003;49:433–439.
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50 Aging of Skin Cells in Culture Suresh I. S. Rattan
Introduction The study of age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. In modern biogerontology, the terms ‘‘cellular aging,’’ ‘‘cell senescence,’’ or ‘‘replicative senescence’’ most commonly imply the study of normal diploid cells in culture, which during serial subcultivation undergo a multitude of changes culminating in the permanent cessation of cell division. This process of cellular aging in vitro is generally known as the Hayflick phenomenon, and the limited division potential of normal cells is called the Hayflick limit, in recognition of the observations first reported by Leonard Hayflick in 1961 [1]. With respect to skin aging, three main cell types have been studied extensively with respect to cellular aging in vitro: dermal fibroblasts, epidermal keratinocytes, and melanocytes [2–7]. The aim of this chapter is to describe the experimental system of aging of skin cells in culture, to provide an overview of the age-related changes in the structural and functional aspects of cells including physiological, biochemical, and molecular changes, and to evaluate the use of such a system in testing and developing effective interventions for maintaining and/or re-achieving a healthy skin during aging.
Experimental Model System of Cellular Aging in Culture Once the primary culture of normal cells is established in culture from the normal tissue (e.g., a skin biopsy), by using any of the standard methods such as the explant growth and enzymic dissociation of cells, the primary culture can then be subcultivated repeatedly at each time it becomes confluent. This repeated subculturing of cells is also known as serial passaging [1]. In a description of the Hayflick phenomenon, Phase I is the period of the establishment of the primary culture from normal tissue; Phase II is a relatively long period of serial passaging,
growth, and cell proliferation at a constant rate; and Phase III, is the final period of slowing-down of growth, which results in the cessation of cell division and end of replicative lifespan of cells. The whole duration of serial passaging is considered as the process of cellular aging and the end-stage irreversible growth arrest in G1 is termed as replicative senescence. After reaching a state of replicative senescence, some cells can still stay alive and be metabolically active at a minimal level for sometime and generally resist undergoing apoptosis [1]. Although the exact culturing conditions, such as the type of the culture medium, the source of growth factors, the use of antibiotics, and the incubation temperature, humidity, and gaseous composition may vary for different cell types, serial subcultivation of normal diploid cells can be performed only a limited number of times. This is in contrast to the high proliferative capacity of transformed, cancerous, and immortalized cells, whose cultures can be subcultivated and maintained indefinitely. The total number of cell divisions, measured as the cumulative population doublings (CPD), which can be achieved by a specific cell type in vitro, depends upon several biological factors. These include the maximum lifespan of the species, developmental and adult age of the donor of the tissue biopsy, the site of the biopsy, and the health status of the donor [8]. For example, for human fibroblasts the range of CPD for the cell strains originating from embryonic tissues is between 50 and 70, whereas for those originating from adult biopsies it is generally less than 50 CPD. A similar range for CPD attained by human keratinocytes and melanocytes has been reported [2–7]. Additionally, gaseous composition, especially oxygen levels, and the quality of the nutritional serum and growth factors added to the culture medium, can significantly affect the proliferative lifespan of cells in vitro. For example, culturing of fibroblasts in vitro in the air with about 20% oxygen levels reduces their replicative lifespan, which could be otherwise achieved at low level (2%) concentration akin to in vivo conditions [9, 10]. Furthermore, the site of the skin biopsy, for example, sun-exposed versus sun-protected area has a significant effect on the CPD levels achieved by cells in culture [1, 11, 12].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_50, # Springer-Verlag Berlin Heidelberg 2010
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The Phenotype of Aging Skin Cells Serial passaging of normal diploid skin cells is accompanied by a progressive and accumulative occurrence of a wide variety of changes before the final cessation of cell replication occurs. The emerging senescent phenotype of serially passaged normal diploid skin cells can be categorized into the structural, physiological, and biochemical and molecular phenotypes, which can be used as biomarkers of cellular aging in vitro, as summarized in > Tables 50.1–50.3. There are more than 200 such structural, physiological, biochemical, and molecular characteristics that have been studied during cellular aging, and a list of major characteristics that appear progressively in cell cultures, and distinguish between young and senescent cells, generally before the end of proliferative lifespan and their irreversible arrest in the G1 phase of the cell cycle, can be found in several publications [13–15]. A summary of such phenotypic changes is given below. . Table 50.1 Structural phenotype of skin cells undergoing aging in culture ● Increased cell size ● Change of shape from thin, long, and spindle-like to flattened and irregular ● Loss of fingerprint-like arrangement in parallel arrays on the cell culture substrate ● Rod-like polymerization of the cytoskeletal actin filaments and disorganized microtubules ● Increased membrane rigidity ● Increased multinucleation ● Increased number of vacuoles and dense lysosomal autophagous bodies
Structural phenotype > Table 50.1 lists the major structural changes observed in aging skin cells in culture. Most commonly, a progressive increase in cell size and the loss of homogenous morphological pattern are the most dramatic and easily identifiable differences in early passage young and late passage old or senescent cells. Other structural changes during aging of skin cells include cytoskeletal and membrane rigidity, accumulation of intracellular debris, and incomplete cytokinesis leading to multinucleation (> Fig. 50.1). In addition to the gross structural alterations listed in > Table 50.1, there are several ultrastructural changes reported by using electron microscopic methods. These include the presence of distorted mitochondria, increased level of chromosomal aberrations, overcondensation of chromatin, increased nucleolar fragmentation, and the accumulation of lipid–protein conjugate lipofuscin in lysosomes [16–18]. Functional phenotype Numerous studies have been performed elucidating changes in various functional and physiological parameters of skin cells undergoing aging. > Table 50.2 lists some of the main changes, which clearly indicate that almost all aspects of cellular function and physiology become impaired during aging. Collectively, these data show that aging skin cells progressively become less active, have reduced ability to maintain various physiological functions, and become more prone to the negative effects of harmful substances.
. Table 50.3 Biochemical and molecular phenotype of skin cells undergoing aging in culture ● Permanent growth arrest in late G1 phase of the cell cycle near the S phase boundary ● Increased mRNA and protein levels of cell cycle inhibitors
. Table 50.2 Physiological phenotype of skin cells undergoing aging in culture ● Altered calcium flux, pH, viscosity, and membrane potential
● Increased mRNA and protein levels of inhibitors of proteases ● Decreased expression, levels, and activities of numerous house-keeping enzymes ● Decreased expression, levels, and activities of macromolecular turnover pathways ● Reduced levels of methylated cytosines in the DNA
● Reduced activity of ionic pumps
● Reduced length of telomeres
● Reduced mobility ● Reduced respiration and energy production
● Increased levels of damage in nuclear and mitochondrial DNAs
● Reduced response to growth factors and other mitogens
● Increased levels of damaged and abnormal proteins
● Increased sensitivity to toxins, drugs, irradiation, and other stresses
● Increased levels of macromolecular cross-linking ● Increased levels of reactive oxygen species
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. Figure 50.1 This figure shows Giemsa-stained light microscopic phase-contrast pictures of serially passaged human skin fibroblasts at various points in their in vitro lifespan. Sparse and confluent cultures at three stages during replicative lifespan are compared: (1) early passage young adult skin fibroblasts with less than 30% lifespan completed; (2) middle-aged cells with 60–80% replicative lifespan completed; and (3) late passage senescent cells with more than 95% lifespan completed
Altered responsiveness of cells during aging is one of the most significant age-related changes, which can be a rate-limiting factor for the use of any potential modulators of aging. Several studies have been performed in order to understand the mechanisms for age-related alteration of responsiveness, and the pathways include unaltered receptor numbers and affinities, ineffective signal transduction, and interrupted networks [8]. Biochemical and molecular phenotype At the biochemical and molecular levels, a large body of data is available, which indicates that skin cells undergo a plethora of changes, which form the mechanistic bases of structural and physiological alterations. > Table 50.3 gives a list of main categories of biochemical and molecular changes that have been reported in aging skin cells in culture.
Depending on the available technologies and the prevailing trends, changes in the amounts and activities of thousands of proteins, and in the levels of thousands of mRNAs have been reported for aging skin cells. Recently, data are beginning to be collected for age-related changes in the so-called epigenome, metabolome, and proteome, including posttranslational modifications [19–21]. All such data will further strengthen the descriptive understanding of the phenomenon of aging of skin cells. Although every single piece of descriptive data for aging skin cells is yet to be collected, a generalized picture of the aging phenomenon has emerged. Therefore, based on the large amount of data collected so far, important inferences and generalizations can already be made, which
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have implications with respect to developing effective interventions for a healthy skin. These are as follows: 1. The rate and phenotype of aging is different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. 2. A progressive accumulation of damage in all types of macromolecules is a universal feature of cellular aging in all cell types. 3. Replicative senescence of cells in culture is not due to the activation of any aging-specific genes, but is an indirect consequence of occurrence and accumulation of molecular damage and molecular heterogeneity. 4. A progressive failure of molecular maintenance and repair pathways is the ultimate cause of cellular aging.
From Cellular Aging In Vitro to Understanding Aging In Vivo The Hayflick system of aging of skin cells in culture has proved to be very useful in developing the cellular and molecular understanding of the overall process of aging. A loss of proliferative capacity of any of the cell types has a deteriorative impact on the functioning and survival of the entire organism. A loss or slowing-down of proliferation of osteoblasts, glial cells, myoblasts, epithelial cells, lymphocytes, and fibroblasts can lead to the onset of many age-related diseases and impairments including osteoporosis, arthritis, immune deficiency, altered drug clearance, delayed wound healing, and altered functioning of the brain. Furthermore, occurrence of fully senescent or near-senescent heterogenous cells in vivo can promote dysfunctioning of the other tissues by producing harmful signals, and can also promote and stimulate the growth of other precancerous and cancerous cells [22–25]. However, the existence of the Hayflick-type senescent cells in vivo is not very well established so far. A commonly used biomarker of senescent cells is the so-called senescenceassociated beta-galactosidase (SABG), which has been used to demonstrate the presence of senescent cells in human skin and some other tissues [23, 26, 27]. However, there are several limitations regarding the use of SABG as a marker of cellular aging in vitro, since SABG can also be detected in immortal cells under various conditions [28]. More and multiple independent markers of senescent cells are needed for this purpose. The correlation between cellular aging in vitro and in vivo is often based on the evidence gathered from studies on the effects of donor age, species lifespan, and
premature aging syndromes on cellular proliferative capacity in culture. These studies indicate that the genetic and intrinsic Hayflick limit of diploid cell strains in culture is a true reflection of what is going on during aging of an organism. However, there are some recent critiques of this based on the replicative potential of stem cells, which in the case of the skin appear to be maintained throughout lifespan [8, 29, 30]. In contrast to this, there is evidence showing that the stem cell population in the skin also undergoes aging, and the number of stem cells declines as a function of donor age and during aging of the skin equivalents in vitro [31].
Modulators of Aging Skin Cells The Hayflick system of cellular aging in culture is primarily a model for the study of slow and progressive accumulation of damage resulting in the arrest of cells in a nonproliferative state [1]. This system has been proved to be very useful for testing various physical, chemical, and biological conditions for their harmful or beneficial effects, and for understanding other aspects of cellular aging with implications in the origin of age-related diseases. For example, irradiation, severe oxidative stress by UV, hydrogen peroxide, or dicarbonyls, and gene transfection have been used to induce a sudden and rapid increase in molecular damage, resulting in premature appearance of the senescent phenotype [32–34]. On the other hand, insertion of catalytically active component of the telomerase gene can completely bypass the Hayflick limit in many cell types including skin cells, and such cells can proliferate indefinitely with or without becoming transformed [35, 36]. Similarly, normal diploid cells can be transformed and immortalized by chemical carcinogens, irradiation, and viral genes. Such approaches are helpful for unravelling the molecular details of cell cycle regulation in normal cells and its dysregulation in cancer cells [36]. The Hayflick system of cellular aging in culture has also been very useful for testing various natural and synthetic molecules as potential anti-aging compounds for the skin. Some of the well-tested examples are cytokinins kinetin and zeatin [37, 38], a dipeptide carnosine [39, 40], and extracts from medicinal plants and some algae [41]. Several of these tests have resulted in the successful development, production, and marketing of various products with pharmaceutical, cosmeceutical, and nutritional applications [42–45]. Another use of the model system of cellular aging in culture has been to test the principle of mild
Aging of Skin Cells in Culture
stress-induced beneficial and anti-aging effects, which is the phenomenon of hormesis [46]. For example, human skin fibroblasts and keratinocytes exposed to repeated mild heat stress (41 C, 1 h, twice a week) show several hormetic effects, such as improved protein degradation pathways, higher levels of chaperones, increased resistance to other stresses, improved differentiation, and increased proliferative lifespan [7, 46–48]. Such studies can form the basis of testing novel hormetic agents, including potential hormetins of natural or synthetic origin, for improved skin care during aging [7, 46–48].
Conclusion In conclusion, it may be reemphasized that the present understanding of the cellular and molecular basis of aging of the skin owes a lot to the use of the Hayflick system of aging of skin cells in culture. Most importantly, studies performed by using this model system have demonstrated that aging of cells is characterized by the accumulation of damage in various molecules, which results in the failure of maintenance and repair systems. Detailed genomic, proteomic, and metabolomic studies using this system can further identify the interacting networks of regulatory pathways, which may be accessible to modulation for the maintenance of the structural and functional integrity of the skin.
Cross-references > The
Use of Reconstructed Skin to Create New In Vitro Models of Skin Aging with Special Emphasis on the Flexibility of Reconstructed Skin
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Metabolism
29 Alterations of Energy Metabolism in Cutaneous Aging Thomas Blatt . Horst Wenck . Klaus-Peter Wittern
Introduction Aging is understood as the result of a complex interaction of biological processes that are caused by both environmental processes (extrinsic aging) and genetic processes (intrinsic aging). Research into the biology of aging has provided detailed insight into the molecular mechanisms of age-related changes in organs, tissues, and cells. Most information relating to intrinsic aging processes comes from tissues other than the skin. This is in part due to the fact that clinically manifest diseases such as Type2 diabetes or neurodegenerative disease are often correlated with aging of cells. In part it is also due to the fact that substantial amounts of primary cells and organelles for biochemical analyses can be more easily isolated from other organs such as muscle, brain, or liver, as compared with skin. Nevertheless, intrinsic aging is based on general biological processes that apply more or less to all proliferating cells and terminally differentiated cells as well. Therefore, general intrinsic aging processes seen in a liver cell, muscle cell, or neuron can be expected also to apply more or less to skin cells. In fact, most of the aging processes identified and studied with other cells could also be confirmed with keratinocytes or dermal fibroblasts, even though some downstream details may be different. Extrinsic aging processes have been intensively studied in the skin though. This applies especially to a process called photoaging, which is induced by the skin’s most dominant stressor – UV light. In contrast to skin, UV light is an irrelevant stressor to other tissues. Researchers have developed a battery of slightly invasive or non-invasive biophysical measurement procedures to study UV-induced stress and aging-related damage in situ even in small skin samples. Furthermore, many in vitro methods are available to study photoaging based on cultured cells or threedimensional cultured skin models. Besides UV light, there are many other extrinsic stressors, encompassing, for example, environmental chemicals, nutritional conditions, or even hormonal imbalances which may induce extrinsic aging of the skin and other organs as well.
This overview will focus on aspects of energy metabolism in cutaneous aging. These aspects are especially important since human skin tissue, being exposed to a plethora of endogenous and environmental stress factors, is highly dependent on energy supply in order to combat cellular deregulation and/or to repair damage. As detailed below, cellular energy levels decline during intrinsic and extrinsic aging as well, and consequently the capacity of the skin to counteract environmental stress declines with aging. Decreased compensation of environmental stress and insufficient repair, in turn, accelerate skin aging, which consequently leads to further decline of cellular energy levels in the skin. Breaking this feedback loop by sustaining cellular energy levels in the skin is thought to decelerate, stop, or even reverse intrinsic and extrinsic skin aging, with compounded interest over time.
Phenomenology of Energetic Factors Associated with Skin Aging The energy demand of skin cells is supplied by two major primary sources – mitochondrial oxidative phosphorylation and glycolysis. In addition to these major primary sources, skin cells may also utilize a major secondary energy store for situations of acute high energy demand – the creatine/phosphocreatine system. As detailed below, all three major energy sources are affected by intrinsic and extrinsic skin aging and offer potential entry points for intervention strategies to decelerate the aging process.
Aging Effects on Mitochondrial Function The mitochondria, as small oxidative power-plants in the cells, play a pivotal role in energy supply. Beyond energy production, mitochondria can perform other pivotal cellular functions such as regulation of programmed cellular death (apoptosis). These organelles contain their own genetic material, mitochondrial DNA (mtDNA), which
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_29, # Springer-Verlag Berlin Heidelberg 2010
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is maternally inherited. Although much smaller than the nuclear genome, mtDNA is equally important, as it has been shown to play a crucial role in aging, as discussed in detail in the section Genetic Damage to Mitochondria of this overview. This central organelle of energy metabolism and control of cell death is supposed to be a target for aging and a promoter of aging as well [1]. Within living cells, mitochondria are observed as small sausage-shaped organelles, longer snake-like tubules, branched reticula, extended filaments and networks or clusters that are connected via intermitochondrial junctions. Mitochondrial morphology is regulated in many cultured eukaryotic cells by fusion and fission, and a tightly controlled balance between fission and fusion events is supposed to ensure normal mitochondrial ultrastructure as well as mitochondrial and cellular functions. Mitochondria of old endothelial cells show a significant and equal decrease of both fusion and fission activity [2], indicating that these processes are sensitive to aging and are likely to contribute to the accumulation of damaged mitochondria during aging [3]. Aging of cells is also associated with aberrations of mitochondrial morphology and ultrastructure. Typical changes of mitochondrial ultrastructure during aging, as can be seen by electron microscopy in cultured fibroblasts, are loss of branched mitochondria in old cells, enlargement of mitochondria, matrix vacuolization, shortened cristae, and loss of dense granules [4]. Furthermore, cystic blebs are evident in mitochondria of some cells with an apparent increase in old cells. These blebs appear to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appears intact. Similar ultrastructural changes of mitochondria as seen in aged cells are also seen in cultivated skin fibroblasts of patients with point mutations in mitochondrial DNA (mtDNA) affecting the energy metabolism of the organelles. The changes encompass partially swollen mitochondria with unusual and sparse cristae, heterogeneity of cristae in size and shapes or their absence, as well as almost complete absence of branched mitochondria [5]. Furthermore, similar changes in mitochondrial ultrastructure as seen in aged fibroblasts are also seen in photoaged keratinocytes chronically exposed to low doses of UV-B irradiation [6]. Thus, ultrastructural changes of mitochondria in intrinsically aged cells are similar to those seen in mitochondria from skin cells damaged by intrinsic genetic defects or damaged by exogenous stressors leading to photoaging. Since morphological features typical of mitochondria from aged cells can also be induced in mitochondria from otherwise normal cultured primary skin fibroblasts by inhibition of energy
metabolism with drugs targeting the respiratory chain [7], changes of the mitochondrial ultrastructure can be hypothesized to be a cause and consequence of altered mitochondrial energy metabolism as well. The central function of the mitochondrial network in the cell is the production of Adenosintriphosphate (ATP) by transfer of electrons from digested food to carrier molecules such as Nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD+), thus generating NADH and FADH, and subsequent delivery of the electrons to the respiratory Oxidative phosphorylation generates mitochondrial ATP by means of five multiple subunit enzyme complexes (I through V) plus the adenine nucleotide translocator (ANT), all localized within the mitochondrial inner membrane. Complexes I–IV constitute the electron transport chain. Reduced NADH is oxidized by Complex I (NADH dehydrogenase), and succinate is oxidized by Complex II (succinate dehydrogenase); the electrons are transferred to ubiquinone (Coenzyme Q10) to yield ubiquinol. The electrons from ubiquinol are transferred to Complex III (ubiquinol:cytochrome c oxidoreductase), then to cytochrome c, then to Complex IV (cytochrome c oxidase), and finally to oxygen. The energy released is used to pump protons out of the mitochondrial inner membrane through Complexes I, III, and IV, and the resulting electrochemical gradient is exploited by Complex V (ATP synthase) to condense Adenosindiphosphate (ADP) and inorganic phosphate to form ATP. Both ATP and ADP are exchanged across the mitochondrial inner membrane by ANT. The five protein complexes of the electron transport chain work as an integrated system, with mitochondrial DNA (mtDNA) encoding 13 of the proteins and nuclear DNA encoding approximately 60. Formation of these complexes is a complicated procedure based on the coordinated transport and assembly of components from two different genomes and compartments [8]. As an organism ages, either by extrinsic or intrinsic aging, there is a significant decline in mitochondrial function and cellular energy balance [9, 10]. This applies especially to mitochondrial membrane potential which is key to mitochondrial function [11]. Assessment methods to monitor the mitochondrial respiration rate and membrane potential, even on a single intact cell level are discussed in the section Energetic effects of Creatine of this overview. Decrease of mitochondrial function during aging has been described as a general feature in many in vitro systems. These descriptions encompass the loss of mitochondrial membrane potential of old and postmitotic human umbilical vein endothelial cells [2], or lower
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respiration rates of spleen lymphocytes isolated from from old mice as compared with lymphocytes from young mice [12], just to mention a few. In fact, the general decline of mitochondrial respiratory functions with age has also been described in human studies, for example in hepatocytes investigated in a study enrolling subjects of 31–76 years old, where a significant negative correlation between age and respiratory control and ADP/O ratios was observed [13]. Decline in skeletal muscle mitochondrial respiratory chain function has also been investigated in isolated intact skeletal muscle mitochondria in a study enrolling subjects aged 16–92 years. State 3 (activated) mitochondrial respiration rates showed a significant negative correlation between respiration rate and age. A similar trend was seen for respiratory enzyme activities assayed in muscle homogenate [14]. These findings demonstrate a substantial fall in mitochondrial function in aging muscle and liver cells, and suggest that a fall in mitochondrial oxidative capacity and membrane potential in aging cells may be an important general contributor to the aging process. Loss of mitochondrial membrane potential has also been reported in human dermal fibroblasts aged in vitro by serial passage [15]. Furthermore, a marked agingrelated decline in efficiency of oxidative phosphorylation was observed in human skin fibroblasts isolated from a large group of subjects ranging in age between 20 weeks fetal and 103 years [16]. In the latter study, the analysis of endogenous respiration rate revealed a significant decrease in the age range from 40 to 90 years, and a tendency to uncoupling in the samples from subjects above 60 years. These findings clearly pointed to a dramatic mitochondrial dysfunction, which would lead to a decrease in ATP synthesis rate in skin fibroblasts with increasing age. The impact of intrinsic aging on the mitochondrial oxidative capacity and mitochondrial membrane potential of skin cells is furthermore supported by a number of indirect studies discussed in this review, addressing the effects of aging on the generation of reactive oxygen species (ROS) in skin cells from young and old donors, or the effect of energy enhancers. Besides intrinsic aging effects on the mitochondrial capacity of skin cells, a substantial decline of the mitochondrial membrane potential can be observed following UV irradiation of keratinocytes in vitro, which is the dominant stressor leading to photoaging of cells [17, 18]. A decline of mitochondrial membrane potential after UV irradiation has also been reported in situ using suction blisters taken from irradiated and non-irradiated skin areas of healthy old volunteers with an average age of 65.2 years [17].
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Aging Effects on Anaerobic Energy Pathways The loss of mitochondrial function represents an inherent part in modern theories trying to explain the cutaneous aging process. The number of damaged mitochondria increases with aging, and as a consequence an impaired mitochondrial ATP synthesis can be observed. To assure survival of a cell, any decrease in mitochondrial energy production due to impaired mitochondrial function has to lead to compensatory actions in cellular metabolism which result in higher energy production via non-mitochondrial pathways such as glycolysis. This goes along with reports from several in vitro studies demonstrating a higher glucose uptake and lactate production at advanced cellular age in fibroblasts [19, 10]. As a speacial feature of the skin, atmospheric oxygen may be directly taken up by the human epidermis, and in theory, the flux of oxygen from the environment should be sufficient to fully cover its oxygen demand [20]. A rather surprising hypothesis, stimulated by a study from Ronquist et al. [21], is that human epidermis works to a substantial extent in an anaerobic manner. In cell culture, keratinocytes contain more lactate than do most other cell types. Their lactate production in vitro is vigorous and independent of oxygen and most of it is released to the medium. During autoincubation of the epidermis under starved conditions, energy charge values are low and comparable with those reported for smooth muscle. Moreover, the overwhelming majority of the keratinocytic mitochondria have an appearance markedly deviating from those in other cells such as Langerhans cells, melanocytes and fibroblasts, and, above all, are characterized by an enormous reduction of the inner membrane. Ronquist concludes from these findings that epidermal energy metabolism is predominantly anaerobic in spite of the formal presence of mitochondria and sufficient oxygen. According to not yet published data generated in at the authors’ institution, significant age-dependent differences in mitochondrial function can be observed in keratinocytes isolated from skin biopsies of young and old donors. The data suggest that energy metabolism shifts to a predominantly non-mitochondrial pathway and is therefore functionally anaerobic with advancing age. Primary keratinocytes derived from old donors show a higher glucose uptake compared to the cells obtained from a young donor panel, and the increased lactate production in keratinocytes from the old age group clearly indicates a suboptimal utilization of glucose and a shift in metabolism towards an increased glycolysis. The data generated so far show no differences in mitochondrial
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content and structure during aging in skin keratinocytes, indicating that the number of mitochondria does not change, but rather their function. This decline of mitochondrial function may explain the observed age-associated glycolytic activity as some kind of compensatory counterregulation. Simulation of mitochondrial dysfunction by inhibition of ATP synthase in keratinocytes from young donors leads to a comparable rise in glucose uptake and lactate production as seen in the basic, unstressed state of keratinocytes from the old age group. In fact, the energy metabolism of keratinocytes is a subject of controversy, and it is unclear why keratinocytes express a metabolic status that is, as compared with fibroblasts or endothelial cells, partially shifted to an anaerobic status. It was found that keratinocytes respire as much oxygen as fibroblasts, even though maximal activities of the respiratory chain complexes are two- to fivefold lower, whereas expression levels of respiratory chain proteins are similar. Congruent with this, superoxide anion levels are much higher in keratinocytes, and keratinocytes display higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress [22]. Thus, it seems that keratinocytes actively use the mitochondrial respiratory chain not only for adenosine 5’ triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity. The reason for this behaviour of keratinocytes is not clear, but it may indicate that superoxide-driven processes might be a prerequisite for keratinocyte differentiation, even though the lack of energy supply via oxidative phosphorylation has to be compensated via glycolysis then.
Aging Effects on Extramitochondrial Energy Stores Precise coupling of spatially separated intracellular ATPproducing and ATP-consuming processes is fundamental to the bioenergetics of living organisms, ensuring a failsafe operation of the energetic system over a broad range of cellular functional activities, thereby securing the cellular economy and energetic homeostasis under stress [23]. Beside the mitochondrial energy supply and glycolysis as primary sources for energy, cells also have a secondary energy storage system named the creatine/phosphocreatine (Cr/PCr) pathway. This is also established in the human skin and is responsible for an extremely fast energy supply in situations of high temporal energy demand when ATP is used up faster than can be produced from primary sources [24, 25, 17], or when energy supply from
primary sources is temporarily interrupted, such as in situations of short term hypoxia or anoxia [26]. The free energy of ATP, which itself cannot be stored efficiently, is stored and transported in the form of PCr from sub-cellular sites of energy production, e.g., mitochondria, to places of high energy requirements, where creatine kinase (CK) activity can rapidly replenish cellular ATP in situ [27, 28, 29]. Predominant isoforms of CK consist of cytosolic mm-CK (muscle-type), mainly found in muscle cells, as well as cytosolic bb-CK (braintype) and ubiquitous mitochondrial mt-CK, with the last two mainly located in the brain but also present in skin [30, 31]. Creatine kinases catalyze the following reversible reaction: Mg ATP þ Cr $ PCr þ Mg ADP þ Hþ : The mt-CKs form octamers assembled from four dimers each, but only the octameric form can interact with both inner and outer mitochondrial membranes through the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). The mt-CK activity couples the oxidative phosphorylation to mitochondrial PCr production by catalyzing the conversion of Cr to PCr at expenses of the intramitochondrially produced ATP. The PCr is exported to the cytosol, whereas the produced ADP is pumped back to the mitochondrial matrix via ANT. Cells attain their physiological levels of creatine, either by biosynthesis from the amino acids arginine, glycine, and methionine in the kidney, liver, and pancreas of vertebrates including humans, and/or alternatively by ingestion of meat and fish. Creatine is transported via the blood circulation and is taken up into cells by the Na- and Cl-dependent creatine transporter (CRT) protein [32, 33]. Once inside a cell, creatine can be stored at high concentrations (e.g., 40 mM for muscle cells). In skin, oxidative damage of cellular and extracellular components activates intrinsic repair mechanisms, which necessarily require ATP for full functionality. The PCr/CK system together with the recently discovered epidermal creatine transporter (CRT) [30] provides human skin with an important tool to cope efficiently with such conditions of high energy demand. In fact, both creatine kinase subtypes, bb-CK and mt-CK, as well as CRT, are expressed in human skin [17], showing high levels in the epidermis but less in the dermis, with the highest enzyme activity found in keratinocytes, which are generally shifted to a more anaerobic state of metabolism, as discussed in the section Aging Effects on Anaerobic Energy Pathways. Results from experiments using skeletal muscle and heart muscle clearly show that both PCr amount [34, 35, 36], and CK activity decrease with age [37, 35].
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Also in skin, a reduction in the cellular concentration of creatine can be determined from the age of about 30 [38], paralleled by a slightly reduced CK activity in skin cells from older donors [17]. This decline in skin CK activity may be caused by the generation of ROS during cutaneous aging. This is supported by the fact that CK, specifically mt-CK, is a primary target for ROS, especially peroxynitrite [39]. Moreover, cutaneous cells may show signs of a declining creatine level, probably caused by a stress and age-related decline of dermal vascularization [40].
Mechanisms of Skin Aging Related to Energy Metabolism Mitochondrial Impairment by Its Own Oxidation By-products Free reactive oxygen species (ROS) are generated in the skin by several different processes [41], with exogenous stress following UV-irradiation being the most dominant generator of ROS in UV-exposed skin. Other important contributions include proteins within the plasma membrane, such as the growing family of NADPH oxidases. Furthermore, generation of H2O2 as a by-product of fatty acid degradation in peroxisomes has to be taken into account as an endogenous source of ROS. The same holds for the generation of ROS by oxidative burst of phagocytes during inflammatory reactions, as well as the activity of various cytosolic enzymes such as cyclooxygenases. Although all these sources contribute to the overall oxidative burden of a cell, the vast majority of cellular ROS (estimated at approximately 90%) which is generated in cells independently of UV-stress can be traced back to the mitochondria as by-products of impaired mitochondrial respiration [42], and oxidants generated by mitochondria are supposed to be the major source of the oxidative lesions that accumulate with age [43]. The continuous threat of oxidant damage to the cell, tissue, and organism as a whole is underscored by the existence of an impressive array of cellular defenses that have evolved to battle these reactive oxidants [44]. The cell is equipped with a variety of defence mechanisms to remove ROS. Superoxide dismutases convert superoxide into hydrogen peroxide, which in turn can be transformed into water by catalase or glutathione peroxidase. The cell also contains nonenzymatic scavengers such as ascorbate, pyruvate, flavenoids, carotenoids and glutathione which may inactivate potentially damaging ROS. However, these defenses are not perfect and, consequently, cellular macromolecules become oxidatively damaged.
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Relevant to mitochondrial function is the efficiency of electron movement through the electron transport chain and its coupling to oxidative phosphorylation to produce ATP. The coupling efficiency can be measured experimentally by determining ADP/O ratio, and by determination whether the mitochondria are in State 3 or State 4, whereby State 3 represents a condition where the rate of oxidative phosphorylation is not limited by ADP concentration, and State 4 a condition where the level of ADP limits oxidative phosphorylation. State 4 is associated with a reduced respiratory chain, leading to an ‘‘electron jam’’ and increased formation of ROS byproducts. When the rate of electron flow is slow, electrons tend to accumulate in the respiratory chain, and electrons escaping from the somewhat ‘‘leaky’’ electron transport chain (ETC) can reduce oxygen to form the highly reactive free radical superoxide anion (O2●), which, in turn, can be further reduced to hydroxyl radical (OH●) and hydrogen peroxide (H2O2). Furthermore, the superoxide anion can initiate the oxidation of sulphite or nitric oxide, resulting in ROS such as sulphur pentoxy anion or peroxynitrite. Overall, mitochondrial ROS generation is high during resting respiration, but when electrons flow quickly through the respiratory chain reducing O2 to water, the rate of ROS production is usually lower. It has been estimated that about 1012 O2 molecules are processed by each cell daily, and that the leakage of partially reduced oxygen molecules is about 2%, yielding about 2 1010 O2● and H2O2 molecules per cell per day [45]. In addition to the toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to a further increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol [46]. High ROS concentrations, resulting from either increased production or decreased detoxification, can cause oxidative damage to various cellular components, ultimately leading to cell death [47]. Beyond damage to mitochondria, which will be discussed in detail below, age-related features often associated with excess ROS are accumulation of oxidized intracellular proteins with age [48], the decrease of fluidity of cellular membranes with age [49], or even malfunctioning of the connective tissue remodeling process due to increased activity of extracellular matrix-degrading metalloproteinases [50], just to mention a few. Toxic reactions exerted by ROS, and imbalances in the production and removal of ROS, significantly contribute to the aging process and are the basis of the ‘‘The Free Radical Theory of Aging’’ [51], postulating that the
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production of intracellular reactive oxygen species is the major determinant of life span [42]. The hypothesis that rate of intracellular ROS production is associated with the rate of aging was tested by Sohal [52], comparing the rate of H2O2 generation by mitochondria in houseflies. The rate of mitochondrial H2O2 release was found to be associated with remaining life expectancy or the physiological age of flies. At the same chronological age, mitochondria from flies with a shorter remaining life expectancy had a markedly higher rate of H2O2 generation than those with a longer life expectancy. In another experimental model of aging – the senescenceaccelerated mouse (SAM) – animals exhibit a shortened life span (about 18 months) as compared with normal mice, as well as early manifestation of various signs of senescence including changes in physical activity, skin, and spinal curvature. In the SAM the respiratory control ratio and the ATP/O – an index of ATP synthesis – decreases more rapidly as compared with the unaffected wild-type mice. Furthermore, uncoupled respiration in liver mitochondria is markedly decreased with aging in SAM [53]. Cultured dermal fibroblasts from SAM produce more ROS within the mitochondria than do cells from wild-type control mice, coinciding with an increase in the mass of the mitochondria, degenerative mitochondrial morphology with longer culture periods, and lower membrane potential as compared with the controls [54]. Another study supporting the hypothesis that rate of intracellular ROS production is associated with the rate of aging used genetically modified mice with impaired mtDNA repair function. Secondary to the impaired mtDNA repair function all such mice exhibit a significant reduction in respiratory chain activity and ATP generation in postmitotic tissue such as heart, as well as a significantly shortened life span and the appearance of a number of age-related phenotypes, including hair loss, kyphosis, and reduced fertility [55]. These findings suggest that damaged mitochondria, which produce an excess of ROS, can accelerate the aging process in a kind of feedback loop. In fact, the mitochondria seem to be highly susceptible to harm exerted by ROS, including those produced by themselves, and increasing levels of oxidative damage in various compartments of the mitochondrium can be observed during cellular aging. Damages to mitochondrial DNA (mtDNA) – which contributes a substantial part of the proteins of the respiratory chain – isolated from rat liver or various human brain regions are at least tenfold higher than those of nuclear DNA. These higher levels of oxidative damage and mutation in mtDNA have been ascribed to location of the DNA near the inner mitochondrial
membrane sites where oxidants are formed. The extent of damage and mutation of mtDNA may be further aggravated by lack of protective histones, and lack of DNA repair activity in mitochondria. Oxidative lesions in mtDNA accumulate as a function of age, which has been well described for human diaphragm muscle [56], human brain [57]), and rat liver [58]. As in the case of oxidative damage to DNA, an ageassociated increase in oxidative damage to mitochondrial protein is also observed [59]. The accumulation of oxidized dysfunctional protein with reactive carbonyl groups can lead to inter- and intramolecular cross-links with protein amino groups and may cause loss of biochemical and physiological function in mitochondria. Thus the age-related accumulation of protein oxidation products in mitochondria may also lead to loss of energy production and increased production of oxidants. Increased oxidants may also contribute to alterations in mitochondrial membrane fluidity and phospholipid composition that occur during aging. These in turn can affect the ability of mitochondria to transport substrates and to generate sufficient proton motive force to meet cellular energy demands. With regard to lipids, part of the increased sensitivity of mitochondria to oxidants appears to be due to peculiarities in membrane lipid composition [60] which is characterized by the presence of cardiolipin. Cardiolipin serves as an insulator and stabilizes the activity of protein complexes important to the electron transport chain, and it also ‘‘glues’’ them together. Because cardiolipin plays a pivotal role in facilitating the activities of key mitochondrial inner membrane enzymes, it would be expected that changes that increase its susceptibility to oxidative damage would be deleterious to normal mitochondrial function [43]. Cardiolipin is solely synthesized in the mitochondria, and is typically present in the membranes of mitochondria, mostly in the inner membrane, which consists roughly 20% of its lipids. Cardiolipin aquires an increasing percentage of polyunsaturated fatty acids with increasing age of an organism (substitution 18:2 to 22:4 and 22:5), which renders it even more susceptible to oxidative damage. In fact, mitochondrial cardiolipin content has been reported to decrease with age in a number of tissues, including heart, liver, nonsynaptic brain mitochondria, and epithelial cells [61, 62, 63, 64], presumably due to oxidative damage. The decrease of cardiolipin with age is associated with a decrease in State 3/State 4 ratio. This loss of cardiolipin by increased susceptibility to oxidation could play a critically important role in the agerelated decrements in mitochondrial function. In addition to the established role of the mitochondria in energy metabolism, regulation of cell death has emerged
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as a second major function of these organelles. This seems also to be intimately linked to their generation of ROS [65]. Mitochondrial regulation of apoptosis occurs by mechanisms, which have been conserved through evolution, involving the release of cytochrome c into the cytoplasm which may be initiated by the oxidation of cardiolipin. Oxidation of cardiolipin, which occurs at higher rates in aged cells, reduces cytochrome c binding to mitochondrial inner membranes and increases the level of soluble cytochrome c in the intermembrane space. Subsequent release of the hemoprotein into the cytoplasm, which starts the apoptotic machinery via activation of caspases, occurs by pore formation mediated by pro-apoptotic Bcl-2 family proteins, or opening of mitochondrial permeability transition pores (MPTP). Various factors enhance the likelihood of MPTP opening, among them are dissipation of the difference in voltage between the inside and outside of mitochondrial membranes (known as permeability transition), or the presence of free radicals, both typical features of mitochondria in aged cells. ROS are also known as signaling molecules under subtoxic conditions which may activate cytoplasmic signal transduction pathways that are related to growth, differentiation, senescence, transformation and tissue degradation [66, 67, 68]. Hydrogen peroxide, for example, has been shown in different cultured cell lines to induce either apoptosis at high concentrations, or features of senescence at subtoxic concentrations [69], with cellular senescence being defined as the loss of proliferative capacity of primary cell lines, characterized by cell cycle arrest, reduced DNA synthesis, increased cell size, granularity and size heterogeneity [70, 71]. Senescent cells enter a terminally nondividing state in which they can stay for long periods before dying. Thus, in the case of stress-induced premature senescence, ROS are considered important intermediates contributing to the phenotype. The data of Zwerschke et al. [10] suggest the occurrence of significant metabolic imbalances in human fibroblasts rendered senescent by exposition to ROS. There is a drastic deregulation of the carbohydrate metabolism in senescent cells, characterized by an imbalance of glycolytic enzyme activities and the failure to maintain ATP levels. This leads to an up-regulation of adenylate kinase activity and the levels of AMP, which acts as a growth-suppressive signal that induces premature senescence. The activities of several glycolytic enzymes are strongly up-regulated. Moreover, the function of the malate–aspartate shuttle is reduced in senescent cells, preventing the transport of hydrogen into the mitochondria, where it could be used for ATP production. Instead, senescent cells activate LDH and take up pyruvate to get rid of hydrogen. Thus
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ATP-consuming steps of glycolysis are enhanced, whereas the ATP-producing steps are inhibited, and this leads to a severe reduction of the intracellular concentration of both ATP and GTP. Senescent keratinocytes and fibroblasts were also described by Campisi [72] to accumulate with age in human skin, likely due to the impact of increased endogenous oxidative stress in aged skin.
Genetic Damage to Mitochondria The mitochondrial genome is very small and economically packed, and the expression of the whole genome is essential for the maintenance of mitochondrial bioenergetic function. Thus, even small genetic alterations may have tremendous effects on mitochondrial function. In the past decade, more than 100 mtDNA mutations have been found in patients with mitochondrial disease, and some of them also occur in aging human tissues [73]. Thus, it may be hypothesized that accumulation of mitochondrial DNA deletions may be an important factor in intrinsic aging. At higher age, several independently acquired types of mtDNA mutations, accumulating clonally in certain cells, can even be found in different tissues of the same subject [74]. The incidence and abundance of mutant mtDNAs are increased with age, and much more than for nuclear DNA mutations [75, 76]. Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues derived from young adult and senescent animals have revealed that about 1% of the native mtDNA population in adult liver and about 5% in senescent liver having deleted/inserted segments [77]. In humans it was found that normal heart muscle and brain from adult human individuals contain low, though substantial levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease. This deletion was not observed in fetal heart or brain [78]. In a further human study focussing on the prevalence of mtDNA deletions in tumorous and surrounding healthy tissue, mtDNA mutations were found to be abundant in margin tissue specimens from older patients and their number correlated with the patient age. Significantly fewer deletions were detected in the tumours than the margins, and the tumours often had no deletions [79], implying a potential selection for full-length mtDNA or perhaps a protective role for mtDNA deletions in the process of tumourigenesis. Concerning photoaging, also photoaged skin is characterized at the molecular level by increased amounts of large-scale deletions of the mitochondrial genome such as
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the 4,977 bp common deletion encompassing deletion of four genes for subunits of complex I, one gene for complex IV, two genes for complex V, and several genes for mitochondrial tRNAs [80, 81]. The common deletion can be generated in dermal fibroblasts through repetitive ultraviolet UV-A irradiation [82]. For example, in a human study, previously unirradiated skin of 52 normal human individuals was repetitively exposed to physiological doses of UV-A light, and repetitive UV exposure led to an approximately 40% increase in the levels of the common deletion in skin tissue. Nine individuals were examined up to 16 mo after cessation of UV exposure and some showed accumulation up to 32-fold [83]. In another human study focussing on extrinsic photoaging, several types of mtDNA length mutations including the common 4,977 bp deletion were investigated in normal human skin tissues. It was found that the incidences of these deletions and tandem duplications of mtDNA in sun-exposed skin were all significantly higher than those in non-exposed skin [84]. Moreover, these mutations started to appear in the third decade of life, and the age at which the mutations could be detected in sun-exposed skin was substantially younger than in non-exposed skin. In another human study focussing on mtDNA aberrations in skin tissue, the frequency of a so-called T414G mutation within the control region of mtDNA, has been demonstrated to accumulate in both chronologically and photoaged skin using cultured dermal fibroblasts from donors of different age [85]. Thus, there is a strong correlation of extrinsic and intrinsic cellular aging with the accumulation of aberrations of mtDNA, and increasing evidence suggests that, due to a negative feedback loop, damaged mtDNA is a cause and consequence of aging as well. The causative role of mtDNA mutations and resulting mitochondrial dysfunction for intrinsic and extrinsic aging has been demonstrated in several experimental models and human studies. Premature aging has, for example, been reported in knock-in mice expressing a defective mitochondrial DNA polymerase. These knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA, associated with reduced lifespan and premature onset of aging-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement [55]. The results thus provide a causative link between mtDNA mutations and aging phenotypes in mammals. It is plausible that the accumulation of mtDNA mutations leads to decreased gene expression, resulting in a decline in
oxidative phosphorylation, and inefficient electron transport, which consequently increases the generation of ROS [86]. A direct link between the amount of mtDNA aberrations and oxidative stress has been demonstrated using a series of the cybrids harboring varying proportions of mtDNA with the common 4,977 bp deletion. The population doubling time was longer for the cybrids containing higher proportions of 4,977 bp-deleted mtDNA. In addition, the respiratory function was decreased with the increase of the portion of aberrant mtDNA in the cybrids. The results also showed that the specific contents of typical cellular oxidation products stemming from ROS in cybrids harboring >65% of the aberrant mtDNA were significantly increased as compared with those of the cybrids containing undetectable mutant mtDNA [80, 87]. In a study on human fibroblasts, gradual largescale deletion of the mtDNA from unirradiated human skin fibroblasts was found to induce a gene expression profile reminiscent of photoaged skin. The modified cells exhibited an altered gene expression profile resulting from intracellular, mitochondria-derived oxidative stress, dominated by high expression of matrix metalloproteinase-1 (MMP-1) which is known to be expressed in response to oxidative stress 95]. Vice versa, the increase in ROS production is a likely promotor for additional mtDNA damage and accumulation of mtDNA mutations. The causative role of oxidative stress for the increased frequency of mitochondrial DNA aberrations has been demonstrated in a variety of experimental models and in human studies. For example, oxidative damage elicited by imbalance of free radical scavenging enzymes and its association with large-scale mtDNA deletions in aging human skin has been suggested using skin tissue derived from donors of different age. In subjects above the age of 60 years, elevated oxidative stress was caused by an imbalance between the production and removal of ROS and free radicals, and was paralleled by an increase of the proportion of mtDNA with the 4,977 bp deletion [88]. The causative role of singlet oxygen in mediating UV-A induced generation of the photoagingassociated mitochondrial common deletion has been demonstrated in a pivotal study by Berneburg et al. [89]. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion. There was a time- and dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the
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common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide direct evidence for the involvement of reactive oxygen species in the generation of agingassociated mtDNA lesions in human cells.
Side Effects of Anaerobic Energy Pathways High glycolytic fluxes and glucose accumulation may be a last resort when oxidative energy capacity of mitochondria declines during aging, but they are also sources of endogenous damage by themselves. In fact, both aging and diabetes are characterized by the formation of socalled advanced glycation endproducts (AGEs), though due to different reasons. Most glycolytic intermediates favour the formation of (AGEs) via reactive carbonyl groups, that are able to modify protein amino groups in the cytosol based on the Maillard reaction. This reaction is termed glycation or non-enzymatic glycosylation. AGEs constitute a heterogeneous group of structures, whereby N’-(carboxymethyl)lysine (CML) adducts are the most prevalent AGEs present in vivo. Methylglyoxal, glyoxal and other autooxidated derivatives of sugars induce AGEs that negatively affect essential features of skin cells and extracellular matrix proteins. It has been reported that AGE formation results in a loss of contractile capacity and cytoskeleton integrity in human skin fibroblasts, which possibly affects tissue cohesion and leads to visible effects of skin aging. Vimentin was identified as a major target in skin glycation besides other longlived proteins such as fibronectin, laminin, collagen, and elastin [90]. Strikingly, the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in skin into strong relationship with loss of organ contractile functions associated with aging. It is also reported that the intracellular concentration of the glycating agents – such as highly reactive methylglyoxyal, which is formed from dihydroxyacetone and glyceraldehyde-3-phosphates, and rapidly glycates proteins – damages mitochondria [91], and AGEs may even induce apoptosis by enhancement of expression of pro-apoptotic genes and stimulation of apoptosis through cytoplasmic and mitochondrial pathways [92]. Furthermore, intracellular AGEs induce oxidative stress, activate NF-kB and heme oxygenase, produce lipid peroxidation products, and cross-link proteins [93].
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Monitoring Methods for Energetic Processes in Skin Cells Monitoring of Mitochondrial and Glycolytic Activity in Skin There is no direct method available to measure mitochondrial or glycolytic activity in skin in situ. In any case, dermal cells have to be isolated from skin biopsies or suction blisters, or cultured dermal cell lines have to be employed. Assessment of the mitochondrial membrane potential is then possible through the availability of redistribution potentiometric radioactive compounds such as tetraphenylphosphonium bromide or redistribution potentiometric dyes such as rhodamine 123 (R123), or tetramethylrhodamine methyl ester (TMRM). These substances are lipophilic cations accumulated by mitochondria in proportion to the membrane potential (DC). Whereas the use of tetraphenylphosphonium bromide requires isolation of the mitochondria for uptale analysis [94], the dyes may be used also with living intact cells. Upon accumulation in the cytoplasm, R123 and TMRM exhibit a red shift in both their absorption and fluorescence emission spectra. The fluorescence intensity is quenched when the dyes are accumulated by mitochondria. These properties can be used to monitor membrane potentials even in single cells or in isolated mitochondria [95]. Rhodamine 123 and TMRM have also been used to monitor the mitochondrial membrane potential of intact human skin fibroblasts [96, 97], down to the single organelle level [98]. Another way to monitor the mitochondrial membrane potential is the use of redox indicators such as safranine, or the tetrazolium dye 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazol-carbocyanine iodide (JC-1). The latter is taken up by cells and exists as a monomer in the cytosol (green). The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded as the mitochondrial membrane becomes more polarized, J-aggregates form, which become fluorescent red and can be monitored photometrically. JC-1 has also been used to monitor the mitochondrial membrane potential on the level of single intact cells, for example by flow cytometry [99]. Another method to determine the energy flux in mitochondria is the estimation of the amount of ATP in relation to the amount of oxygen consumed, also called the P:O ratio. If mitochondria or permeabilized cells are incubated in an oxygraph apparatus (oxygen electrode) in
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an isotonic medium containing substrate and phosphate, then addition of ADP causes a sudden burst of oxygen uptake as the ADP is converted into ATP. The actively respiring state is refered to as State 3 respiration, while the slower rate after all the ADP has been phosphorylated to form ATP is refered to as State 4. The ratio [State 3 rate]: [State 4 rate] is called the respiratory control index and indicates the tightness of the coupling between respiration and phosphorylation. It is possible to calculate P:O ratio by measuring the decrease in oxygen concentration during the rapid burst of state 3 respiration after adding a known amount of ADP [16]. In parallel, the degree of glycolytic activity in a given cell suspension can be assessed by comparing the glucose uptake of cells with the amount of lactate produced.
Monitoring of Reactive Oxygen Species Although many enzymatic and chemical methods have been developed for evaluating ROS, in cell homogenates and in cell suspensions down to the single cell level, evaluation methods for ROS generation in situ are quite limited. Intracellular ROS production in single cells, for example, can be measured by a fluorometric assay based on deacetylation and oxidation of non-fluorescent dichlorodihydrofluoresceindiacetat (DCHF-DA), which penetrates the plasma membrane followed by enzymatic cleavage of the acetate groups, and which specifically reacts with peroxides to the fluorescent Dichlorofluorescein (DCF). Dihydrocalcein (H2-calcein) is another probe for intracellular ROS detection. In contrast to dichlorodihydrofluorescein, its fluorescent oxidation product calcein is thought not to leak out of cells. Other methods to monitor ROS in cells rely on luminescence enhancers such as luminol-, coelenterazine-, or lucigenin-enhanced chemiluminescence, which have been used extensively as indicators of O2● generation in intact cells and homogenates. Luminescence in this case is based on the activation of the probes by O2● and subsequent release of photons which can be measured in a luminometer. In situ, oxidative modification of dermal biomolecules induces ultraweak photon emission (UPE) as a by-product. Such light signals, which can be recorded in a non-invasive way, are supposed to contain valuable information regarding the extent of chemical damage, the nature of the oxidative modifications, and might be employed as a sensitive tool to monitor the efficacy of cosmetic or dermatological antioxidative intervention regimens. Data generated in indicate that UPE is induced by oxidative damage especially in deeper (living) skin layers, where antioxidants must be active in order to interfere with accelerated skin aging [100, 101].
Energetic Entry Points for Intervention There are several entry points for cosmetic intervention with intrinsic and extrinsic skin aging. Many of them refer to the topical application of antioxidants aimed at neutralizing free ROS, which are the central nocious effector molecules in both intrinsic and extrinsic aging processes. Typically used antioxidants such as Vitamin C, Vitamin E, Vitamin A and carotenes, have only indirect effects on energy metabolism, as they may reduce the ROS exposition of skin cells. These typical antioxidants will not be discussed in detail in this chapter because they are discussed extensively elsewhere in this book. There are, however, two compounds that may be topically used in cosmetic applications and dermatology, which interfere directly with the energy metabolism of skin cells – Creatine and Coenzyme Q10. Both compounds are in the very scope of this chapter and deserve in-depth discussion.
Energetic Effects of Creatine Creatine (Cr), a body-inherent amino acid derivative, is known to play a pivotal role in organ energy supply, because it acts like an energy store which can fastly provide energy in situations of high energy demand. After cellular uptake, creatine is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as a secondary energy source, serving not only as an energy buffer but also as an energy transport vehicle. In humans, the major part of the total creatine content is located in skeletal muscle, of which approximately a third is in its free form. The remainder is present in the phosphorylated form. It is supposed, for example, that the energy required for a 100-m sprint is entirely delivered from the creatine/phospho-creatine battery. Numerous scientific studies indicate that nutritional creatine supplementation favourably affects long-endurance exercise Vandenberghe et al. [102] and exerts protective effects in many clinical disorders, presumably caused by its energetic capacity. Creatine is normally metabolised to creatinine which is cleared via renal excretion, and daily turnover of creatine to creatinine for a 70-kg male has been estimated to be around 2 g. Cells attain their physiological levels of creatine, either by biosynthesis from the amino acids arginine, glycine, and methionine in the kidney, liver,
Alterations of Energy Metabolism in Cutaneous Aging
and pancreas of vertebrates including humans, and/or alternatively by ingestion of meat and fish. Creatine is transported via the blood circulation and is taken up into cells by a Na- and Cl-dependent CRT protein [103, 32, 33]. Once inside a cell, creatine can be stored at high concentrations (e.g., 40 mM for muscle cells). Creatine can be synthesized in human cells, but from the age of about 30, a reduction in the cellular concentration in the skin can be determined [38]. Highly interesting with regard to aging processes is that mitochondrial creatine kinase activity prevents reactive oxygen species generation due to a kind of ‘‘antioxidant’’ role of mitochondrial kinase-dependent ADP re-cycling activity. Of course, creatine is not a radical scavenger in itself. Activation of the mitochondrial creatine kinase (mt-CK) by creatine and ATP or ADP rather induces a State 3-like respiration in mitochondria, which represents a condition where the rate of oxidative phosphorylation is not limited by ADP concentration [104]. Thus, supplementation of cells with creatine may reduce the electron ‘‘jam’’ in the respiratory chain by providing a sink for free ATP-coupled energy by building-up phosphocreatine (PCr) stores and consequently increasing the pool of available ADP for phosphorylation. Supplementation of skin cells with creatine has been described to reduce the amount of cellular damage induced by UV-A irradiation or chemical oxidants per se [17]. This protective effect is most likely due to the general energy-recharging effect of creatine and may have further implications in modulating processes which are involved in premature skin aging and skin damage. In contrast to the protective effect of creatine supplementation, inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells, as performed at the authors’ institution, affects cell viability and mitochondrial morphology negatively [105]. Besides its role on energy metabolism it has recently been demonstrated that activation of mt-CK by creatine inhibits the mitochondrial permeability transition (MPT), a process that is involved in apoptosis [106]. The postulated protective mechanism of mt-CK activity against MPT pore opening lies on the one hand on functional coupling between the mt-CK reaction and oxidative phosphorylation. It is known that MPT can be directly induced by mitochondrial ROS and it is conceivable that the protective role of mt-CK activity against MPT may occur through reduction of ROS generation by keeping ADP phosphorylation. On the other hand, octamer-dimer transitions of mt-CK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Kinetic
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analyses suggested a functional interaction between the mt-CK, outer membrane pore protein, and inner membrane adenylate translocator (ANT). Permeability transition-pore-like functions are not observed unless the creatine kinase octamer is dissociated, which is facilitated in the absence of creatine [107, 108, 109]. As a zwitter-ion, creatine is also able to penetrate skin remarkably well, and is able to replenish energy stores of epidermal cells. Several research data clearly demonstrate that only when enough creatine is available, skin cells function perfectly, all repair and protection systems work faultlessly, and the metabolism runs at full performance. For example, supplementation of keratinocytes in vitro with creatine has marked protective effects against oxidative stress, and in clinical trials conducted at the authors’ institution, exogenous supplementation of the skin with creatine in a topical formulation had marked protective against UV-induced damage [17]. In these studies, healthy old volunteers with an average age of 65.2 years were topically treated with a stabilized creatine formulation twice a day for 4 weeks on their upper arm. Afterwards, epidermal cells were isolated via suction blister to examine the mitochondrial membrane potential in response to UV irradiation. Epidermal cells from placebo-treated skin sites showed a substantial and statistically significant decline in their mitochondrial membrane potential compared with non-irradiated control cells. In contrast, cells from the creatine/creatinine-treated skin sites showed a statistically significant maintenance of their mitochondrial membrane potential even after irradiation compared with the irradiated placebo control cells. Thus, topically applied creatine significantly protects human epidermal cells from a UV-induced decline in mitochondrial energy metabolism. Moreover experimental results indicate that creatine promotes protection and repair of mitochondrial DNA to ensure the mainainance of healthy cells. As discussed in the section Genetic Damage to Mitochondria of this overwiew, mitochondrial mutations are thought to be mediated by ROS and persist in human skin as long-term biomarkers of UV exposure. In a pivotal study by Berneburg et al. [110], UV-induced mitochondrial mutagenesis of skin cells, as assessed by the frequency of the common deletion, as well as functional consequences on mitochondrial energy metabolism, could be normalized by increasing intracellular creatine levels. All these data clearly indicate that human skin cells energetically recharged with creatine both in vitro and in vivo are better protected against a variety of cellular stress conditions by reversing deficiencies in cutaneous energy supply [25]. So far there are no reports of harmful side effects of topical Cr loading of human skin.
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Energetic Effects of Coenzyme Q10 Coenzyme Q10 (CoQ10, ubiquinone) is a lipophilic vitamin-like substance which is present in most eukaryotic cells, primarily in the mitochondria [111]. The CoQ10 found in humans is a benzoquinone with a polyisoprene chain containing ten isoprene units. It is a component of the electron transport chain, where CoQ10 has a unique function since it transfers electrons from the primary substrates to the oxidase system at the same time that it transfers protons to the outside of the mitochondrial membrane, resulting in the proton gradient across the mitochondrial membrane which is employed by Comples IV to generate ATP. It is known that Complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinone reductase) are found to be the predominant generators of ROS during prolonged respiration under uncoupled conditions, whereas complex III (ubiquinol:cytochrome c reductase) plays a less dominant role for mitochondrial ROS production. Complex II, in particular, appears to contribute most to the basal production of ROS in cells [112]. It is obvious that reduction of CoQ10 in a cell impairs the respiratory chain in mitochondria, resulting in electron ‘‘jam’’ and excess production of ROS. By reduction of the quinone to quinol, a carrier of protons and electrons is produced. Thus, in addition to its ‘‘antioxidative’’ role in the respiratory chain, the reduced form of CoQ10, ubichinol-10, is itself a real lipid-soluble radical scavenger molecule. It could be demonstrated in this context, that ubiquinone reduced to ubiquinol through the electron transport chain strongly inhibits lipid peroxidation in isolated mitochondria [113, 114]. Thus, reduced CoQ10 interferes also as an antioxidant with some of the basic age associated processes in mitochondria such as destruction on cardiolipin. Ubiquinol10 is about as effective in preventing peroxidative damage to lipids as Vitamin E, which is considered the best lipidsoluble antioxidant in humans. In contrast to Vitamin E, ubiquinol-10 is not recycled by ascorbate. However, it is known that ubiquinol-10 can be recycled by electron transport carriers present in various biomembranes and possibly by some enzymes [115]. In addition to direct antioxidant radical scavenging, the quinol can rescue tocopheryl radicals produced by reaction with lipid or oxygen radicals by direct reduction back to tocopherol (Vitamin E). Without CoQ10 in a membrane, regeneration of tocopherol is very slow. The regeneration of tocopherol can also be observed in low density lipoprotein
where a small amount of CoQ10 protects a larger amount of tocopherol [111]. In normal healthy individuals CoQ10 is synthesized in all cells from tyrosine (or phenylalanine) and mevalonate, and supplementation with CoQ10 does not increase tissue levels above normal. However, CoQ10 contents in cells decline during aging, which has been shown for example in muscle cells [116]. Previously it has beenshown that levels of CoQ10 are also lowered in skin cells from aging donors [117], suggesting that a decrease in mitochondrial CoQ10 content is an integral aspect of skin aging. In some tissue, such as aged skin, supplemental CoQ10 can restore normal levels. In fact, it was demonstrated that CoQ10 penetrates into the viable layers of the epidermis and reduces the basal level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was shown in clinical studies [117]. It is further known that UVirradiation depletes CoQ10 as well as other antioxidants in skin and causes oxidative damage [118]. In studies using nude mice, supplementation with CoQ10 was found to reduce the acute oxidative stress response following UV-irradiation, as characterized by reduced induction of manganese superoxide dismutase and glutathione peroxidase following irradiation in the presence of topical CoQ10 supplementation [119]. In studies by the authors, supplemental CoQ10 was also shown to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, modulation of specific phosphotyrosine kinases and prevention of oxidative mtDNA damage [117]. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. In another study, conducted in the author’s institution, healthy volunteers were topically treated with a CoQ10-containing creme formulation twice a day in a 7-day period. Afterwards, epidermal primary keratinocytes were isolated via suction blister, irradiated and examined for mitochondrial membrane potential. CoQ10 application clearly resulted in a significant amelioration (+44%) of mitochondrial membrane potential after irradiation compared to the untreated control. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging and has general ‘‘energizing’’ effects in skin [117, 120].
Conclusion Cutaneous aging is characterized by a decline in energy metabolism of skin cells partially caused by detrimental
Alterations of Energy Metabolism in Cutaneous Aging
changes in mitochondrial respiration. The processes involved seem to be predominantly mediated by free radical actions known to be generated either by exogenous noxes such as UV light, or by endogenous processes such as impaired mitochondrial respiration associated with electron ‘‘jam’’ and generation of ROS by leakage of electrons from the respiratory chain. It is widely accepted that alterations in mitochondrial respiration can be regarded as both a reason as well as an important consequence for aging. Any lack of mitochondrial function impairs cellular ATP synthesis, reducing the ‘‘fuel supply’’ for repair mechanisms. It does further induce the formation of ROS as byproducts of an impaired mitochondrial respiration. Accumulation of ROS may, in turn, damage neighbouring mitochondrial complexes, membranes and mtDNA and further accelerate the aging process in a kind of feedback loop. Once the damage of macromolecules has reached the level of mtDNA, leading to mutations, the energetic age of a mitochondrium, and thus of a cell, is carved in stone. Basically, any loss of mitochondrial energetic capacity is attempted to be compensated by energy generation from other sources. This is either the exploitation of intracellular energy stores for high energy demands in a short term, or the swith to anaerobic pathways for energy supply, such as glycolysis, as a last resort in a long term. In this context glycolysis, used by a cell as last resort, is associated with the generation of reactive glycolytic intermediates which favour the formation of advanced glycation endproducts (AGEs) via reactive carbonyl groups. These AGEs may harm a cell by processes ranging from the generation of infunctional cytoskeletal proteins up to the induction of apoptosis. Thus, it is important to keep tissues from anaerobiosis by keeping mitochondrial energy generation upright. Furthermore, it is important to supply a cell with substantial energy stores to be filled with energy in phases of baseline activity, which can be used in situation of high energy demand without the need to swith to glycolysis. There are several entry points to affect skin aging from an energetic perspective. Besides sunscreens to protect the skin from the most important ROS-generating insult, UV light (> Fig. 29.1A), the first and most obvious entry point is to keep the antioxidant system of a cell working. This will result in protection against ROS from whatever sources and thus targets the key effector molecules of skin aging (> Fig. 29.1B). Antioxidants are beyond the scope of this overview. However, it should be mentioned that one antioxidant which has been proven to be especially potent in pathological dermatological situations characterized by excessive deregulation of ROS generation, such as
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polymorphous light eruption (PLE), is a-glucosylrutin [121, 122, 123], a potent plant-derived antioxidant with excellent bioavailability in skin. The next energetically relevant entry point with regard to skin aging is to keep the respiratory chain in skin cells working, in order to avoid electron ‘‘jam’’ and subsequent generation of ROS. This can be done by supplementation with Coenzyme Q10, which is a key component of the respiratory chain. CoQ10 declines with age but can be effectively replenished in skin by topical formulations (> Fig. 29.1C). In addition to its central role as a structural component of the respiratory chain, CoQ10 is also a potent radical scavenger which protects important membrane proteins of the respiratory chain, such as cardiolipin, from oxidative damage. In addition to this, supplementation of skin with creatine is an important entry point for intervention with aging processes. Creatine levels and activities of creatine kinases in skin decline with age, reducing the available energy store for short term demand. Skin cells that are energetically re-charged with sufficient creatine are better protected against a variety of cellular stressors, age-dependent deficiencies in cellular functions, or oxidative and free-radical-induced cell damage. This is also due to a kind of ‘‘antioxidant’’ activity of the creatine/phosphocreatine system, which results from its capacity to form a sink for ATP, keeping the amount of available ADP for phosphorylation by the mitochondrial respiratory chain high (> Fig. 29.1D). This, in turn, keeps the mitochondrium in a kind of unrestricted State 3 respiration. At first sight, it might be tempting to speculate that supplementation of skin with additional oxygen might also enforce the capacity of the respiratory chain to generate energy. This, however could not yet be proven in experimental systems or clinical trials, and it might be due to the fact that skin can directly take up atmospheric oxygen in sufficient amounts, thus is generally not in a situation of hypoxia as other organs might be [20]. Nevertheless, beneficial effects of short term oxygen pulses on the activity of skin cells have been observed, but these are supposed to be related to some yet unknown signal transduction effects of oxygen, rather than on enhanced energy metabolism (publication in preparation). The energetic pathways leading to skin aging, as well as the possible intervention strategies, as discussed in this overview, are summarized in > Fig. 29.1. In conclusion, biological science has advanced the ability to directly target skin aging. The declining energy
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. Figure 29.1 Pathways leading to aging of skin cells. Processes (A)–(D) are described in detail in the Conclusion of this overview. Blue arrows marked with a (+) sign represent beneficial processes for the cell with anti-aging efficacy. Red arrows marked with a () sign represent detrimental processes for a cell, which are associated with accelerated aging. ROS = reactive oxygen species; AGE = advanced glycation endproduct; RC = respiratory chain; CoQ10 = Coenzyme Q10; mtDNA = mitochondrial DNA; Cr = creatine; PCr = phosphocreatine
Alterations of Energy Metabolism in Cutaneous Aging
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. Figure 29.1 (Continued)
metabolism has turned out as a high priority field of anti-aging interventions such as creatine and CoQ10.
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49. Huber LA, Xu QB, Jurgens G, et al. Correlation of lymphocyte lipid composition membrane microviscosity and mitogen response in the aged. Eur J Immunol. 1991;21:2761–2765. 50. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med. 2004;37:768–784. 51. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11:298–300. 52. Sohal RS. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech Ageing Dev. 1991;60:189–198. 53. Mori A, Utsumi K, Liu J, Hosokawa M. Oxidative damage in the senescence-accelerated mouse. Ann N Y Acad Sci. 1998; 854:239–250. 54. Chiba Y, Yamashita Y, Ueno M, et al. Cultured murine dermal fibroblast-like cells from senescence-accelerated mice as in vitro models for higher oxidative stress due to mitochondrial alterations. J Gerontol A Biol Sci Med Sci. 2005;60:1087–1098. 55. Trifunovic A, Wredenberg A, Falkenberg M, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429:417–423. 56. Hayakawa M, Torii K, Sugiyama S, Tanaka M, Ozawa T. Ageassociated accumulation of 8-hydroxydeoxyguanosine in mitochondrial DNA of human diaphragm. Biochem Biophys Res Commun. 1991;179:1023–1029. 57. Mecocci P, MacGarvey U, Kaufman AE, et al. Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann Neurol. 1993;34:609–616. 58. Ames BN, Shigenaga MK, Gold LS. DNA lesions, inducible DNA repair, and cell division: three key factors in mutagenesis and carcinogenesis. Environ Health Perspect. 1993;5(Suppl 101):35–44. 59. Sohal RS, Dubey A. Mitochondrial oxidative damage, hydrogen peroxide release, and aging. Free Radic Biol Med. 1994;16:621–626. 60. Laganiere S, Yu BP. Modulation of membrane phospholipid fatty acid composition by age and food restriction. Gerontology. 1993;39:7–18. 61. Dumas M, Maftah A, Bonte F, et al. Flow cytometric analysis of human epidermal cell ageing using two fluorescent mitochondrial probes. C R Acad Sci III. 1995;318:191–197. 62. Paradies G, Ruggiero FM. Age-related changes in the activity of the pyruvate carrier and in the lipid composition in rat-heart mitochondria. Biochim Biophys Acta. 1990;1016:207–212. 63. Paradies G, Ruggiero FM. Effect of aging on the activity of the phosphate carrier and on the lipid composition in rat liver mitochondria. Arch Biochem Biophys. 1991;284:332–337. 64. Ruggiero FM, Cafagna F, Petruzzella V, Gadaleta MN, Quagliariello E. Lipid composition in synaptic and nonsynaptic mitochondria from rat brains and effect of aging. J Neurochem. 1992;59:487–491. 65. Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol. 2007;47:143–183. 66. Ha MK, Chung KY, Bang D, Park YK, Lee KH. Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells. Proteomics. 2005; 5:1507–1519. 67. Scharffetter-Kochanek K, Wlaschek M, Brenneisen P, et al. UVinduced reactive oxygen species in photocarcinogenesis and photoaging. Biol Chem. 1997;378:1247–1257. 68. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med. 1997;22:269–285. 69. Davies KJ. The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress. IUBMB Life. 1999;48:41–47.
Alterations of Energy Metabolism in Cutaneous Aging 70. Bladier C, Wolvetang EJ, Hutchinson P, de Haan JB, Kola I. Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis? Cell Growth Differ. 1997;8:589–598. 71. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA. 1994;91:4130–4134. 72. Campisi J. The role of cellular senescence in skin aging. J Invest Dermatol Symp Proc. 1998;3:1–5. 73. Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992;256:628–632. 74. Pang CY, Lee HC, Yang JH, Wei YH. Human skin mitochondrial DNA deletions associated with light exposure. Arch Biochem Biophys. 1994;312:534–538. 75. Linnane AW, Marzuki S, Ozawa T, Tanaka M. Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet. 1989;1:642–645. 76. Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995;27:647–653. 77. Piko L, Hougham AJ, Bulpitt KJ. Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging. Mech Ageing Dev. 1988;43:279–293. 78. Cortopassi GA, Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res. 1990;18:6927–6933. 79. Eshaghian A, Vleugels RA, Canter JA, et al. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J Invest Dermatol. 2006;126:336–344. 80. Porteous WK, James AM, Sheard PW, et al. Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion. Eur J Biochem. 1998;257:192–201. 81. Shoffner JM, Lott MT, Voljavec AS, et al. Spontaneous Kearns-Sayre/ chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy. Proc Natl Acad Sci USA. 1989;86:7952–7956. 82. Schroeder P, Gremmel T, Berneburg M, Krutmann J. Partial depletion of mitochondrial DNA from human skin fibroblasts induces a gene expression profile reminiscent of photoaged skin. J Invest Dermatol. 2008;128:2297–2303. 83. Berneburg M, Plettenberg H, Medve-Konig K, et al. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. 84. Yang JH, Lee HC, Wei YH. Photoageing-associated mitochondrial DNA length mutations in human skin. Arch Dermatol Res. 1995;287:641–648. 85. Birket MJ, Passos JF, von Zglinicki T, Birch-Machin MA. The relationship between the aging- and photo-dependent T414G mitochondrial DNA mutation with cellular senescence and reactive oxygen species production in cultured skin fibroblasts. J Invest Dermatol. 2009;129(6):1361–1366. 86. Bandy B, Davison AJ. Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radic Biol Med. 1990;8:523–539. 87. Wei YH, Lee CF, Lee HC, et al. Increases of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4,977 BP-deleted mitochondrial DNA. Ann N Y Acad Sci. 2001;928:97–112. 88. Lu CY, Lee HC, Fahn HJ, Wei YH. Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin. Mutat Res. 1999;423:11–21.
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89. Berneburg M, Grether-Beck S, Kurten V, et al. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274:15345–15349. 90. Kueper T, Grune T, Prahl S, et al. Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging. J Biol Chem. 2007;282:23427–23436. 91. Hipkiss AR. Does chronic glycolysis accelerate aging? Could this explain how dietary restriction works? Ann N Y Acad Sci. 2006; 1067:361–368. 92. Alikhani Z, Alikhani M, Boyd CM, et al. Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Biol Chem. 2005;280:12087–12095. 93. Kasper M, Funk RH. Age-related changes in cells and tissues due to advanced glycation end products (AGEs). Arch Gerontol Geriatr. 2001;32:233–243. 94. Rugolo M, Lenaz G. Monitoring of the mitochondrial and plasma membrane potentials in human fibroblasts by tetraphenylphosphonium ion distribution. J Bioenerg Biomembr. 1987;19: 705–718. 95. Scaduto RC Jr., Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999;76:469–477. 96. Koopman WJ, Visch HJ, Smeitink JA, Willems PH. Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts. Cytometry A. 2006;69:1–12. 97. Plasek J, Vojtiskova A, Houstek J. Flow-cytometric monitoring of mitochondrial depolarisation: from fluorescence intensities to millivolts. J Photochem Photobiol B. 2005;78:99–108. 98. Distelmaier F, Koopman WJ, Testa ER, et al. Life cell quantification of mitochondrial membrane potential at the single organelle level. Cytometry A. 2008;73:129–138. 99. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun. 1993;197: 40–45. 100. Hagens R, Khabiri F, Schreiner V, et al. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin. Skin Res Technol. 2008;14:112–120. 101. Khabiri F, Hagens R, Smuda C, et al. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)measurement. I: mechanisms of UPE of biological materials. Skin Res Technol. 2008;14:103–111. 102. Vandenberghe K, Goris M, Van Hecke P, et al. Long-term creatine intake is beneficial to muscle performance during resistance training. J Appl Physiol. 1997;83:2055–2063. 103. Daly MM, Seifter S. Uptake of creatine by cultured cells. Arch Biochem Biophys. 1980;203:317–324. 104. Meyer LE, Machado LB, Santiago AP, et al. Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity. J Biol Chem. 2006;281:37361–37371. 105. Lenz H, Schmidt M, Welge V, et al. Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3cells affects cell viability and mitochondrial morphology. Mol Cell Biochem. 2007;306:153–162.
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106. O’Gorman E, Beutner G, Dolder M, et al. The role of creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett. 1997;414:253–257. 107. Brdiczka D, Beutner G, Ruck A, Dolder M, Wallimann T. The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition. Biofactors. 1998;8:235–242. 108. Dolder M, Walzel B, Speer O, Schlattner U, Wallimann T. Inhibition of the mitochondrial permeability transition by creatine kinase substrates. Requirement for microcompartmentation. J Biol Chem. 2003;278:17760–17766. 109. Dolder M, Wendt S, Wallimann T. Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage. Biol Signals Recept. 2001;10:93–111. 110. Berneburg M, Gremmel T, Kurten V, et al. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences. J Invest Dermatol. 2005;125:213–220. 111. Crane FL. Biochemical functions of coenzyme Q10. J Am Coll Nutr. 2001;20:591–598. 112. McLennan HR, Degli Esposti M. The contribution of mitochondrial respiratory complexes to the production of reactive oxygen species. J Bioenerg Biomembr. 2000;32:153–162. 113. Lopez-Lluch G, Barroso MP, Martin SF, et al. Role of plasma membrane coenzyme Q on the regulation of apoptosis. Biofactors. 1999;9:171–177. 114. Mellors A, Tappel AL. The inhibition of mitochondrial peroxidation by ubiquinone and ubiquinol. J Biol Chem. 1966; 241:4353–4356.
115. Frei B, Kim MC, Ames BN. Ubiquinol-10 is an effective lipidsoluble antioxidant at physiological concentrations. Proc Natl Acad Sci USA. 1990;87:4879–4883. 116. Lass A, Kwong L, Sohal RS. Mitochondrial coenzyme Q content and aging. Biofactors. 1999;9:199–205. 117. Hoppe U, Bergemann J, Diembeck W, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. Biofactors. 1999;9:371–378. 118. Podda M, Traber MG, Weber C, Yan LJ, Packer L. UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med.1998;24:55–65. 119. Kim DW, Hwang IK, Yoo KY, et al. Coenzyme Q_{10} effects on manganese superoxide dismutase and glutathione peroxidase in the hairless mouse skin induced by ultraviolet B irradiation. Biofactors. 2007;30:139–147. 120. Stab F, Wolber R, Blatt T, Keyhani R, Sauermann G. Topically applied antioxidants in skin protection. Methods Enzymol. 2000;319:465–478. 121. Hadshiew IM, Treder-Conrad C, v Bulow R, et al. Polymorphous light eruption (PLE) and a new potent antioxidant and UVAprotective formulation as prophylaxis. Photodermatol Photoimmunol Photomed. 2004;20:200–204. 122. Rippke F, Wendt G, Bohnsack K, et al. Results of photoprovocation and field studies on the efficacy of a novel topically applied antioxidant in polymorphous light eruption. J Dermatolog Treat. 2001;12:3–8. 123. Wolber R, Stab F, Max H, et al. Alpha-glucosylrutin, a highly effective flavonoid for protection against oxidative stress. J Dtsch Dermatol Ges. 2004;2:580–587.
3 Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging Peter Helmbold
Introduction Chronic ultraviolet (UV) light exposure of skin leads to typical effects: changes in the collagen and elastic tissue matrix is considered the characteristic histological finding in aged skin, followed by visible wrinkling and pigmentary changes. Changes in the epidermis include thinning to atrophy, hyperplasia of melanocytes, and disturbances in the texture of keratinocytes. Assessment of the degrees of photoaging by a grading system with low interobserver coefficient of variation seems to be of special interest. Different clinical methods have been proposed including descriptive grading clinical scales, visual analogue scales, and photographic grading scales [1]. Some of these methods like ‘‘skin surface topography grading’’ [2] were compared with histological changes like actinic elastosis. Other studies used histological scoring of dermal aging independent of a noninvasive scoring system. The following approaches were used: quantification of elastic tissue [3], type III procollagen, type III to type I procollagen ratio, quantification of the grenz zone (a wide band of eosinophilic material just beneath the epidermis, devoid of oxytalan fibers) [4], activated fibroblasts with positive procollagen staining [5], acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen [6], quantification of changes in the epidermis (thinning of the stratum corneum, granular layer enhancement, and epidermal thickening) [7]. One disadvantage of most of these methods is that actinic and instrinsic aging cannot be distinguished from one another. Bhawan et al. [8] systematically investigated histological effects of photoaging. The following features proved to be significantly changed in photoaged skin: increase in melanocytes, increase in melanocytic atypia and epidermal melanin, reduced epidermal thickness, more compact stratum corneum, increased granular layer thickness,
increased solar elastosis, dermal elastic tissue, melanophages, perivascular inflammation, and perifollicular fibrosis but no change in the number of mast cells or dermal mucin in the photoexposed skin. Of these, actinic elastosis (basophilic degeneration of the dermis) was the single most reliable factor. Basophilic degeneration is very consistent with the clinical sign of wrinkling and with dermal microvasular aging (see Chapter 2). Thus, a single-factor scoring system of dermal aging regarding dermal basophilic degeneration (DBD) was developed. It should be mentioned that the knowledge of dermal fiber degeneration is not new and the use of a scoring system is the result of previous work [9, 10]. After first experiments with a five-level system, it was found that best interobserver agreement was obtainable with a three-level model (> Table 3.1) together with a histological atlas of the different levels (> Fig. 3.1). This model was tested in 120 biopsies from normal skin of 87 patients (42 females, 45 males, 27.9 23.7 years [mean SD]) from surplus areas (i.e., Burow’s triangle) of routinely excised and histologically controlled benign nevus cell nevi of normal skin. Each specimen was
. Table 3.1 Histological scoring of dermal basophilic degeneration (DBD) [11] No actinic damage (Level 0): No fiber degeneration (> Fig. 3.1a, b). Moderate actinic damage (Level 1): Fragmentation of fibers of the upper dermis and presence of single basophilic fibers (> Fig. 3.1c, d). High actinic damage (Level 2): Spotted or band-like basophilic degeneration with conglomerates of basophilic masses in the upper and/or mid-dermis (> Fig. 3.1e, f).
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_3, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 3.1 Scoring of dermal basophilic degeneration (DBD). (a, b) No fiber degeneration (DBD level = 0). (c, d) Fragmentation of fibers (arrows) of the upper dermis and presence of single basophilic fibers (DBD level = 1). (e, f) Spotted (e) or band-like (f) basophilic degeneration (arrows) with conglomerates of basophilic masses in the upper and mid-dermis (DBD level = 2). Scale bar: a, c–f = 200 mm, b = 100 mm (Helmbold et al. [11]. Reprinted with permission of J Invest Dermatol)
characterized by a set of clinical data: age, sex of the patient, and body location of the biopsy with regard to typically solar-exposed skin areas. The interobserver reliability (agreement among four independent observers) of this technique was 92.2 4.6%
in all biopsies. There was no disagreement of more than one level between the investigators. Correlations were found between DBD and the age of the patient (Spearman r = 0.662, p < 0.001) as well as DBD and body regions with typical chronic solar exposure (Spearman r = 0.244,
Basophilic (Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging
p = 0.005). Sixty-eight biopsies revealed no visible DBD (37 from female, 31 from male patients; age: 19.8 18.4 years), 36 biopsies showed moderate ‘‘level 1’’ DBD (28 females, 10 males, 39.9 19 years), and 16 had a high ‘‘level 2’’ DBD (six females, ten males, 64.4 11.9 years). DBD was not observable in patients younger than 15 years.
Conclusion The advantages of this approach are easy application, use of HE-stained routine sections, fast determination, and sure results with high interobserver agreement. Disadvantages are that this approach cannot ‘‘measure’’ minimal differences and that it reflects only the dermal component of photoaging. Thus, the mean application fields are the studies that need reliable classification if there is actinic degeneration (or not). On the other hand, this approach is not suitable for quantification of the effects of an anti-aging product or similar studies.
Cross-references > Histology
of Microvascular Aging of Human Skin
References 1. Kappes UP. Skin ageing and wrinkles: clinical and photographic scoring. J Cosmet Dermatol. 2004;3:23–25.
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2. Battistutta D, Pandeya N, Strutton GM, et al. Skin surface topography grading is a valid measure of skin photoaging. Photodermatol Photoimmunol Photomed. 2006;22:39–45. 3. Chiu AE, Chan JL, Kern DG, et al. Double-blinded, placebocontrolled trial of green tea extracts in the clinical and histologic appearance of photoaging skin. Dermatol Surg. 2005;31:855–860; discussion 860. 4. Seite S, Bredoux C, Compan D, et al. Histological evaluation of a topically applied retinol-vitamin C combination. Skin Pharmacol Physiol. 2005;18:81–87. 5. Rostan E, Bowes LE, Iyer S, et al. A double-blind, side-by-side comparison study of low fluence long pulse dye laser to coolant treatment for wrinkling of the cheeks. J Cosmet Laser Ther. 2001;3:129–136. 6. Ditre CM, Griffin TD, Murphy GF, et al. Effects of alpha-hydroxy acids on photoaged skin: a pilot clinical, histologic, and ultrastructural study. J Am Acad Dermatol. 1996;34:187–195. 7. Newman N, Newman A, Moy LS, et al. Clinical improvement of photoaged skin with 50% glycolic acid. A double-blind vehiclecontrolled study. Dermatol Surg. 1996;22:455–460. 8. Bhawan J, Andersen W, Lee J, et al. Photoaging versus intrinsic aging: a morphologic assessment of facial skin. J Cutan Pathol. 1995;22: 154–159. 9. Suwabe H, Serizawa A, Kajiwara H, et al. Degenerative processes of elastic fibers in sun-protected and sun-exposed skin: immunoelectron microscopic observation of elastin, fibrillin-1, amyloid P component, lysozyme and alpha1-antitrypsin. Pathol Int. 1999;49: 391–402. 10. Lund HZ, Sommerville RL. Basophilic degeneration of the cutis; data substantiating its relation to prolonged solar exposure. Am J Clin Pathol. 1957;27:183–190. 11. Helmbold P, Lautenschlager C, Marsch W, et al. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging. J Invest Dermatol. 2006;126: 1419–1421.
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35 Biological Effects of Estrogen on Skin Zack Thompson . Howard I. Maibach
Introduction Changes in skin aging and function occur at variable rates, and are influenced by environmental, hormonal, and genetic factors unique to each individual. Skin aging involves progressive degenerative changes, such as gradual dryness, thinning, fragility, atrophy, and wrinkling. Over time, the skin experiences a progressive increase in extensibility and a reduction in elasticity, thereby becoming more frail and susceptible to trauma. This in turn leads to an increased risk of skin injury (e.g., lacerations, tears, ulcerations, bruising), and an impairment of wound healing. Improvements in nutrition, sanitation, quality and provision of healthcare, and other related factors have led to a dramatic increase in life expectancy for human beings over the past century. The average life expectancy of women in the United States has increased from roughly 50.7 years in 1900 [1], to an estimated 80.7 years by 2009 [2]. As the developing world catches up, a similar but much more significant trend in average global longevity is projected to occur. The number of women age 60 and over worldwide is expected to increase from about 336 million in 2000, to just over 1 billion by 2050 [3]. Since the average woman in a developed nation spends about one third of her life after the onset of menopause, the benefits and risks of estrogen replacement therapy (ERT) – also known as hormone replacement therapy (HRT), and menopausal hormone therapy (MHT) – have become major areas of focus for research. ERT has been regularly prescribed by physicians for postmenopausal women since the 1940s to reduce symptoms associated with menopause, such as hot flashes, night sweats, vaginal dryness, and sleep disturbances [4]. In the intervening period, the risks and benefits of ERT have been and continue to be debated. In terms of benefits to ERT, estrogen has been demonstrated to ameliorate menopausal maladies such as: vasomotor symptoms, mood changes, atrophy of reproductive organs, and sleep disturbance [5]. More recently, the Women’s Health Initiative (WHI) studies were conducted to assess the risks and benefits from ERT. However, the WHI did not perform any
analysis of the effect of ERT on skin in terms of cosmetic appearance, morbidity, or mortality. Additionally, the WHI studies examined only oral conjugated equine estrogens (CEE) 0.625 mg and medroxyprogesterone acetate 2.5 mg, not topical or transdermal ERTs, which could have a more focused impact on the skin. Particularly in light of the increasing life expectancy of women throughout the world, health workers need to more fully understand the physiology and treatment of menopause. To this end, this chapter focuses on the biological effects of estrogen on the skin of postmenopausal women with regard to skin thickness, moisture, wrinkling, wound healing, and scarring, and briefly discusses future estrogen therapies, such as selective estrogen receptor modulators (SERMs). Studies have uncovered various mechanisms by which estrogen may affect skin aging and function. Research indicates that topical and systemic ERT lead to a statistically significant improvement in many aging skin problems [6]. ERT increases skin collagen content and preserves thickness, thereby reducing wrinkling. Skin moisture content improves with ERT, as it increases the skin’s hyaluronic acid, acid mucopolysaccharides, and sebum levels, and possibly maintains stratum corneum barrier function. Beyond its impact on aging, topical ERT accelerates and improves cutaneous wound healing in elderly individuals, possibly by regulating the levels of a cytokine. Conversely, a lack of estrogen (i.e., hypoestrogenism) or addition of tamoxifen – the first SERM developed – may improve the quality of scarring, though the relationship between estrogen and scarring is more ambiguous.
Thickness and Collagen Collagen is the primary protein of connective tissue in mammals, comprising about 25–35% of total body protein content. Collagen has a high tensile strength, and is a major component of fascia, cartilage, ligaments, tendons, bone, and skin. At least 30 different collagen genes have been identified and described. These collagen genes combine to form over 20 different types of collagen fibrils, of
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which Types I, II, and III are the most common [7]. Human skin contains over 14 types of collagen, of which 80% is Type I – the collagen that is responsible for skin strength – and 15% is Type III – the collagen that is responsible for the elastic properties of skin. Collagen becomes progressively sparse, disordered, and atrophied as skin ages – one of the chief reasons for the skin transformations resulting from aging. Copious studies establish menopause leads to estrogen deficiency, and research over the past 60 years demonstrates skin
thickness, estrogen content, and skin collagen are closely correlated (> Table 35.1). In 1941, Albright et al. first noticed elderly women with osteoporotic fractures – an injury closely correlated with menopause – had visibly thinner skin. Correlation between skin thickness and estrogen content was first noted by Bullough’s 1941 study on mice [8]. McConkey et al. posited in 1963 that the ‘‘transparent skin’’ described by Albright was caused by a decrease in dermal collagen Type I [9]. In 1970, Black et al. [10] demonstrated skin
. Table 35.1 Selected studies on skin thickness and ERT Date
Study
1969 Rauramo and Punnomen
Type of measurement
Hormones used
Skin biopsy analysis Estradiol succinate (measured by calipers) 2.0 mg
Duration
Results
6 months
Improvement in skin thickness
1987 Brincat et al. Skin biopsy analysis
Estradiol implant and percutaneous estradiol gel
2–10 years
Increase in skin thickness by 30%; average linear decline of 1.13% skin thickness and 2.1% collagen per year in the first 15–18 postmenopausal years without ERT
1992 CasteloSkin biopsy analysis Branco et al.
Conjugated equine estrogens or transdermal 17bestradiol
12 months
Increase in skin collagen by 1.8–5.1%
1994 Maheux et al.
Skin thickness (measured by ultrasonagraphy)
Conjugated estrogen 0.625 mg
12 months
Increase in skin thickness by 11.5%
1995 Varila et al.
Skin biopsy analysis
Topical 17b-estradiol
3 months
Increase in hydroxyproline by 38%
1996 Callens et al. Skin thickness 17b-estradiol gel or (measured by estradiol patches ultrasonagraphy at five skin locations)
Mean of Increase in skin thickness by 7–15% in all 4.8 years locations
2000 Sauerbronn et al.
Skin biopsy analysis
2.0 mg valerate estradiol, cycled with 1.0 mg cyproterone acetate
6 months
Increase in skin collagen by 6.49%
2007 Sator et al.
Sebumeter, the Corneometer, and high-frequency ultrasound
2.0 mg 17b-estradiol/ 10 dydrogesterone
7 months
Significant improvements in skin elasticity, skin hydration, and skin thickness
1993 Sawwas and Skin biopsy analysis Laurent
Subcutaneous estradiol and testosterone
3–14 years
Significantly greater levels of collagen Type III
1997 Haapasaari et al.
17b-estradiol and norethisterone acetate; estradiol valerate
12 months
No significant change in skin collagen or thickness
Skin biopsy analysis
Biological Effects of Estrogen on Skin
thickness is in fact proportional to collagen content – as McConkey et al. had suggested – utilizing the 1964 radiographic technique for measuring skin thickness employed by Meema et al. [11]. The above studies established that the estrogen deficiency associated with menopause leads to skin collagen degradation and decreasing skin thickness. Thus, future studies focused on deciphering the effects of reproductive hormones on skin collagen. In 1969, Rauramo and Punnomen noted ERT had a favorable effect on human skin [12]. Their study involved 6 months of treatment with 2.0 mg estradiol succinate, and showed improvements in skin thickness of biopsies measured using calipers. A subsequent 1987 study by Brincat et al. corroborated Rauramo and Punnomen’s findings, showing a decrease in skin thickness and collagen content after menopause. Their study demonstrated an increase in skin thickness for women receiving an estradiol implant or percutaneous estradiol gel. Using radiographic techniques, Brincat et al. found an average increase in skin thickness by 30% for those on ERT. Additionally, Brincat et al. found 30% of collagen is lost within the first 5 years of menopause, and witnessed an average linear decline of 1.13% in skin thickness and 2.1% in collagen per year in the first 15–18 postmenopausal years for those not on ERT. The study also noted that skin collagen decline was correlated specifically to the duration of estrogen deficiency (i.e., postmenopausal years), and not chronological age [13]. In a 1992 study, Castelo-Branco et al. similarly found that ERT – both oral and transdermal – increased skin collagen content. Their study used conjugated equine estrogens and transdermal 17b-estradiol over a 12 month period, and showed an increase in skin collagen content of 1.8–5.1%, varying based on the type of ERT administered. Unlike Brincat et al., Castelo-Branco et al. observed a higher correlation between skin collagen content and chronological age, though they still recognized a statistically significant correlation between skin collagen content and time since the onset of menopause [14]. Subsequent studies by Affinito et al. and others have achieved the same results as Brincat et al., and showed postmenopausal years to be the determining factor [15]. The likely cause of this aberration in the Castelo-Branco et al. study was that many participants were in their initial postmenopausal years, with a short history of estrogen deprivation. This problem with study participants has plagued other studies (e.g., [16, 17]), and has contributed to some of the controversy surrounding the effects of ERT on skin aging. A 1994 study by Maheux et al. controlled for some of the factors which complicate the measurement of skin aging
35
(e.g., smoking, exposure to solar radiation), thereby focusing more specifically on the effects of conjugated estrogens. Maheux et al. employed a randomized, double-blind, placebo-controlled study of postmenopausal nuns. Their study used conjugated estrogen 0.625 mg over a 12 month period, and showed an increase in skin thickness, as measured by ultrasonography, of 11.5% for the group receiving ERT [18]. Varila et al. studied the effect of topical ERT on collagen content, as measured by skin hydroxyproline, in a 1995 study. Hydroxyproline is a major component of the protein collagen, and is found in few proteins other than collagen. The only other mammalian protein that includes hydroxyproline is elastin. For this reason, hydroxyproline content has been used as an indicator to determine collagen or gelatin amount. Via skin biopsy analysis, Varila et al. measured an increase in hydroxyproline of 38% following administration of topical 17b-estradiol for 3 months. The study also observed increased levels of the carboxyterminal propeptide of human type I procollagen and of the aminoterminal propeptide of human type III procollagen, thus showing that estrogen increases collagen synthesis [19]. Subsequent studies by Callens et al. [20], Sauerbronn et al. [21], Sator [22] and others have further substantiated the aforementioned studies’ claims. Callens et al. found ERT increased skin thickness by 7–15% in postmenopausal women utilizing estradiol gel patches or an estradiol transdermal system. Sauerbronn et al. focused on skin collagen rather than thickness. Following 6 months of treatment with estradiol valerate and cyproterone acetate, their study observed a 6.49% increase in collagen fibers in the dermis, and no significant change in epidermal thickness. Sator noted significant improvements in skin elasticity, skin hydration, and skin thickness after 7 months of treatment. This corroborates a 2005 Sumino et al. study, which showed after menopause, skin elasticity declined 0.55% per year, and that 12 months of ERT increased elasticity by 5.2% [23]. While ERT increases skin thickness, there are limits to the potential for ERT to reverse skin aging. Savvas and Laurent conducted research in 1993 which, in accordance with the 1987 Brincat et al. study, suggests ERT produces no additional increase in skin collagen content after 2 years. Their study focused specifically on collagen Type I and Type III, and found increased collagen content in postmenopausal women receiving estradiol and testosterone implants. However, beyond 3 years they found no increase in the proportion of collagen Type III [24]. At least one notable study from 1997 appears to dispute the evidence that ERT increases collagen content and
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Biological Effects of Estrogen on Skin
skin thickness. Utilizing immunohistochemistry and colorimetric methods, Haapasaari et al. detected no increase in skin collagen content following 1 year of systemic estrogen therapy. The investigators hypothesized estrogen affects collagen turnover rather than skin collagen [18]. A more likely explanation, however, is that the low median postmenopausal age of 12 months amongst the study participants did not provide adequate time to show the effects of low estrogen levels. Lastly, as seen earlier by the wide range of improvement, it is difficult to quantify the real effect of ERTs on skin thickness and collagen content. Some factors such as individual skin history are extremely difficult to correct for when studied. For example, Lee et al. highlighted the need to standardize the method of measuring skin atrophy and thickness so that results are more comparable across studies [25].
Moisture Studies show the loss of moisture, which leads to dry skin, is another age-related skin condition. In 1996, Schmidt et al. noted an increase in skin moisture amongst perimenopausal and postmenopausal women after using topical estradiol 0.01% and estriol 0.3% (systemic) for 6 months [26]. Dunn et al. compiled results from a much larger population based cohort study in 1997 – the first National Health and Nutrition Examination Survey (NHNES), a Centers for Disease Control and Prevention (CDC) program of studies designed to assess the health and nutritional status of adults and children in the United States – and showed postmenopausal women on ERT were significantly less likely to experience dry skin [27]. Research provides the following possible explanations as to why topical and systemic estrogen therapies preserve skin moisture: increased acid mucopolysaccharides and hyaluronic acid in the dermis, higher sebum levels, increased water-holding capacity of the stratum corneum, and changes in the corneocyte surface area. First, Grosman et al. observed increased acid mucopolysaccharides and hyaluronic acid in the skin of mice treated with estrogen in the early 1970s. Hyaluronic acid is known to have a high water holding capacity, which supports an increase in dermal water content [28, 29]. Second, the 1996 study by Callens et al. demonstrated a 35% increase in sebum levels amongst women on estrogen [19]. This study suggests that ERT may prevent the decrease in glandular secretions Pochi et al. noted in postmenopausal women in their 1979 study [30].
Third, the ability of skin to retain water is largely associated with the stratum corneum lipids, which maintain skin barrier function. In 1995, Pierard-Franchimont et al. suggested estrogen may play a role in stratum corneum barrier function, noting women on transdermal ERT showed increased water-holding capacity of the stratum corneum [31]. Similarly, Paquet et al. demonstrated in 1998 that estrogen also improves the ability of the stratum corneum to prevent water loss, by observing a decrease in the rate of water accumulation in postmenopausal women [32]. Lastly, ERT may lead to changes in the corneocyte surface area, thereby further enhancing the epidermal barrier function.
Wrinkling Skin wrinkling is the result of lost skin elasticity [33], dermal thickening, and elastic deterioration caused by a variety of degenerative environmental, hormonal, and genetic factors. Histological studies of wrinkles by ContetAudonneau et al. [34] and Bosset et al. [35] showed alterations of dermal collagen and elastic fibers, as well as a marked decrease in glycosaminoglycans. Numerous studies have shown ERT can improve fine wrinkles, prevent development of skin wrinkles, and decrease existing wrinkle depth. Creidi et al. studied the effect of conjugated estrogen cream on postmenopausal women in a double-blind, placebo-controlled study utilizing clinical evaluation by dermatologists. This study found significant improvement in fine wrinkles [36]. Dunn et al. employed the NHNES to control for age, body mass index, and sun exposure, and found postmenopausal women on ERT were less likely to develop wrinkles in the first place [26]. Schmidt et al. further demonstrated that existing wrinkle depth can be decreased using topical ERT [25]. In contradiction to these studies, a 2008 study by Phillips et al. found no improvement in age-related skin changes from topical ERT. However, this study was limited in that it utilized low-dose estrogen (i.e., 1 mg norethindrone acetate and 5–10 mg ethinyl estradiol) for only 48 weeks, and in women with an average of only 5 postmenopausal years [17]. The impact of ERT on wrinkling likely relates partially to its impact on collagen; namely its capacity to increase the proportion of collagen III in skin. Punnonen et al. noted the elastic fibers in the papillary dermis following local estriol treatment had thickened, increased slightly in number, and become better oriented [37].
Biological Effects of Estrogen on Skin
Furthermore, the increase in hyaluronic acid noted by Grosman increases water capacity and thus skin turgor, thereby reducing the ability to develop wrinkles and the appearance of any wrinkles present [28]. An interesting side note, however, is that a study by Castelo-Branco et al. found ERT does not appear to reduce wrinkling in those with a history of over 10 years smoking. Thus, it seems ERT cannot reverse some of the damage done by smoking, such as destruction of the ground substance, decreased blood flow in the skin, and direct toxic effects [38].
Wound Healing Natural aging significantly impacts wound healing by increasing susceptibility to trauma, bruising, and chronic wounds. The role of estrogen and effect of ERT on wound healing is only recently becoming understood, largely because most early studies were performed using animal models that produced conflicting results. Differences in species, duration of treatment, and methodologies employed likely led to the inconsistent findings in animal studies conducted during the 1960s and 1970s [39]. Recent studies on wound healing have focused primarily on the molecular role of estrogen on the cells and metabolic processes involved in wound repair. Age-related impairment of wound healing has been partially attributed to low levels of transforming growth factor-b1 (TGFb1), decreased collagen synthesis, and increased presence of proteases (specifically elastase). Furthermore, estrogen’s presence on fibroblasts – the main cell type involved in wound healing – indicates it may directly modify their function [39]. Studies by Ashcroft et al. suggest estrogen positively affects wound repair by causing TGF-b1 secretion by fibroblasts – not by increasing fibroblast production in a wound – increasing collagen content, and reducing collagenolysis [40–42]. The 1997 Ashcroft et al. study used rats to demonstrate topical ERT was associated with significantly accelerated acute wound healing as shown by decreased re-epithelialization time, decreased wound width, and increased collagen deposition. The 1999 and 2003 Ashcroft et al. studies demonstrated topical ERT reduces the activity of protease elastase in cutaneous wounds in humans, thereby improving healing as shown by decreased wound sizes, faster increases in collagen levels, increased fibronectin levels, and enhanced strength. Furthermore, the latter study suggested topical ERT may even be useful on a prophylactic basis, though more work needs to be done in this area.
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Scarring The cellular and subcellular sites and mechanisms involved in the scarring process are poorly understood. Whitby et al. conducted a study comparing the differences between fetal and adult wound healing. Fetal scars tend to be superior in that they are pale and flat, rather than pigmented and everted as in adults. Their study found fetal wounds deficient in the inflammatory cytokine TGFb1, whereas adult skin typically had a large amount of TGF-b1 [43]. As mentioned in the section on wound healing, postmenopausal women in a hypoestrogenic state tend to be deficient in TGF-b1; thus, they should produce scars that are better both macroscopically and microscopically. A study by Shah et al. supports this supposition, and suggests estrogen antagonists may be effective in limiting scarring [44].
Selective Estrogen Receptor Modulators (SERMs) SERMs act at the level of the estrogen receptors, either mimicking positive estrogen effects or blocking negative estrogen effects, depending on the tissue. This tissue specificity allows for targeted ERT treatments. For example, tamoxifen has an antiestrogenic effect on breast tissue but an estrogenic effect on bone, and is used to treat breast cancer and prevent postmenopausal osteoporosis simultaneously. Hu et al. demonstrated tamoxifen also inhibits collagen wound contraction, and indicated it affected fibroblast morphology. A potential mechanism for the inhibition of wound contraction could be the inhibition of fibroblasts or fibroblast proliferation [45]. Surazynski et al. found that in fibroblasts, a SERM currently used for the treatment of postmenopausal osteoporosis known as raloxifene has a stronger positive stimulating effect on collagen synthesis than estradiol [46]. Intensive research is currently underway to develop new SERMs specifically for the purpose of targeting the skin without incurring systemic side effects [47]. Thus, these drugs will likely become an important means of controlling scarring and other effects of skin aging.
Conclusion New studies continue to support the above conclusions that both topical and systemic ERT have positive impacts
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Biological Effects of Estrogen on Skin
on skin aging as it relates to thickness, moisture, wrinkling, wound healing, and scarring [48–50]. ERT’s ability to slow skin aging is largely due to estrogen’s ability to repair and prevent a decline in skin collagen, increase skin turgor and water holding capacity, improve epidermal barrier function, and decrease changes in skin elasticity over time. Other areas remain to be explored further, such as optimal methods for administering ERT, determining the impact of ERT on men and premenopausal women, understanding the effect of estrogen on melanocyte function, and ascertaining the role of estrogen in skin cancer prevention, if any. ERT also affects skin and skin appendages in ways not discussed in this chapter, such as hair growth during pregnancy, and hair loss during menopause [51–53]. When determining if ERT is appropriate, the benefits and risks of ERT must be weighed by patients and healthcare workers in light of the potential side effects, particularly from systemic ERT. At this point in time, treatment of skin aging should not be the sole basis for systemic ERT, at least until it is understood what minimum concentration of estrogen can achieve the best local effects without systemic hormonal side effects. Most topical ERTs (e.g., estradiol creams) may not be significantly systemically absorbed [54]. Since topical ERT may present a safe and effective treatment for skin aging, menopausal women not receiving systemic ERT are candidates for this treatment when it is administered by a dermatologist educated in endocrinology. Finally, SERMs may present another future means of achieving the benefits of estrogen therapy without the systemic risks. Taken together, current experimental data suggests a careful prospective examination of topical estrogens to ameliorate skin aging. Today’s experimental tools should provide reliable efficacy and toxicity metrics.
Cross-references > Aging
Genital Skin and Hormone Replacement Therapy Benefits
References 1. World Health Organization Fact Sheet Number 252. http://www. who.int/mediacentre/factsheets/fs252/en/. June 2000. 2. Censtral Intelligence Agency. The World Fact Book. https://www.cia. gov/library/publications/the-world-factbook/geos/US.html. Updated Apr 30, 2009. 3. Women, Ageing and Health: A Framework for Action. World Health Organization. http://www.who.int/ageing/publications/ Women-ageing-health-lowres.pdf. 2007. 4. Shah M, Maibach H. Estrogen and skin, an overview. Am J Clin Dermatol. 2001;2(3):143.
5. Palacios S. Current perspectives on the benefits of HRT in menopausal women. Maturitas. 1999;33:S1–S3. 6. HRT/ERT refers to supplements of hormones, such as estrogen or estrogen with progesterone (progestin in its synthetic form). ERT will be used going forward to prevent confusion with other hormone therapies. 7. King, M. The Medical Biochemistry Page. IU School of Medicine. http://themedicalbiochemistrypage.org/extracellularmatrix.html. Last modified June 11, 2008. 8. Bullough HF. Cyclical changes in the skin of the mouse during estrous cycle. J Endocrinol. 1943;3:280–287. 9. McConkey B, Fraser GM, Bligh AS, et al. Transparent skin and osteoporosis. Lancet. 1963;I:693–695. 10. Black MM, Bottoms E, Shuster S. Changes in skin collagen and thickness in endocrine disease. Eur J Clin Invest. 1970;1:127. 11. Meema HE, Sheppard RH, Rapoport A. Roentgenographic visualization and measurement of skin thickness and its diagnostic application in acromegaly. Radiology. 1964;82:411. 12. Rauramo L, Punnomen R. Wirkung einer oralen ostrogenotherapie mit ostriolsuccinat auf die Hautt kastrierter Frauen. Z Haut Geschl Kr. 1969;44:463–470. 13. Brincat M, Yuen AW, Studd JW, et al. Response of skin thickness and metacarpal index to estradiol therapy in postmenopausal women. Obstet Gynecol. Oct 1987;70(4):538–541. 14. Castelo-Branco C, Duran M, Gonzales-Merlo J. Skin collagen and bone changes related to age and hormone replacement therapy. Maturitas. 1992;14:113–119. 15. Affinito P, Palomba S, Sorrentino C, et al. Effects of postmenopausal hypoestrogenism on skin collagen. Maturitas. 1999;33:239–247. 16. Phillips TJ, Symons J, et al. Does hormone therapy improve agerelated skin changes in postmenopausal women? A randomized, double-blind, double-dummy, placebo-controlled multicenter study assessing the effects of norethindrone acetate and ethinyl estradiol in the improvement of mild to moderate age-related skin changes in postmenopausal women. J Am Acad Dermatol. Sept 2008;59(3):397–404, e3. 17. Haapasari K, Raudaskoski T, Kallioinen M, et al. Systemic therapy with estrogen or estrogen with progestin has no effect on skin collagen in postmenopausal women. Maturitas. 1997;27:153–162. 18. Maheux R, Naud F, Rioux M, et al. A randomized, double-blind, placebo-controlled study on the effect of conjugated estrogens on skin thickness. Am J Obstet Gynecol. 1994;170:642–649. 19. Varila E, Rantala I, Oikarinen A, et al. The effect of topical estradiol on skin collagen of postmenopausal women. Br J Obstet Gynaecol. Dec 1995;102(12):985–989. 20. Callens A, Valliant L, Lecomte P, et al. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193:289–294. 21. Sauerbronn AVD, Fonseca AM, Bagnoli VR, et al. The effects of systemic hormone replacement therapy on the skin of the postmenopausal women. Int J Gynecol Obstet. 2000;68:35–41. 22. Sator PG, Sator MO, Schmidt JB, et al. A prospective, randomized, double-blind, placebo-controlled study on the influence of a hormone replacement therapy on skin aging in postmenopausal women. Climacteric. 2007;10:320–334. 23. Sumino H, Ichikawa S, et al. Effects of aging, menopause, and hormone replacement therapy on forearm skin elasticity in women. J Am Geriatr Soc. 2004;52:945–949. 24. Savvas M, Laurent G. Type III collagen content in the skin of postmenopausal women receiving estradiol and testosterone implants. Br J Obstet Gynaecol. Feb 1993;100:154–156.
Biological Effects of Estrogen on Skin 25. Lee JY, Maibach HI. Corticosteroid skin atrophogenicity: assessment methods. Skin Res Technol. 1998;4:161–166. 26. Schmidt J, Binder M, Demschik G, et al. Treatment of skin aging with topical estrogens. Int J Dermatol. 1996;35:669–674. 27. Dunn L, Damesyn M, Moore A, et al. Does estrogen prevent skin aging? Results from the First National Health and Nutritional Examination Survey. Arch Dermatol. 1997;133:339–342. 28. Grosman N, Hridbey E, Schon J. The effect of estrogenic treatment on the acid mucopolysaccharide pattern in the skin of mice. Acta Pharmacol Toxicol. 1971;30:458–464. 29. Grosman N. Studies on the hyaluronic acid protein complex the molecular size of hyaluronic acid and the exchangeability of chloride in skin of mice before and after estrogen treatment. Acta Pharmacol Toxicol. 1973;33:201–208. 30. Pochi PE, Strauss JS, Downing D. Age related changes in sebaceous gland activity. J Invest Dermatol. 1979;73:108–111. 31. Pierard-Franchimont C, Letawe C, Goffin V, et al. Skin waterholding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 32. Paquet F, Pierard-Franchimont C, Fuman I, et al. Sensitive skin at menopause; dew point and electrometric properties of the stratum corneum. Maturitas. 1998;28:221–227. 33. Sumino H, Ichikawa S, Abe M, et al. Effects of aging and postmenopausal hypoestrogenism on skin elasticity and bone mineral density in Japanese women. Endocrine J. 2004;51:159–164. 34. Contet-Audonneau JL, Jeanmaire C, Pauly G. A histological study of human wrinkle structures: comparison between sun-exposed areas of the face, with or without wrinkles, and sun-protected areas. Br J Dermatol. 1999;140:1038–1047. 35. Bosset S, Barre P, Chalon A, et al. Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol. 2002;12(3):247–252. 36. Creidi P, Faivre B, Agache P, et al. Effect of a conjugated estrogen (Premarin) cream on ageing facial skin. A comparative study with a placebo cream. Maturitas. 1994;19:211–223. 37. Punnonen R, Vaajalahti P, Teisala K. Local estriol treatment improves the structure of elastic fibers in the skin of postmenopausal women. Ann Chir Gynaecol. 1987;202(Suppl):39–41. 38. Castelo-Branco C, Figueras F, Martinez de Osaba M, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 39. Brincat MP, Muscat Baron Y, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123.
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40. Ashcroft GS, Dodsworth J, van Boxtel E, et al. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-b1 levels. Nat Med. 1997;3:1209–1215. 41. Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson MW. Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response. Am J Pathol. 1999;155:1137–1146. 42. Ashcroft GS, Ashworth JJ. Potential role of estrogens in wound healing. Am J Clin Dermatol. 2003;4(11):737–743. 43. Whitby DJ, Ferguson MWJ. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol. 1991;147:207–215. 44. Shah M, Foreman DM, Ferguson MWJ. Control of scarring in adult wounds by neutralizing antibody to transforming growth factor B. Lancet. 1992;339:213–214. 45. Hu D, Hughes MA, Cherry GW. Topical tamoxifen – a potential therapeutic regime in treating excessive dermal scarring? Br J Plast Surg. Sept 1998;51(6):462–469. 46. Surazynski A, Jarzabek K, Haczynski J, Laudanski P, Palka J, Wolczynski S. Differential effects of estradiol and raloxifene on collagen biosynthesis in cultured human skin fibroblasts. Int J Mol Med. 2003;12:803–809. 47. Osborne K, Zhao HH, Fuqua SAW. Selective estrogen receptor modulators: structure, function, and clinical use. J Clin Oncol. 2000;18:3172–3186. 48. Hall GK, Phillips TJ. Skin and hormone therapy. Clin Obstet Gynecol. 2004;47(2):437–449. 49. Kanda N, Watanabe S. Regulatory roles of sex hormones in cutaneous biology and immunology. J Dermatol Sci. 2005;38(1):1–7. 50. Schmidt JB. Perspectives of estrogen treatment in skin aging. Exp Dermatol. 2005;14(2):156. 51. Thornton MJ. Estrogen functions in skin and skin appendages. Expert Opin Ther Targets. 2005;9(3):617–629. 52. Lynfield YL. Effect of pregnancy on the human hair cycle. J Invest Dermatol. 1960;35:323–327. 53. Whiting DA. Diagnosis of Alopecia. Current Concepts. Kalamazoo: A Scope Publication, The Upjohn, 1990. 54. Burger H. Hormone replacement therapy in the post-Women’s Health Initiative era. Report of a meeting held in Funchal, Madeira, February 24–25. Climacteric. 2003;6(Suppl 1):11–36.
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40 Biology of Stratum Corneum: Tape Stripping and Protein Quantification Ali Alikhan . Howard I. Maibach
Introduction Stratum corneum (SC) adhesive tape stripping has been utilized in the measurement of stratum corneum mass, barrier function, drug reservoir, and percutaneous penetration of topical substances. The process involves a methodical, relatively noninvasive layer-by-layer removal of the SC, which comprises the outermost epidermal cell layers. Complete SC removal may require over 70 tape strips [1, 2]. The quantity of SC harvested diminishes with each sequential strip, possibly due to increased SC cohesiveness in deeper layers. Thus, the mass of any single strip depends on the mass removed by the prior strip [3]. SC removal may rely on the interaction between the adhesive stripping force and the cohesive intercellular force [3].
Tape Stripping Studies Tape stripping was first devised in the 1940s, and examined by Pinkus in 1951. Pinkus demonstrated a remarkable burst of mitotic epidermal activity post-stripping, concluding that the lost horny layer is replaced by basal mitotic division [4]. The degree of hyperplasia correlates with the level and duration of barrier disruption [5]. Nevertheless, mitotic rate may remain five times greater than baseline six days after stripping [6]. Keratinocyte hyperproliferation may be a response to water barrier disruption or cytokine release secondary to epidermal injury [5, 6]. Adhesive stripping increases: epidermal lipid synthesis, lamellar body production/secretion in the stratum granulosum, epidermal DNA synthesis, epidermal cytokine production, dermal inflammation, and presence of TNF and IL-1a in skin [6]. Conversely, occlusion of stripped human skin via adhesive application suppresses mitotic activity; adhesive occlusion may provide artificial restoration of the lost barrier [6]. Similar experiments in mice do not support these findings [6]. The SC is essential to life, protecting the human body from desiccation and external penetration of deleterious agents. The SC is composed of a nucleated, keratin-rich
corneocytes embedded in an extracellular multilamellar lipid matrix organized into membrane-like bilayers; intercorneocyte communication occurs through desmosomes [7]. Many other SC models exist, but none of them fully integrate all aspects of the skin barrier function. The SC is thin, less than 20 mm thick, and composed of about 10–15 tightly stacked layers, depending on the location [8]. Ceramides, cholesterol, and free fatty acids comprise the lipid matrix of the SC, providing invaluable roles in the barrier structure and function [7]. Their synthesis is required for barrier homeostasis; as with DNA, a burst of lipid synthesis (due to synthesis of their rate-limiting enzymes) occurs following barrier perturbation [9]. Lipid levels decrease in aged human skin, possibly due to SC pH increases and subsequent lipid processing impairment; this is described further, below [7]. The SC provides the rate determining step for the passage of most molecules across skin [10]. Therefore, topical agent concentration within the SC is directly related to that in the epidermis and dermis, the typical target sites. Additionally, corneocytes and intercellular lipids are responsible for preventing insensible water loss [11]. The transepidermal water loss can be measured with an evaporimeter, and frequently used to assess skin barrier integrity [11]. Anatomically, regional SC variations in percutaneous drug absorption, lipid composition, TEWL measurements, mean thickness, and number of cell layers have been described. Despite its structural heterogeneity, each layer of SC equally contributes in preventing water loss [11]. In doing so, the SC behaves as a membrane compatible with Fick’s laws of passive diffusion [11]. TEWL increase as a function of tape strip number depends on the factors including: anatomical site, pressure, pressure duration, and tape removal rate [12]. Loffler et al. demonstrated that TEWL increased fastest on the forehead, followed by the back, and finally, the forearm [12]. These findings may be explained by the differences in SC thickness, differences in spontaneous desquamation (SC cohesion), and pressure resistance because of inherent viscoelasticity and type of tissue underlying the skin [12]. Rapid removal (vs. slow),
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_40, # Springer-Verlag Berlin Heidelberg 2010
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Biology of Stratum Corneum: Tape Stripping and Protein Quantification
shorter pressure duration (2 vs. 10 s), and higher pressure (330 g cm 2 vs. 165 cm 2) all produced earlier TEWL increases [12]. A similar study by Breternitz et al. revealed the highest rise of TEWL on the cheek, compared to the back, upper arm, and forearm [13]. Interestingly, the cheek also demonstrated the greatest increase in SC hydration after stripping [13]. Breternitz et al. further established greater, earlier TEWL increase with higher pressure (7 N stamp vs. 2 N) and longer application (10 vs. 2 s) [13]. Moreover, using the thumb, stretching the skin, and utilizing a roller or stamp all result in varying quantities of harvested SC [13]. The use of thumb removed most of SC and produced the highest TEWL, even when compared with usage of a roller or skin stretching [13]. Occlusion of the test site prior to the stripping procedure resulted in higher TEWL values [13]. Occlusion results in water retention and degradation of the intercellular proteins [13]. In conclusion, reliable, reproducible results depend on standardization of the aforementioned variables. Kalia et al. found that initial tape strips removed thicker layers of the SC, relating this to decreased number of desmosomes closer to the skin surface [10]. Kalia et al. demonstrated decreased impedance with increasing depth achieved, theorizing that removal of the upper corneocyte layers and lipid matrix diminishes structural opposition to ion flow, facilitating ion transport [10]. In addition, TEWL increased disproportionally with later tape strips; removing only the upper SC layers was insufficient to significantly enhance the water loss [10]. Removal of 6–8 mm of SC (deeper layers) typically resulted in significant TEWL increases [10]. Removal of the outermost layers affected impedance more than TEWL, with a 40% decrease in impedance after removal of only 4 mm of SC. Nonetheless, a correlation between TEWL increase and impedance decrease was observed. Upon completion of the tape stripping experiment, full return to the basal values of impedance occurred after 3 days, while TEWL recovery time was 5–6 days [10]. External layers, more crucial in impedance, are formed prior to deep compact layers [10]. The aforementioned findings suggest a gradation in water-regulating ability within the SC, with the deepest layers most responsible for controlling water flux [10]. However, via simple mathematical deduction, these results, in fact, support a Fickian model [10]. Though structurally heterogeneous and complex, the SC behaves as a homogenous barrier to water in vivo [10]. The water transport route may be homogeneous throughout SC, with each layer contributing equally to the barrier [10]. The best fit curve plotting experimental values of TEWL
as a function of tape stripping frequency closely resembled a theoretical curve based on Fick’s first law of diffusion [14]. The first half of the theoretical curve fit the actual curve; in the second half, experimental data show slightly higher TEWL values than Fick’s theoretical values [14]. The authors of the study offer plausible explanations for this discrepancy [14]. In contrast to most studies, Schwindt et al. demonstrated that quantity of harvested SC was constant with each strip in a given anatomical site and volunteer [11]. Schwindt et al. found a linear relationship (in all anatomical sites) between 1/TEWL and the total mass of removed SC, further establishing that the SC acts as a Fickian membrane for steady state water diffusion [11]. It also appears that intercellular lipids, not corneocytes, are the determining factor for SC water diffusion [11]. This linear relationship was also described by another group, plotting 1/TEWL as a function of SC thickness (13 subjects examined) [15]. > Table 40.1 summarizes the results from three studies quantifying SC thickness. Tape construction influences outcome [13]. Three brands of adhesive tapes, utilized in vivo, displayed statistically equivalent mean water diffusion coefficients, SC permeability, and SC mass/thickness removal [3]. After 40 strips, however, a proprietary adhesive stripped the most, while a rayon adhesive stripped the least [3]. TEWL increased significantly as deeper SC layers were reached with proprietary and polyethylene adhesives, but not with rayon tape [3]. Tape properties, subject properties, or a combination may account for variation
. Table 40.1 Calculations of SC thickness in vivo in man
Authors
No. of Anatomical subjects site
No. of strips
Mean total SC thickness (mm)
Kalia et al. [10]
3
Forearm
22–28
12.7 3.3
Schwindt et al. [11]
6
Lower back
Up to 35
11.2
Pirot et al. [15]
13
Abdomen
Up to 35
7.7
Thigh
Up to 35
13.1
Forearm (ventral)
Up to 35
12.3 3.5
Forearm (ventral)
15
12.6 5.3
Thickness appears to be a function of anatomical site
Biology of Stratum Corneum: Tape Stripping and Protein Quantification
in barrier disruptive properties. Variation may also be accounted for by unique adhesive systems; adhesives of different tape brands may bind similarly to cellular SC, but differently to extracellular components of the SC barrier. These extracellular components (e.g., free fatty acids, ceramides, and lipids) are essential to barrier function. Furthermore, apparently 5–7 mm of SC removal resulted in significant TEWL elevations, a depth unobtainable by the rayon tape (> Table 40.2) [3]. This implies that structural elements of the water barrier may not be homogeneously distributed. In some subjects, neither the proprietary adhesive nor the polyethylene adhesive disrupted the water barrier; these individuals experienced no barrier disruption at any of six tested sites, suggesting variation of water barrier disruption to be a function of the individual. Demonstrating that removal of the same amount of SC from different individuals does not result in similar increases in TEWL, Kalia et al. asked whether this variation was secondary to inter-individual differences in intact membrane thickness [16]. Kalia et al. demonstrated that once inter-individual differences in the thickness of the intact SC are corrected for (by normalizing the SC thickness removed with respect to calculated total SC thickness), the same degree of barrier disruption induces the same increase in TEWL in each individual [16]. Stated differently, removal of the same percentage of SC in two individuals results in equivalent barrier disruption. TEWL rises considerably only after about 75% of the SC has been removed, presenting a very consistent barrier to water loss in the healthy human population [16].
. Table 40.2 Relationship between protein removal and TEWL, from Bashir et al. [3]
Tape type Proprietary
No. Location of (forearm) strips
TEWL (g m 1 h 1) 30.33
Dorsal
40
8.10
Ventral
40
5.83
30.80
40
7.25
31.98
Ventral
40
4.96
30.83
Dorsal
40
4.99
13.4
Ventral
40
2.99
11.95
Polyethylene Dorsal Rayon
Mean thickness removed (mg)
Note, there are significant differences in TEWL and mean thickness removed depending on tape construction. The dorsal forearm, in all cases, had greater SC thickness removed than the ventral forearm
40
Tape Stripping and Aging Aged skin demonstrates increased susceptibility to the xerosis, exogenous, and environmental insults, and diminished ability to recover from these insults, indicating a suboptimal epidermal barrier. It is believed that no definitive studies have compared aged vs. normal SC thickness; nonetheless, some authors believe aged SC to be thicker, with decreased lipid content and deficient water-binding capacity [8]. TEWL is decreased in the aged, as is topical absorption [8]. The aging barrier was elegantly examined by Ghadially et al.; results are summarized below. Aged humans (>80 years) have prolonged barrier recovery rates after tape stripping or acetone application compared to control subjects (20–30 years) [17]. 24 h after acetone treatment, 50% recovery occurred in control subjects compared to 15% in aged subjects [17]. Photoaging, in combination with this chronologic aging, may further delay recovery [9]. Furthermore, delays in SC lipid reappearance after barrier disruption have been described in aged murine epidermis [17]. Additionally, tape stripping studies have revealed decreased cohesiveness in aged skin [9]. In fact, barrier perturbation (TEWL 20 g m 2 h 1) occurred after 18 2 strippings in aged skin versus 31 5 strippings in control skin [17]. Fortunately, topical lipid formulations, containing predominantly cholesterol, may accelerate barrier recovery in aged human skin [18]. The above findings may be explained by reduced delivery of secreted lipids to the epidermal surface in the elderly. There is a global diminution ( 30%) of ceramide, cholesterol, and free fatty acid contents in the aged murine skin [17]. This reduction could be due to the decreased production and/or increased destruction; cytokines (e.g., IL-1a) and growth factors may play a role [9]. Additionally, decreased secretion of lamellar body contents (at stratum granulosum-stratum corneum interface) with fewer extracellular lamellar bilayers (at stratum corneum interstices) contributes to a more porous extracellular SC matrix [17]. Ghadially et al. further examined the effect of lipids on SC barrier function [19]. As described previously, SC of aged mice displays decreased lipid content and extracellular bilayers. This may result in impaired barrier recovery after a tape stripping insult (18.7 vs. 60.8% recovery by 24 h in aged vs. young mice). Upon further examination, Ghadially et al. determined that cholesterol synthesis is decreased significantly under basal conditions. Furthermore, sterologenesis fails to reach absolute levels obtained in young epidermis following tape stripping perturbation.
403
404
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Biology of Stratum Corneum: Tape Stripping and Protein Quantification
A 40% decrease in activity of HMG-CoA reductase, the rate-limiting enzyme in sterologenesis, was observed under basal conditions in aged mice. Despite a greater than 100% increase in HMG-CoA reductase activity after barrier perturbation in aged mice, absolute levels did not attain those reached in treated, young epidermis. Ghadially et al. also supplemented aged murine SC with an equimolar mixture of SC physiological lipids (cholesterol:ceramide:linoleic acid:palmitic acid) or cholesterol alone [19]. Either mixture applied once enhanced the recovery after barrier disruption. Additionally, after four applications of either mixture, electron microscopy demonstrated repletion of extracellular spaces with normal lamellar bilayer structures. Further work examining the role of aging on the SC remains to be done. Tape stripping and TEWL studies of aged skin are currently underway.
Protein Quantification After harvesting of SC onto adhesives is complete, protein can be measured via several methods. For decades, weighing (gravimetry) was the preferred method, despite its inherent inconvenience (weighing before and after stripping under constant hydration conditions). Additionally, results were subjected to inflation secondary to absorption of exogenous (topically applied) or endogenous (sebum, sweat, and interstitial fluid) substances within the SC. Initial strips were most affected by this absorption. One decade ago, a novel colorimetric method was developed and validated by Dreher et al. [20]. This colorimetric method relies on a protein assay similar to one developed by Lowry et al. over half a century ago. Lowry’s method involved measurement of protein with a folin phenol reagent after alkaline copper treatment [21]. It was demonstrated to be simple, sensitive, specific, and easily adaptable to small scale analyses, making it suitable for measurement of miniscule absolute protein amounts [21]. Dreher’s method relies on spectrophotometry and colorimetry, based on the calibration of stained SC proteins to the corneocyte mass [22]. Drawbacks include time-consuming preparation of tape strips with necessary destruction of the original strips. The Bradford dye reaction, which relies on Coomassie Brilliant Blue G-250 dye, is similar to Dreher’s method. The dye binds protein, resulting in ionic and hydrophobic reactions, with a spectral shift from reddish-brown to blue. Maximal absorption for the bound form of the dye is 595 nm, the optimal wavelength for colorimetric measurement once the reaction has occurred. Despite
disadvantages (e.g., serial dilutions), it is a fast and generally reliable method for protein quantification. Dreher’s colorimetric method has been successfully adapted to 96-well microplates, effectively shortening analysis time [23]. Note that limited areas of adhesive tape are not predictive of SC removal on the entire tape [23]. Alternatively stated, SC distribution on tape is not homogeneous [23]. A pivotal study examined direct spectroscopic SC protein quantification via absorption in the visible range (595 and 600 nm), with and without staining of corneocyte aggregates, and the UV range (278 nm) [24]. Correlation coefficients R2 were 0.71 and 0.74, respectively. The results demonstrated weak SC protein absorption with immense light scattering [24]. The Coomassie brilliant blue protein coloring did not increase light absorption by SC proteins, and thus, could not decrease the interference secondary to light scattering [24]. The absorption techniques utilized in this study cannot accurately predict corneocyte aggregate quantity. Latter studies utilizing wavelengths of 430 nm have established optical spectroscopy in the visible range as a sensitive and reproducible method of protein quantification [2]. Absorbance in this range depends exclusively on quantity of corneocyte aggregates, and adequately reflects SC mass [2]. Corneocyte aggregates, adhering to tape strips, decrease transmission of visible light by scattering, reflection, and diffraction. The resulting pseudoabsorption has been successfully correlated with mass of removed SC particles [2]. Absorbance measurement allows facile determination of absolute mass from corneocyte aggregates harvested via tape stripping. Topically applied substances do not interfere with the spectroscopic measurements as they do with gravimetric measurements, explaining mass differences in the most superficial strips (when compared with gravimetry) [25]. Practically comparing spectrally measured quantity (absorbance) with corneocyte aggregate weight requires correction for: topical applications in upper SC layers, interstitial fluid in deeper SC layers, the ‘‘stack effect’’ which decreases absorbance, and the tape stripping procedure itself (e.g., nonhomogeneous removal of tape or incomplete tape contact with skin) [2]. Once these factors are corrected for (primarily by excluding analysis of the most superficial and deep strips), R2 = 0.93, demonstrating proportionality between quantification methods [2]. A multicenter study involving 24 subjects found a correlation coefficient of R2 = 0.94 when comparing UV/VIS spectroscopy (430 nm) with conventional weight determination [25]. Superficial (first five) and deep (19–23) strips were excluded on the basis of weight-enhancement;
Biology of Stratum Corneum: Tape Stripping and Protein Quantification
application on an oil-water emulsion (part of the study) inflated superficial strip weight, and intrinsic interstitial fluid increased deep strip weight [25]. Nonhomogeneous strips and those subjected to handling errors were excluded [25]. Only 66% of total strips were utilized to determine the correlation coefficient [25]. Weigmann et al. explain that pseudo-absorption/weight correlation can be extrapolated to the deepest layers of the SC [25]. A recent study demonstrated strong correlation (R2 = 0.92 and R2 = 0.95) between pseudo-absorption at 430 nm and both protein absorption at 278 nm and absorption of Trypan blue-stained proteins at 652 nm [22]. However, protein absorption at 278 nm was characterized by a weak band, implying application limited to tape strips with high amounts of corneocytes [22]. Mass determination based on the UV absorption is further limited by the potential superpositioning of strong absorption bands from exogenous substances and/or tape components in the same spectral range. Unlike the previous study, correlation was described using all the tape strips (superficial and deep), regardless of adherent exogenous or endogenous components [22]. Lademann et al. tested an inexpensive, easily reproducible optical device (‘‘corneocyte density analyzer’’), based on a slide projector, which also measures corneocyte pseudoabsorption at 430 nm [1]. When compared with standard UV-visible spectrometric measurements, a correlation factor of R2 = 0.95 was demonstrated [1]. The device may simplify calculation of removed SC, without messy chemistry or an expensive spectrometer; it includes a mechanical autofeed system, well suited for the handling of tape strips [1].
Colorimetric Bioassay of Keratolytic Efficacy The desquamating effects of three keratolytics are presented in table-format (> Table 40.3) using the data
40
obtained from colorimetric protein assays described by Dreher et al. (mentioned above) [20]. The process begins with cutaneous application of the agent; the agent is placed on a patch and taped onto the subject’s skin for a predetermined number of hours. After this period, placement and removal of tape strips (number varies by study) onto the site of topical treatment are performed. The assay involves immersion and shaking of SC adhering tapes in sodium hydroxide solution resulting in extraction of the soluble SC protein fraction. The solution, now containing SC protein, is neutralized with hydrogen chloride, as the assay is ineffective under strongly alkaline conditions. The protein assay is performed using the Bio-Rad Detergent Compatible Protein Assay Kit and following the prescribed microassay procedure. This assay is similar to the Lowry assay, and is based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent. Finally, absorbance at 750 nm is measured using a Hitachi U-2001 UV-vis Spectrophotometer. This method allows for quantification of microgram amounts of SC, diminishing confounding factors, namely vehicle and water uptake by the SC. The protein measured using the assay described can be compared amongst groups, with statistical analysis allowing determination of strong and weak keratolytics. SC removal via tape stripping in treatment and control groups is attributable to keratolytic mechanisms, which loosen SC cohesion. The disintegrated SC is subsequently collected by the adhesive. In the first keratolytic bioassay using this technique, salicylic acid was examined [26]. Keratolytic efficacy of salicylic acid was determined as a function of pH. The test preparations were: aqueous vehicle control of pH 7.4, 2% SA aqueous solution of pH 3.3, 2% SA aqueous solution of pH 3.3 with menthol, and 2% SA aqueous solution of pH 6.95 [26]. A statistically significant mass of SC was removed after 6 h and 20 tape strips in all three
. Table 40.3 Studies using colorimetric protein assay to measure keratolytic potential Authors
Drug
Result
Bashir et al. (2005)
Aqueous solution 2% Salicylic Acid – 3 formulations
Statistically significant mass of SC removed after 6 h and 20 tape strips in all three experimental groups (salicylic acid pH 3.3, salicylic acid pH 3.3 w/ menthol, salicylic acid pH 6.95) compared to vehicle, untreated, and untreated but occluded groups.
Waller et al. (2006)
Aqueous solutions of 0.05 % all-trans RA, 2% BPO, and 2% SA
Statistically significant mass of SC removed after 6 h and 25 tape strips in all three experimental groups compare to vehicle, untreated, and occluded groups. The first 10 tape strips from SA group removed more protein than the other groups; at 10–15 strips, treatments were comparable; at 16–25 strips, protein removed from BPO sites was greatest.
All agents tested demonstrated significant efficacy in SC removal. SA had superior superficial removal, while BP had superior deep removal
405
406
40
Biology of Stratum Corneum: Tape Stripping and Protein Quantification
experimental groups compared to vehicle, untreated, and untreated but occluded groups [26]. However, after 10 strips, the SA pH 3.3 solution with menthol and the SA pH 6.95 solution removed significantly more SC than any other group, including the SA pH 3.3 solution [26]. These data suggest that a neutral preparation of SA results in a pronounced keratolytic effect. Moreover, the neutral preparation was associated with the least skin irritation among treatment groups [26]. This finding differs from that of a previous study, which demonstrated superior SA skin penetration in an acidic solution compared to neutral solution [27]. In the second bioassay using the aforementioned technique, salicylic acid, benzoyl peroxide (BPO), and retinoic acid were examined [28]. The test preparations were: 0.05% all-trans retinoic acid, 2% salicylic acid at pH 6.95, 2% BPO, vehicle, untreated skin, and occluded but untreated skin [28]. After 3 h of treatment, only BPO treatment removed significantly more SC on 25 strips than untreated skin, while the other treatments did not achieve statistical significance [28]. At 3 h, SA had greater SC amounts removed in the first 10 (superficial) strips, while deeper strips (11–25) demonstrated BPO to have the greatest SC removal [28]. Statistically significant masses of SC were removed after 6h and 25 tape strips in all three experimental groups when compared to vehicle, untreated, and occluded groups [28]. At 6 h, the first 10 tape strips from the SA group removed more protein than the other groups; at 10–15 strips, all treatments were comparable; at 16–25 strips, BPO removed the most protein [28]. These in vivo human results indicate that all treatments tested are effective keratolytics, which may account for their effectiveness against acne vulgaris. Furthermore, it appears that salicylic acid may be a more suitable treatment for mild, superficial acne while BPO may be optimal for deeper, inflammatory acne. BPO’s ability to loosen SC at deeper levels complements its antimicrobial/ anti-inflammatory properties, resulting in an effective anti-inflammatory agent for papulo-pustular acne. Additionally, BPO appears to be effective even with short-term administration. RA had inferior SC disruption at 3 h but significant disruption at 6 h, indicating time-dependent keratolytic effects, consistent with its complex nuclear receptor interactions and alteration of gene transcription.
Conclusion Taken together, the SC is beginning to reveal some of its secrets. Much remains to be done.
Cross-references > Corneocyte
Size and Cell Renewal: Effects of Aging and Sex Harmones > Stratum Corneum Cell Layers > The Stratum Corneum and Aging
References 1. Lademann J, Ilgevicius A, Zurbau O, Liess HD, Schanzer S, Weigmann HJ, Antoniou C, Pelchrzim RV, Sterry W. Penetration studies of topically applied substances: optical determination of the amount of stratum corneum removed by tape stripping. J Biomed Opt. 2006; 11(5):054026. 2. Weigmann H, Lademann J, Meffert H, Schaefer H, Sterry W. Determination of the horny layer profile by tape stripping in combination with optical spectroscopy in the visible range as a prerequisite to quantify percutaneous absorption. Skin Pharmacol Appl Skin Physiol. 1999;12(1–2):34–45. 3. Bashir SJ, Chew AL, Anigbogu A, Dreher F, Maibach HI. Physical and physiological effects of stratum corneum tape stripping. Skin Res Technol. 2001;7(1):40–48. 4. Pinkus, H. Examination of the epidermis by the strip method of removing horny layers. I. Observations on thickness of the horny layer, and on mitotic activity after stripping. J Invest Dermatol. 1951;16(6):383–386. 5. Denda M, Wood LC, Emami S, Calhoun C, Brown BE, Elias PM, Feingold KR. The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss. Arch Dermatol Res. 1996;288 (5–6):230–238. 6. Fisher LB, Maibach HI. Physical occlusion controlling epidermal mitosis. J Invest Dermatol. 1972;59(1):106–108. 7. Jungersted JM, Hellgren LI, Jemec GB, Agner T. Lipids and skin barrier function – a clinical perspective. Contact Dermatitis. 2008;58(5):255–262. 8. Tagami H. Functional characteristics of the stratum corneum in photoaged skin in comparison with those found in intrinsic aging. Arch Dermatol Res. 2008;300(Suppl 1):1–6. 9. Elias PM, Ghadially R. The aged epidermal permeability barrier: basis for functional abnormalities. Clin Geriatr Med. 2002;18 (1):103–120, vii. 10. Kalia YN, Pirot F, Guy RH. Homogeneous transport in a heterogeneous membrane: water diffusion across human stratum corneum in vivo. Biophys J. 1996;71(5):2692–2700. 11. Schwindt DA, Wilhelm KP, Maibach HI. Water diffusion characteristics of human stratum corneum at different anatomical sites in vivo. J Invest Dermatol. 1998;111(3):385–389. 12. Loffler H, Dreher F, Maibach HI. Stratum corneum adhesive tape stripping: influence of anatomical site, application pressure, duration and removal. Br J Dermatol. 2004;151(4):746–752. 13. Breternitz M, Flach M, Prassler J, Elsner P, Fluhr JW. Acute barrier disruption by adhesive tapes is influenced by pressure, time and anatomical location: integrity and cohesion assessed by sequential tape stripping. A randomized, controlled study. Br J Dermatol. 2007;156(2):231–240.
Biology of Stratum Corneum: Tape Stripping and Protein Quantification 14. van der Valk PG, Maibach HI. A functional study of the skin barrier to evaporative water loss by means of repeated cellophane-tape stripping. Clin Exp Dermatol. 1990;15(3):180–182. 15. Pirot F, Berardesca E, Kalia YN, Singh M, Maibach HI, Guy RH. Stratum corneum thickness and apparent water diffusivity: facile and noninvasive quantitation in vivo. Pharm Res. 1998;15 (3):492–494. 16. Kalia YN, Alberti I, Sekkat N, Curdy C, Naik A, Guy RH. Normalization of stratum corneum barrier function and transepidermal water loss in vivo. Pharm Res. 2000;17(9):1148–1150. 17. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95(5):2281–2290. 18. Zettersten EM, et al. Optimal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin. J Am Acad Dermatol. 1997;37(3 Pt 1):403–408. 19. Ghadially R, Brown BE, Hanley K, Reed JT, Feingold KR, Elias PM. Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice. J Invest Dermatol. 1996;106(5):1064–1069. 20. Dreher F, Arens A, Hostynek JJ, Mudumba S, Ademola J, Maibach HI. Colorimetric method for quantifying human Stratum corneum removed by adhesive-tape stripping. Acta Derm Venereol. 1998;78 (3):186–189. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193 (1):265–275.
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22. Lindemann U, Weigmann HJ, Schaefer H, Sterry W, Lademann J. Evaluation of the pseudo-absorption method to quantify human stratum corneum removed by tape stripping using protein absorption. Skin Pharmacol Appl Skin Physiol. 2003;16(4):228–236. 23. Dreher F, Modjtahedi BS, Modjtahedi SP, Maibach HI. Quantification of stratum corneum removal by adhesive tape stripping by total protein assay in 96-well microplates. Skin Res Technol. 2005;11 (2):97–101. 24. Marttin E, Neelissen-Subnel MT, De Haan FH, Bodde HE. A critical comparison of methods to quantify stratum corneum removed by tape stripping. Skin Pharmacol. 1996;9(1):69–77. 25. Weigmann HJ. UV/VIS absorbance allows rapid, accurate, and reproducible mass determination of corneocytes removed by tape stripping. Skin Pharmacol Appl Skin Physiol. 2003;16(4):217–227. 26. Bashir SJ, Dreher F, Chew AL, Zhai H, Levin C, Stern R, Maibach HI. Cutaneous bioassay of salicylic acid as a keratolytic. Int J Pharm. 2005;292(1–2):187–194. 27. Leveque N, Makki S, Hadgraft J, Humbert P. Comparison of Franz cells and microdialysis for assessing salicylic acid penetration through human skin. Int J Pharm. 2004;269(2):323–328. 28. Waller JM, Dreher F, Behnam S, Ford C, Lee C, Tiet T, Weinstein GD, Maibach HI. ‘‘Keratolytic’’ properties of benzoyl peroxide and retinoic acid resemble salicylic acid in man. Skin Pharmacol Physiol. 2006;19(5):283–289.
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14 Buffering Capacity Considerations in the Elderly Jacquelyn Levin . Howard I. Maibach
Introduction The acidic character of skin was first mentioned by Heuss [1] and later by Schade and Marchionini [2] who introduced the term ‘‘acid mantle’’ for skin’s acidic outer surface pH. It has been recognized as playing a crucial role in permeability barrier homeostasis, skin integrity/cohesion and immune function [3–5]. Given this, it is important for skin to be able to resist acidic/alkaline aggression to some extent (i.e., have buffering capacity) [6]. The pH of skin increases and the ability to buffer the change in skin pH decreases with age [7]. This increase in pH and decreased buffering capacity in elderly skin results in impaired barrier homeostasis and skin integrity/cohesion, increased likelihood for skin infection, and increased sensitivity/irritation to topically applied products [8]. The chapter briefly reviews the basic science of pH and buffering capacity and the deleterious effects of increased pH in the skin of the elderly. The decrease in buffering capacity in elderly skin will be discussed, firstly by discerning which components of the stratum corneum are most likely responsible for the buffering capacity in skin of all ages and secondly by reviewing the physiologic changes of the stratum corneum that may contribute to the decrease in the buffering capacity detected clinically in elderly skin.
Defining and Measuring the pH and Buffering Capacity of Skin When dilute aqueous acid or alkaline solutions come into contact with skin, the change in pH is generally temporary and the original skin pH (a measure of the hydronium ion concentration) is rapidly restored, indicating that skin has significant buffering capacity. A buffer is a chemical system that can limit changes in pH when an acid or a base is added. Buffer solutions consist of a weak acid and its conjugate base. The system has its optimum buffering capacity when about 50% of
buffer is dissociated, or in other words at a pH approximately equal to its pKa [6, 9]. The pKa is the negative of the common logarithm of the acid dissociation constant (Ka) and is a measure for the strength of the acid. The buffer capacity is further dependent on the concentration of the system. An acid/alkali aggression test is one way to measure the acid/alkali resistance (i.e., buffering capacity) of skin. Alkali/Acidic resistance tests were commonly used in the 1960s to detect workers that may likely develop occupational diseases in certain chemical work environments [6]. A mild variation of the alkali/acidic resistance tests, also called acid/alkali neutralization test, assesses how quickly the skin is able to buffer applied acids/bases without the occurrence of skin corrosion. Repetitive applications of acid or base demonstrate that the skin’s buffering capacity is limited and may be overcome, as illustrated by the long time required for neutralization [10–13].
Effect of Increase in pH on Elderly Skin Function and Defenses In a multicenter study on measurement of the natural pH of the skin surface, the values of skin surface pH were 4.9 (arithmetic mean) with a 95% confidence interval between 4.1 and 5.8 [9]. Ideal acidity for the stratum corneum is a pH of approximately 5.4 [14]. It is well known that an increased skin pH is detected in the elderly skin starting anywhere from age 50 to 80 years [14–18]. Most likely this decreased acidity is due to less efficient mechanisms for skin acidification and more specifically decreased NA + /H + antiporter (NHE1) expression. The NHE1 is one of three highly studied mechanisms for maintaining skin acidity and is assumed to be the predominate mechanism for maintaining skin acidity [18]. Elevation of the skin pH in the elderly alters multiple functions. Those discussed here include impairment of permeability barrier homeostasis, decreased skin integrity/cohesion, and increased susceptibility for microbial infection.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_14, # Springer-Verlag Berlin Heidelberg 2010
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Buffering Capacity Considerations in the Elderly
Impaired Permeability Barrier Homeostasis An acidic pH is critical for permeability barrier homeostasis, in part because of two key lipid-processing enzymes, B-glucocerebrosidase and acid sphingomyelinase which generate a family of ceramides from glucosylceramide and sphingomyelin precursors and exhibit low pH optima [19]. An increased skin pH results in defective lipid processing and delayed maturation of lamellar membranes [18]. These lipids form multi-lamellar sheets amidst the intracellular spaces of the stratum corneum critical to the stratum corneum’s mechanical and cohesive properties, enabling it to function as an effective water barrier [18]. This delayed barrier function allows easier penetration of topically applied products and delays barrier recovery after injury or insult to the skin [18, 19].
Decreased Skin Integrity and Cohesion An acidic pH also clearly promotes stratum corneum integrity and cohesion. In a neutral pH environment there is an enhanced tendency for the stratum corneum to be removed by tape stripping (integrity) as well as increased amount of protein removed per stripping (cohesion) [18, 20]. The impaired stratum corneum integrity/cohesion is due to pH dependent activation of serine proteases which exhibit neutral pH optima [21]. Serine proteases become activated in the increased pH of elderly skin and lead to the premature degradation of corneodesmosomes and hence increased desquamation [18, 19, 22].
Increased Susceptibility for Skin Infections The acidic pH of the stratum corneum restricts colonization by pathogenic flora and encourages persistence of normal microbial flora. Pertinently elderly skin, intertriginous areas, and chronically inflamed skin display an increased skin pH [2] and hence reduce resistance to pathogens [14]. In summary, elderly skin commonly has abnormalities in stratum corneum integrity/cohesion, permeability barrier homeostasis, and immune function due to increased skin pH. These abnormalities are attributable to the pH mediated increase in serine protease mediated degradation of corneodesmosomes, defect in lipid processing, and decrease in antibacterial activity, respectively.
Effect on Buffering Capacity of Skin in the Elderly and Elderly Skin Both the skin surface pH and reduced buffering capacity have been documented for skin of elderly. The reduced buffering capacity contributes to the increased sensitivity of skin to contact irritants and cleansing procedures [8]. The next section focuses on the aggression tests aimed at discerning which components of the epidermis are responsible for skin’s buffering capacity.
Free Fatty Acids/Sebum Early experimentation hypothesized that the sebum contributes to the buffering capacity of skin in two ways: first, it protects the epidermis against the influence of alkali by slowing down the exposure and penetration of acids or alkalis applied to the skin [23–25] and second, the fatty acids in sebum may act as buffer system [26, 27]. Later Lincke et al. [28] refuted the second hypothesis by demonstrating that the sebum had no relevant acid and a negligible alkali buffering capacity around pH 9. Further challenging the hypothesis, a quicker neutralization was observed on delipidized skin than untreated skin [23, 26]. Vermeer concluded similarly when comparing the neutralization on soles and forearm with and without sebum removal respectively [27]. However, when comparing these different skin regions, differences in sebum content may have also contributed to the observed effect. Vermeer [25] and Neuhaus [29] believed that the increased rate of neutralization after sebum removal may have been due to increased amounts of carbon dioxide (CO2) diffusion. This theory, discussed later in detail, is generally not accepted and also not clearly substantiated. After lipid removal, skin starts to increase acid production which may account for the faster neutralization. The same investigators also found that the increase in neutralization after lipid removal is temporary and limited to the first few minutes, which is probably related to the activity of sebaceous glands to produce relevant amounts of sebum. Due to the negligible buffering capacity of sebum and to standardize experimentation (limit inter- and intraindividual variability), today most neutralization experiments are performed after cleansing the skin with solvents which remove most of the sebum including fatty acids.
Buffering Capacity Considerations in the Elderly
Epidermal Water-Soluble Constituents
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Vermeer et al. [27] first demonstrated the importance of water-soluble constituents to the skin’s buffering ability. Water soaked skin, where the water-soluble constituents were extracted, demonstrated a significantly reduced neutralization capacity indicating that water-soluble substance constituent(s) of the skin are major contributors to the buffering capacity [12, 30, 31]. The significance of water-soluble constituents of the epidermis to the buffering capacity of skin further supports the theory of minimal contribution from the sebum of skin due to its lipid soluble nature [27].
in the water-soluble portion of the epidermis. However, the AA composition of keratin [41, 42] does not correspond with the composition of free AA found in the water-soluble portion of the stratum corneum [34], which implies that keratin is not a major contributor to the pool of free AA. Despite little evidence of keratin’s role in the buffering capacity, a modifying action of keratin is assumed [28]. Without an intact keratin layer, neither a physiological surface pH nor normal neutralization capacity can be maintained [43]. Further research remains to be conducted to determine keratin’s role in the buffering capacity of the epidermis.
Sweat
CO2
Eccrine sweat initially accelerates the neutralization of alkalis [10–13, 27, 30, 32, 33]. Spier and Pasher [34] suggest that the main buffering agents of sweat are lactic acid and amino acids. The lactic acid-lactate system in sweat has a highly efficient buffering capacity between pH 4 and 5 [24]. However, it has not been completely demonstrated that lactic acid is the main buffering agent in sweat or at the surface of the skin. Conversely, the contribution of amino acids (AA) to the buffering capacity of sweat and of the horny layer surface has been investigated thoroughly [27, 30, 33]. By comparing sweating and non-sweating persons, Vermeer [27] found that AA play a significant role in neutralization during the first 5 min while lactic acid does not. This confirms that AAs are key elements contributing to the buffering capacity of skin.
Little is known about the role of CO2/HCO3 participating in skin’s buffering capacity. Burckhardt’s studies were the first to suggest that the CO2 diffusing from the epidermal layer may be responsible for neutralizing alkali in contact with skin [10–13]. He demonstrated that when a 5 min alkali-neutralization experiment is repeated subsequently several times on the same skin area, the neutralization times became longer and finally reach an approximately constant time. He suggested that the shorter neutralization times at the beginning were due to acids present on the skin surface rapidly neutralizing the alkali. He further suggested that after successive alkali exposure, the endogenous acids were no longer present on the skin surface resulting in longer neutralization times and diffusing carbon dioxide would take over the role of neutralizing the alkali. At this time, Burckhardt’s hypothesis of the role of carbon dioxide as a buffering agent was accepted by others despite the rather weak experimental evidence [28, 36, 44, 45]. The decreased neutralization time after lipid removal of the skin surface with the help of soaps or neutral detergents was believed by Burckhardt and others to be the consequence of greater diffusion of CO2 although this has never been quantified [24, 44, 45]. It was also postulated that a more hydrated stratum corneum retains a greater amount of CO2 by limiting its diffusion. Therefore a moderate hydration level was regarded better for effective alkali neutralization; however, this has also never been analyzed in further detail [45]. Knowing that several authors considered CO2 a relevant contributor in alkali neutralization without having quantitative data to sustain their hypothesis, Vermeer et al. [19] demonstrated that CO2 is unlikely of great
Keratin The contribution of keratin to the buffering capacity of skin remains questionable. Keratin is an amphoteric protein with the ability to neutralize acids and alkalis in vitro [10– 13, 28, 35–37] and hence may participate in skin’s buffering capacity. Scales scraped from normal skin bind small amounts of alkali in vitro [38, 39]. However, Vermeer and coworkers showed that water-soluble constituents of the epidermis participate more in skin’s buffering capacity than the insoluble constituents of the skin such as keratin. While insoluble keratin filaments on the skin may have only little buffering capacity [27, 40], keratin hydrolysates (free amino acids) may contribute to the free AAs
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importance for alkali neutralization on skin. His experiment was focused on the first minutes of the neutralization process in contrast to the previous experiments mentioned [29, 36, 44, 45], which paid attention to the later neutralization process. For example, Piper [36] analyzed the neutralization process for up to 1 h and concluded that, for the first ½ hour, alkalis are neutralized on the skin by the skin’s own amphoteric substances (such as amino acids) but that in the second half-hour diffusing carbon dioxide takes over. Piper’s conclusions are not necessarily contradictory to the results obtained by Vermeer above and may actually be in agreement. According to Piper, ‘‘the longer the contact between skin and alkali, the greater the importance of CO2.’’ This statement is supported by the recent discoveries of relatively low level of carbon dioxide production in the epidermis and the limited activity of the Kreb’s Cycle, suggesting that a minimal amount of CO2 would be available for neutralization [41]. It seems likely that CO2 does not significantly contribute in the alkali neutralization process. Further studies should further help to clarify the relevance of CO2 in skin’s buffering capacity. The above studies fail to provide quantitative support for their conclusions concerning CO2 as relevant buffering agent. More likely, the constant neutralization time after successive alkali exposure may be related to the destruction of the ‘‘skin barrier’’ and unlimited penetration of the applied alkali as suggested by others [29, 30].
Free Amino Acids Free amino acids in the water-soluble portion of the epidermis seem to play a significant role in the neutralization of alkalis within the first 5 min of experimentation [27, 36, 37]. Piper [36] found a good buffering capacity of skin between pH 4 to pH 8 with an optimum at 6.5 well corresponding to the pKa of AA. This observation further indicates that lactic acid may be less relevant in the buffering capacity of skin. Despite the general agreement about the role of amino acids in the neutralization of alkalis, which amino acids are the key buffering agents remains an open question. The AA composition of the upper stratum corneum was reported by Spier and Pascher [33]. Spier and Pascher reported that the free AA account for 40% of the water-soluble substances extracted from the stratum corneum removed by tape stripping [34, 40]. Of the amino acids present the composition was as follows: 20–32% serine, 9–16% citrulline, 6–10% aspartic acid, glycine, threonine, and alanine, and 0.5–2% glutamic acid.
The water-soluble, free AA on the skin surface may originate from five possible sources. 1. Eccrine sweat Sweat contains 0.05% amino acids which remain on the surface of the skin after evaporation. The specific AA found in sweat was not investigated. 2. Degradation of skin proteins Degradation of skin proteins including proteins constituting the desmosomes may be a source for AA such as serine, glycine, and alanine. 3. Hair follicles Citrulline is recognized as a constituent of protein synthesized in the inner root sheath and medulla cells of the hair follicle. Specific proteases release citrulline. Citrulline is also found in proteins in the membrane of the corneocytes [41]. 4. Keratin hydrosylates Although as discussed above the AA composition of Keratin does not correspond with the composition of free AA found the stratum corneum [34, 41, 42]. 5. Keratohyalin granule histidine-rich protein The pool of free amino acids, urocanic acid and pyrrolidone carboxylic acid in mammalian stratum corneum has been shown to be derived principally or totally from the histidine-rich protein of the keratohyalin granules. The time course of appearance of free amino acids and breakdown of the histidine-rich protein are similar, as are the analyses of the free amino acids and the histidine-rich protein. Quantitative studies show that between 70% and 100% of the total stratum corneum-free amino acids are derived from the histidine-rich protein [46, 47]. These results strongly suggest that the free amino acids and/or their metabolites of the stratum corneum might be the final products of a degradation of the histidine-rich protein. Further research needs to be completed in order to identify which of these AAs contribute to the buffering capacity of skin.
Specific Physiologic and Structural Changes in the Stratum Corneum of Elderly Skin: Impact on the Buffering Capacity of Elderly Skin Lipid Content/Sebum Production The brick and mortar model is often used to describe the stratum corneum’s protein-rich corneocytes embedded in a matrix of ceramides, cholesterol, and fatty
Buffering Capacity Considerations in the Elderly
acids, and smaller amounts of cholesterol sulfates, gucosylceramides, and phospholipids. As stated earlier, these lipids form multi-lamellar sheets amidst the intracellular spaces of the stratum corneum critical to the stratum corneum’s mechanical and cohesive properties, enabling it to function as an effective water barrier [18, 48]. Many authors agree that the overall lipid content of human skin decreases with age [48–50], although the proportion of different lipid classes seems to remain fairly constant [7]. Sebaceous gland function is decreased in association with concomitant decrease in endogenous androgen production [51]. This is the likely cause of decreased surface lipid levels in the elderly. In males, sebum levels remain essentially unchanged until the age of 80 years. In women, there is a gradual decrease in sebaceous secretion from menopause through the seventh decade, after which no appreciable change occurs [52]. As discussed in the previous section the lipid layer is presumed to slow down the exposure of any topical insult. Therefore with decreased amount of lipid, any topical insult will more easily overwhelm the buffering system of the stratum corneum.
Water In young skin, most of the water is bound to proteins and appropriately termed bound water [53]. Bound water is important for the structure and mechanical properties of many proteins and their mutual interactions. Water molecules that are not bound to proteins bind to each other and are called tetrahedron or bulk water [53]. In aged skin water is mostly found in the tetrahedron form, bound to itself rather than to other molecules [54]. The lack of interaction between water and surrounding molecules in aged skin leads to variation in the water-soluble portion of the stratum corneum and likely contributes to decreased buffering capacity found in elderly skin. In addition, this chemical change in the water explains why although aged stratum corneum has higher total water content than younger skin it is often dry and weathered [7].
Proteins The majority of proteins in young skin are in helical conformation. This is in contrast to aged skin which can show markedly altered protein conformation such as increased protein folding resulting in less exposure to aliphatic residues to water [53, 54]. Increased protein folding and
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decreased interaction of proteins with water effects the concentration of AA in the stratum corneum which as discussed previously, likely plays an important role in the buffering capacity of skin. In addition the AA composition of proteins and free amino acids in aged skin also differ significantly from that of young skin. There is an increase in the overall hydrophobicity of amino acid in the elderly [55, 56]. As free amino acids are believed to play a key role in stratum corneum buffering capacity this shift in composition, combined with evidence of altered protein tertiary protein structure, provides insight into the diminished buffering capacity in aged individuals. In addition, it should also be noted that the increase in pH of aged skin will also change the fraction of AA in the stratum corneum that are associated or disassociated. Free AA work best as a buffer at pH that is equal to their pKa (i.e., the pH at which 50% of the AA associated and 50% disassociated). Because of the increased baseline pH found in elderly skin the percentage of associated to disassociated AA changes, hence changing the effectiveness of the buffer.
Eccrine Sweat Glands With aging the number of active eccrine sweat glands is reduced and sweat output per gland is diminished in both in rate and amount. Morphologically, the secretory cells flatten and become atrophic. A progressive accumulation of lipofuscin is found in the cytoplasm of the glandular epithelium [51, 57]. Therefore any contribution of eccrine sweat to the buffering capacity would be decreased in aged skin due to the decreased output of sweat overall.
Conclusion Skin’s exquisite buffering capacity has been widely studied in vitro and in vivo, yet further research is required to better understand the exact mechanisms responsible for the buffering capacity of skin. Experimentation reviewed here suggests that AAs are primarily responsible for the neutralization capacity of skin. The exact sources of the amino acids as well as the types of AA that are primarily responsible for the neutralization capacity remain still rather speculative. In addition, it seems that a sweat component increases the neutralization capacity of the epidermis. Whether the buffering component of sweat is additional AA or lactic acid remains unknown.
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While additional components of the epidermis such as sebum, keratin, and CO2 seem not to significantly participate as buffering agents of the epidermis, they still may play a role in the protection of skin from the harm of acids and bases. Sebum may slow down the initial penetration of applied substances. Keratin is important for the hydration of the skin and may contribute to the free AA pool responsible for buffering of applied acids/alkalis. Finally, CO2 may play a role in the buffering capacity of certain compounds under certain circumstances such as after prolonged or repetitive exposure to an alkali. Elderly skin has an increased pH and decreased buffering capacity. These two changes in the physiochemical nature of elderly skin arguably contribute to the fragility of elderly skin by influencing barrier homeostasis, skin integrity/cohesion, susceptibility to infection, and skin sensitivity to topical acids and alkalis. After thorough review of studies investigating the buffering capacity of skin and studies investigating the endogenous mechanisms for restoring and maintaining skin pH, it is interesting that the two topics have been investigated separately without looking for a commonality. It would not be surprising if the mechanisms responsible for maintaining skin pH influence the processes responsible for maintaining skin buffering capacity. The above rationale may shed light on clinical correlation of increased pH and decreased buffering capacity that is seen in certain skin disease [58] and in elderly skin [7]. This theory is supported by the discovery that 70–100% of AAs of the stratum corneum are derived from the degradation of histidine-rich protein in keratohyalin granules which is also one of the essential pathways involved in maintaining skin pH [3, 46, 47]. This theory is further supported by the fact that decreasing skin pH in the elderly via acidic topical products has lead to an increased buffering capacity and reduced skin sensitivity. One study in particular used a preparation acidified with citric acid/ammonium citrate buffer and demonstrated a significant shortening of the alkaline neutralization time in aged skin from 5.3 0.6 to 4.9 0.5 min after 4 weeks application [59]. While more research needs to be conducted on the benefit of topical acidic therapy for aged individuals, this application seems reasonable as many authors have demonstrated the use of acidic topic products or washes on patients with increased pH to help restore integrity/cohesion [22] and barrier recovery [22]. The rich experimental literature, even if old at times, leads the way to utilizing several contemporary methods to further refine insights into skin’s buffering capacity and aging. This capacity, when fully understood, may lead not
only to the potential for decreasing threat of exogenous acids and bases to aged skin, but also for establishing an experimental bases for optimal pH in many cosmetic, pharmacologic, metabolic and toxicologic situations in elderly individuals.
References 1. Heuss E. Die Reaktion des Scheisses beim gesunden Menschen. Monatsh. Prakt. Dermatol. 1892;14:343. 2. Schade H, Marchionini A. Zur physikalischen Cheme der Hautoberflache. Arch Dermatol Syphil. 1928;154:690. 3. Kim M, Patel R, Shinn A. Evaluation of gender difference in skin type and pH. J Dermatol Sci. 2006;41:153–156. 4. Greener B, Hughes A, Bannister N, Douglas J. Proteases and pH in chronic wounds. J Wound Care. Feb 2005;14(2):59–61. 5. Hachem J, Crumrine D, Fluhr J, Brown B, Feingold K. Elias P. pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol. 2003;121: 345–353. 6. Agache P. Measurement of skin surface acidity. In: Agache P, Humbert P, Maibach H (eds) Measuring Skin. Springer, 2004, pp. 84–86. 7. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (II): protein, glycosaminoglycan, water, and lipid content and structure. Skin Res Technol. 2006 Aug;12 (3):145–154. 8. Raab W. Skin cleansing in health and idsease. Wien Med Wschr. 1990;141(108):4–10. 9. Segger D, Abmus U, Brock M, Erasmy J, Finkel P, Fitzner A, Heuss H, Kortemeier U, Munke S, Rheinlander T, et al. IFSCC Magazine. 2007;10(2):107–110. 10. Burckhardt W. Beitrage zur Ekzemfrage. II. Die rolle des alkali in Pathogenese des ekzems speziell des Gewerbeekzems. Arch f Dermat U Syph. 1935;173:155–167. 11. Burckhardt W. Beitrage zur Ekzemfrage. III. Die rolle des alkalischadigung der haut bei der experimentellen Sensibilisierung gengen Nickel. Arch f Dermat U Syph. 1935;173:262–266. 12. Burckhardt W. Neure untersuchungen uber die Alkaliempfindlicjkeit der haut. Dermatologica. 1947;94:73–96. 13. Burckhardt W, Baumle W. Die Beziehungen der saurempfindlichkeit zur Alkaliempfindlicjkeit der haut. Dermatologica. 1951;102:294–300. 14. Fore-Pfliger J. The epidermal skin barrier: implications for the wound care practitioner, part I. Adv Skin Wound Care. 2004;17(8): 417–425. 15. Zlotogorski A. Distribution of skin surface pH on forehead and cheek of adults. Arch Dermatol Res. 1987;279:398–401. 16. Thune P, Neilsen T, Hnastad IK, et al. The water barrier function of skin in relation to water content of the stratum corneum, pH and skin lipids. Acta Derm Venerol. 1988;68:277–283. 17. Laufer A, Dikstein S. Objective measurement and self-assessment of skin care treatments. Cosmet Toiletires. 1996;111:91–98. 18. Choi EH, Man MQ, Xu P, Xin S, Liu Z, Crumrine DA, Jiang YJ, Fluhr JW, Feingold KR, Elias PM, Mauro TM. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol. 2007;127(12):2847–2856.
Buffering Capacity Considerations in the Elderly 19. Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias P. pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol. 2003;121:345–353. 20. Fluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol. 2001;117:44–51. 21. Ekholm E, Egelrud T. Expression of stratum corneum chymotrypic enzyme in relation to other markers of epidermal differentiation in skin explant model. Exp Dermatol. 2000;9:65–70. 22. Leveque JL, Corcuff P, de Rigal J, Agache P. In vivo studies of the evolution of physical properties of the human skin with age. Int J Dermatol. 1984 Jun;23(5):322–9. 23. Dunner M. Der Einfluss des Hauttalges auf die Alkaliabwehr der Haut. Dermatologica. 1950;101:17–28. 24. Fishberg E, Bierman W. Acid base balance in sweat. J Biol Chem. 1932;97:433–441. 25. Vermeer D. The effect of sebum on the neutralization of alkali. Dederl Tijdschr V Geneesk. 1950;94:1530–1531. 26. McKenna B. The composition of the surface skin fat (Sebum) from the human forearm. J Invest Dermatol. 1950;15:33–37. 27. Vermeer D, Jong J, Lenestra J. The significance of amino acids for the neutralization by the skin. Dermatologica. 1951;103:1–18. 28. Lincke H. Beitrage zur Chemie und Biologie des Hautoberflachenfetts. Arch f Dermat U Syph. 1949;188:453–481. 29. Neuhaus H. Fettehalt und Alkalineutralisationskahigkeit der haut unter Awendung alkalifrier waschmittel. Arch f Dermat U Syph. 1950;190:57–66. 30. Vermeer D, Jong J, Lenestra J. The significance of amino acids for the neutralization by the skin. Dermatologica. 1951;103:1–18. 31. Schmidt P. Uber die Beeinflussung der Wasserstoffionenkonzentration der Hautoberflache durch Sauren. Betrachtungen uber die Funktionen des ‘‘Sauremantels’’. Arch f Dermat U Syph. 1941;182:102–26. 32. Vermeer D. Method for determining neutralization of alkali by skin. Quoted in Yearbook: Dermat & Syph. 1951;415. 33. Wohnlich H. Zur Kohlehydratsynthase der Haut. Arch f Derm Syph. 1948;187:53–60. 34. Spier H, Pascher G. Quantitative Untersuchungen uber die freien aminosauren der hautoberflache. – Zur frage Ihrer Genese. Klinische Wochenchrift. 1953:997–1000. 35. Sharlit H, and Sheer M. The hydrogen ion concentration of the surface on healthy intact skin. Arch Dermat Syph. 1923;7: 592–598. 36. Piper H. Das Neutralisationsvermogen der haut gegenuber Laugen und seine Beziehung zur Kohlensauteabgabe. Arch f Dermat U Syph. 1943;183:591–647. 37. Jacobi O. Uber die Reaktiosfagigkeit und das Neutralisationsvermogen der lebenden menschlichen Haut. Dermat Wchnschr. 1942;115: 733–741. 38. Lustig B, Perutz A. Ube rein einfaches Verfahren zur Bestimmung der Wasserstoffionenkonzentration der normalen menschlichen Hautoberflache. Arch f Dermat U Syph. 1930;162:129–134. 39. Steinhardt J, Zaiser E. Combination of wool protein with cations and hydroxyl ions. J Biol Chem. 1950;183:789–802.
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40. Green M, Behrendt H. Patterns of Skin pH from Birth to Adolescence with a Synopsis on Skin Growth. Springfield: Charles C Thomas, 1971, pp. 93–100. 41. Peterson LL, Wuepper KD. Epidermal and hair follicle transglutaminases and crosslinking in skin. Mol Cell Biochem. 1984;58 (1–2):99–111. 42. Steinhert P, Freedberg I. Molecular and Cellular biology of Keratins. In: Goldsmith L (eds) Physiology and Molecular Biology of the Skin, 2nd ed. Oxford University Press, 1991, pp. 113–14732. 43. Arnold D. The self disinfecting power of skin. Am J Hyg. 1934;19:217–228. 44. Szakall A. Uber die physiologie der obersten Hautschichten und ihre Bedeutung fur die Alkaliresistenz. Arbeitsphysiol. 1941;11:436–452. 45. Szakall A. Die Veranderungen der obersten Hautschichten durch den Dauergebrauch einiger Handwaschmittel. Arbeitsphysiol. 1943;13:49–56. 46. Scott IR, Harding CR, Barrett JG. Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim Biophys Acta. 1982;719(1):110–117. 47. Horii I, Kawasaki K, Koyama J, Nakayama Y, Nakajima K, Okazaki K, Seiji M. Histidine-rich protein as a possible origin of free amino acids of stratum corneum. Curr Probl Dermatol. 1983;11:301–315. 48. Rogers J, Harding C, mayo A, Banks J. Rawlings A. Stratum corneum lipids: the effects of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 49. Roskos KV. The effect of skin aging on the percutaneous penetration of chemicals through human skin. Dissertation, UCSF, CA. 50. Saint Leger D, Francois AM, Leveque JL, Stoudemayer TJ, Grove GL, Kligman AM. Age-associated changes in the stratum corneum lipids and their relation to dryness. Dermatologica. 1988;177:159–164. 51. Pollack SV. The aging skin. J Fla Med Assoc. 1985;72(4):245–248. 52. Pochi PE, Strauss JS, Downing DT. Age-related changes in sebaceous gland activity. J Invest Dermatol. 1979;73:108–111. 53. Gniadecka M, Nielsen OF, Christensen DH, Wulf HC. Structure of water, proteins, and lipids in intact human skin hair nail. J Invest Dermatol. 1998;110:393–398. 54. Gniadecka M, Nielsen OF, Wessel S, Heidenheim M, Christensen DH, Wulf HC. Water and protein structure in photoaged and chronically skin. J Invest Dermatol. 1998;11:1129–1133. 55. Jacobson T, Yuksel Y, Geesin JC, Gordon JS, Lane AT, Gracy RW. Effects of aging and xerosis on the amino acid composition of human skin. J Invest Dermatol. 1990;95:296–300. 56. Jacobson TM, Umit Yuksel K, Geesin JC, et al. Effects of aging and xerosis on the amino acid composition of human skin. J Invest Dermatol. 1990;95:296–300. 57. Selmanowitz VJ, et al. Aging of the skin and its appendages. In Finch C, Hayflick (eds) Handbook of the Biology of Aging. New York: van Nostrand Reinhol Company, 1977, pp. 496–509. 58. Kurabayahi H, Tamura K, Machida I, Kubota K. Inhibiting bacteria and skin pH in hemiplegia: effects of washing hands with acidic mineral water. Am J Phys Med Rehabil. 2002;81:40–46. 59. Meigel W, Sepehrmanesh M. Untersuchung der pflegenden wirkung und der vertraglichkeit einer cre`me/loti bei alteren patienten mit trockenem hautzustand. Dtsch Derm. 1994;42:1235–1241.
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30 Cellular Energy Metabolism and Oxidative Stress Regina Hourigan
Introduction The objective of this chapter is to provide an overview of how changes to the skin’s energy metabolism systems lead to a decline in function and hence contribute to skin aging. This chapter first discusses how the skin uses energy to maintain its appearance followed by a background on energy production in cells. Defects in energy production are part of the mitochondrial theory of aging, which will be introduced next. Skin-specific examples which support this theory of aging will be given as well as evidence that questions this theory. The examples are divided into chronological skin aging and extrinsic skin aging. Lastly, examples of anti-aging therapies which improve or maintain metabolic functions of the skin are given. This chapter intends to also provide areas for discussion or debate, as there is a cyclic nature to the role of mitochondria in aging. It is not clear if aging causes mitochondrial defects or if mitochondrial defects cause aging. Similarly, while oxidative stress can cause mitochondrial defects, mitochondrial defects can also generate oxidative stress. It is in fact this cyclic nature which may progressively lead to more damage.
Energy Metabolism and the Role of Mitochondria in the Skin Skin is in essence a sacrificial protective coating of the body. It is continually shed to maintain an effective barrier to outside insults. As such, the skin is composed of many types of proliferating cells which have a high energy demand. For example, suprabasal layers of the epidermis and cells within the root of the hair follicle have high metabolic activity associated with the synthesis of keratin and the cornified envelopes. Adenosine triphosphate (ATP) is a vital source of energy for these metabolic activities of skin cells. It is required for proliferation as a result of mitogenic stimuli, collagen synthesis, and DNA repair. It supports functions that maintain skin turnover and the extracellular matrix. A decline or dysfunction in ATP
production impacts the skin’s functions, and ultimately its appearance. Mitochondria can also have an impact on the skin through keratinocyte differentiation. In cell cultures, mitochondria-mediated cell death can trigger keratinocyte differentiation. Characteristics of differentiation (flattened morphology, stratification, and keratin 10 expression) are detected after an reactive oxygen species (ROS)-induced release of cytochrome c and apoptosisinducing factor (AIF) [1].
Background on Mitochondria Structure and Function This section briefly describes the mitochondrial mechanisms for energy production. This provides a brief background for the later discussions. Mitochondria generate ATP, which is used as chemical energy for most eukaryotic cells. They also control cell functions related to cell death, differentiation, and cell signaling. They contain their own genetic code, similar to the nucleus of the cell. Their genetic material is referred to as mitochondrial DNA (mtDNA). The mitochondria are composed of several compartments, enclosed by an inner and outer membrane. The outer membrane contains a protein called porin, which forms aqueous channels allowing for protein transport through the membrane. The inner membrane contains an important mechanism for energy production, the electron transport chain (ETC). The ETC is a major source of ATP production in the cell. It is a series of enzymatic complexes, called complexes I, II, III, IV, and V. It uses molecules derived from fuel sources to produce oxygen. The starting molecules are electron donators, reduced nicotinamide adenine dinucleotide (NADH), and reduced flavin adenine dinucleotide (FADH2). The NADH enters at complex I and FADH2 enters at complex II. At the end of complex IV the output is oxygen, which combines with the electrons and protons to form water. Electron transfer through complexes I–IV is managed by the complexes and electron carriers, such as
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_30, # Springer-Verlag Berlin Heidelberg 2010
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coenzyme Q10 (CoQ10) and cytochrome c. The flow of electrons and the ATP synthesis by OxPHOS is continuous within tissues containing mitochondria [2]. The exchange of electrons from a high-energy state to a lower-energy state generates an electrochemical proton gradient. This gradient provides the energy to drive the phosphorylation of adenosine diphosphate (ADP) to ATP. This process, in which ADP is converted to ATP using inorganic phosphate, is called oxidative phosphorylation (OxPHOS). It occurs in complex V. The ATP is then available for use as chemical energy for the cell [2].
ROS and Mitochondria The majority of electrons travel successfully to successive enzymatic complexes, but some electrons can leak from the ETC. These electrons readily react with the available oxygen to form the ROS superoxide. It is thought that about 1–3% of the O2 reduced in the mitochondria may form superoxide [2]. The mitochondria contain antioxidant defenses to control the level of ROS. Superoxide is converted to hydrogen peroxide and water by the antioxidant enzyme superoxide dismutase. The hydrogen peroxide is converted to water and molecular oxygen by catalase or glutathione peroxidase. Although at times detrimental, ROS is also used for cell signaling [2], for example, for apoptosis of the cell [3]. Damage to the mitochondria increases ROS, which triggers the release of cytochrome c and apoptosis-inducing factor (AIF). Their release initiates the caspase-dependent and caspase-independent cell death pathways to remove the damaged cell [3]. ROS leakage from the ETC increases unchecked ROS, which directly damage the mitochondria. Specific damage will be discussed in the later sections. The damaged mitochondria decline in function, which in turn generates more ROS. This is known as the ‘‘vicious cycle’’ where ROS are both a cause and a consequence of mtDNA mutations. The ‘‘vicious cycle’’ creates an amplifying feedback loop which sustains the damaging effects, even with a small amount of initial insult. The ‘‘vicious cycle’’ is believed to create ongoing elevated levels of stress, such as those found with aging [4, 5]. The ROS and its resulting ‘‘vicious cycle’’ are the foundations of the mitochondrial theory of aging.
Mitochondrial Theory of Aging The free radical theory of aging (FRTA) was formed by Harman, in 1956 [6]. The FRTA proposes that the
underlying source of aging is the accumulation of oxidative damage in macromolecules and tissues. Later, Harman proposed that the mitochondria’s production of the superoxide may be central to the FRTA [7]. This is called the mitochondrial theory of aging. A fundamental part of this theory is that mtDNA is at particular risk for ROS damage. This is proposed because of the proximity of mtDNA to the ROS-producing mitochondrial matrix. The mitochondrial theory of aging is supported by evidence of age-related ROS accumulation and mitochondrial changes. There is an increase in ROS produced from the ETC with aging [8, 9]. Complexes I, II, and III are considered to be the sites of excessive ROS [10–13]. As tissues age, there is lower flux through the ETC and reduced ATP production [14]. The lower flux causes more free electrons to be lost and form ROS [15, 16]. The increased ROS with age can directly damage the structures of the mitochondria itself, such as proteins, lipids, and mtDNA [17–19]. ROS damages mtDNA by creating strand breaks within the mtDNA. Studies of the respiration-dependent mitochondrial processes conclude that mtDNA damage is related to a decline in respiratory processes. These processes include mitochondrial protein synthesis, oxygen consumption, and ATP generation. For example, as the amount of mtDNA damage increases, the mitochondria membrane potential lowers and cannot be maintained. Maintaining membrane potential is critical to the electrochemical proton gradient and OxPHOS. In parallel, cytochrome c is released into the cytoplasm, which activates caspases leading to premature apoptosis [20]. The compromised respiratory processes elevate ROS levels further, creating the above-mentioned ‘‘vicious cycle.’’ It has been debated whether the accumulation of such oxidative damage is a cause or a consequence of aging. In 2007, Muller et al. reviewed the topic of whether oxidative stress determines life span. They conclude that the case for oxidative stress as a life span determinant may be tentatively made for Drosophila melanogaster, but is not certain in humans or mice [21]. Kujoth et al. have proposed accumulation of mtDNA mutations, which promote that apoptosis may be a mechanism driving mammalian aging [22]. Recent work by Doonan et al. found that there is no impact on basal life span with increased levels of superoxide dismutase (through gene manipulation of Caenorhabditis elegans) [23]. While basal life span was not changed, improving organisms’ ability to cope with elevated oxidative stress can lengthen life span. Supplying C. elegans with antioxidant mimetics extended their life span, and normalized prematurely aged organisms’ life spans, during exposure to oxidative stress [24].
Cellular Energy Metabolism and Oxidative Stress
Coping with stresses is critical to survival and longevity. It should be noted that other factors related to coping with stresses, such as inflammation or repair mechanisms are also critical determinants of longevity. In the skin, stressful environments can clearly cause oxidative damage that leads to extrinsic aging and there is likely a place for the mitochondrial theory of aging.
Skin Energy Metabolism and Chronological Aging Skin is aged through two mechanisms: chronological aging (a function of the passage of time) and extrinsic aging (a function of external stress, i.e., photoaging from ultraviolet radiation [UVR]). This section discusses the chronological aging of skin, related to energy metabolism, and will be followed by a separate discussion on extrinsic skin aging and energy metabolism. One approach to studying chronological aging is harvesting the skin cells from variously aged donors and comparing their functions. When skin cells are collected in this manner, there are differences in the metabolic functions of age groups. According to Greco et al., in 2003, human dermal fibroblasts from 51 donors aged 1–103 years showed a clear reduction in mitochondrial processes with age. These included mitochondrial protein synthesis, respiration rate, and coupling of respiration to ATP production. In individuals above 40 years there was a significant decline in the mitochondrial protein synthesis. There was also a significant decrease in endogenous native respiration rate within the age range of 40–90 years. Human skin fibroblasts also had a significant agedependent decrease in the efficiency of respiration and phosphorylation. The ratio of skin cell’s rate of respiration in the presence of ADP to that in the absence of ADP (RCR) is a measure of the OxPHOS’s control of respiration efficiency. This ratio significantly decreased with the age of the donor [25]. In the skin cells, particularly from donors over the age of 40, metabolic functions decline as a function of age. The loss of function of donated cells may have been due to multiple causes experienced over their life span. Another study looked at the targeted influence of older mtDNA on cell functions. In this study, mitochondria from fibroblasts of 21 individuals between the ages of 20 weeks and 103 years were inserted into human mtDNA-less cells. An age-dependent decrease in growth rate and a decline in respiratory rate were detected in the cells receiving the older mitochondria [26]. This observation supports that chronologically aged mitochondria
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can cause characteristics of aging, such as reduced cell growth and respiratory functions. The amount of available energy metabolites in the skin may be thought to be related to the age of the skin, that is, younger skin contains more energetic materials – yet that is not the case observed in vivo. There are no differences in the skin’s basal levels of energy metabolites with age [27]. Using 31P nuclear magnetic resonance spectroscopy, young and old skin (ventral aspect of the wrist) from panelists did not show differences in baseline levels of phosphocreatine, inorganic phosphate, adenosine triphosphate, phosphomono, and phosphodiesters. However, what is significantly different is how the age groups respond to stress. After single exposure to a low, sub-erythema level of UVA irradiation, there were significant differences in the response and recovery of energy metabolism. The older skin showed slower response and recovery than younger skin [27]. This is an indication of the importance of evaluating skin aging characteristics in basal as well as stressed conditions. This is an important consideration when evaluating therapies related to energy metabolism. An anti-aging material may not influence the basal characteristics of the skin but, may be influential in reducing stresses or up-regulating repair, thereby preventing extrinsic aging. The mitochondria and its functions play both causative and effective roles in cell dysfunctions and senescence. Senescent human cell cultures are a model for chronological aging [28]. The loss of mitochondrial functions, as passage number increases, can cause premature senescence in skin cells. It induces a senescent phenotype likely with the increase in ROS. This has been demonstrated by a reduction in the level of OxPHOS in fibroblasts causing a reduction in cell proliferation and premature senescence in human fibroblasts [29]. On one hand, the mitochondria affect senescence but, on the other, changes that occur with senescence can effect mitochondrial respiration. With increasing passage number, senescent fibroblasts show a loss of membrane potential [30] and a decline in ATP production [31]. This may be due to inefficient removal of mitochondrial damage in the cells as proteasomes activity declines. Proteasome inhibition is emerging as a common factor, based on in vitro and in vivo experiments, in aging and age-related diseases. Proteasomes are part of the protein removal system for most eukaryotic cells. They contain proteases, which degrade damaged or unnecessary proteins from the cell. Proteasome activity declines with age in the human epidermis [32]. Keratinocytes that undergo replicative senescence are known to have a reduction in proteasome levels [33]. While oxidative
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damage has been known to cause proteasome dysfunction during aging, Torres and Perez have recently shown that proteasome inhibition is a mediator of oxidative stress and ROS production by affecting mitochondrial function. They proposed that a progressive decrease in proteasome function during aging can promote mitochondrial damage and ROS accumulation [34].
Skin Energy Metabolism and Extrinsic Aging The change in skin appearance from external insults is called extrinsic aging. These insults can include UVR, pollution, and smoking. The major cause of extrinsic aging is UVR, referred to as photoaging. Photoaged skin has many well-accepted characteristics, such as loss of elasticity, reduced hydration, greater barrier damage, to mention a few. The UVR induces theses changes by interacting with and damaging skin structures; i.e., the proteins, DNA, and lipids of the skin. mtDNA can also be damaged by UVR. Greater accumulations of mtDNA damage are found in sun-exposed skin compared to protected skin [35–42]. It is thought that the strand breaks in mtDNA caused by UVR are mediated by ROS, particularly superoxide. The 4,977-base pair (bp) deletion and 3,895-bp deletion mtDNA are among the recognized mtDNA deletions related to skin photoaging [37, 40, 41, 43]. The 4,977-bp deletion is called the common deletion because it is the most prevalent marker of mtDNA damage in humans and is found in several types of tissues. The 3,895-bp deletion, which is less reported, may also play a role in skin photoaging [41]. The 4,977-bp deletion occurs in both in vitro and in vivo skin studies and relates to UVR exposure. The common deletion was found in human skin fibroblasts treated with a repeated, sublethal dose of UVA [36]. Studies with keratinocytes are less numerous, but have also indicated a link between UVR and mitochondrial damage. A single dose of UVB exposure to kertatinocytes induced two mtDNA deletions: the 4,977-bp and a novel 5,128-bp deletion [43]. In vivo, the common deletion can be induced in the dermal tissue of living skin after repeated exposure to UVA radiation. UVA induced a 40% increase in the common deletion in the dermis, but not in the epidermis of the irradiated skin [37]. There are differing findings on the presence of the 4,977-bp deletion in chronologically aged skin. There is evidence for an age-related increase in the 4,977-bp type of deletion in skin mtDNA. The 4,977-bp deletion was not
found in skin samples from donors under the age of 60 years. The frequency of this deletion in skin did increase with age for individuals who were 60–90 years old [37]. Keratinocytes-induced 4,977-bp deletion has, in other cases, not corresponded to the chronological age of the donors (30–78 years). Koch et al. noted slower cell proliferating body sites, that is, blood, brain, and skeletal tissues, and showed correlation between chronological age and increasing mtDNA deletion [40]. The 3,895-bp deletion corresponds to broadspectrum UV exposure in both in vitro and in vivo studies. HaCaT cells (line derived from keratinocytes) exposed to repeated UVA/UVB doses were found to have the mtDNA 3,895-bp deletion [41]. Among skin samples from 42 skin donors, the 3,895-bp deletion was found at a higher amount in ‘‘usually’’ sun-exposed body sites (face, ears, neck, and scalp) compared with ‘‘occasionally’’ exposed sites (shoulders, back, and chest). The deletion was not detected in the body sites that were ‘‘rarely’’ exposed to sunlight. The 3,895-bp deletion induction was in both the epidermis and the dermis of the exposed sites. In the usually exposed sites, the level was almost equal in the epidermis and the dermis. Therefore, mitochondrial damage may serve as a biomarker for cumulative sun exposure [41]. In summary, UV-induced ROS can cause mtDNA damage, which serves as a marker of skin damage. These genetic changes also lead to a decline in mitochondrial function (i.e., ATP production). The UVA-induced common deletion in human dermal fibroblasts corresponds to decreases in oxygen consumption, mitochondrial membrane potential, and ATP content [44]. This leads to compromised mitochondrial respiration. Once compromised in this manner, the mitochondrial respiration causes an increase in ROS produced by the ETC. The intracellular mitochondrial oxidative stress generated under these conditions upregulates matrix metalloproteinase-1 (MMP-1) [44]. MMP-1 is an accepted indicator of aged and damaged skin. MMP-1 is responsible for collagen degradation in photoaged skin [45], the skin of tobacco smokers [46], and chronologically aged (sun-protected) skin [47]. Skin fibroblast studies have shown that in the absence of UVA, a deletion of 4,977-bp causes an increase of MMP-1. UVA-induced common deletion also corresponds to an increase in the expression of MMP-1 without an increase of the tissuespecific MMP inhibitors [44]. The cascade of UV stress, ROS generation, mtDNA damage, and elevated ROS from compromised respiration leads to collagen degradation, a direct factor in the appearance of aged skin. While this cascade has a linear sequence of events, there is also a feedback loop (the ‘‘vicious cycle’’
Cellular Energy Metabolism and Oxidative Stress
mentioned earlier) created by the ROS. The ROS that cause mitochondrial respiratory dysfunction lead to more ROS being produced by the cell. There has been recent evidence of the ‘‘vicious cycle’’ in the skin as measured by sustained mtDNA damage. The common deletion was found to remain in UV-exposed skin for up to 16 months after experimental treatment (in vivo). In some cases, the deletion continued to increase after UV exposure has ceased [37]. This increase after exposure and sustained level of mtDNA deletion may be due to the ongoing cycle of ROS generation and mtDNA mutations. This ‘‘vicious cycle’’ may be a source of chronic oxidative stress within the skin. The chronic stress may be a factor in premature aging as the ROS may then interact with skin proteins, initiate inflammation, and promote extracellular matrix degradation (> Fig. 30.1). While ultraviolet radiation has been well studied for its impact on skin photoaging, recent studies are now showing an emerging role for infrared (IR) radiation in photoaging.
. Figure 30.1 The potential vicious cycle of mitochondrial damage and skin aging
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IR accounts for more than half of the solar energy that reaches the human skin [48]. It is divided into IRA, IRB, and IRC. IRB and IRC do not penetrate deeply into the skin, while more than 65% of IRA reaches the dermis [48]. Near IRA (760–1,440 nm) can induce MMP-1 expression in ex vivo fibroblast cells [49]. The production of ROS from IRA stress originates from the mitochondrial ETC. Cultured fibroblasts treated with antioxidants are protected from the IRA radiation and do not upregulate MMP-1 expression [49]. In vivo studies also show that skin responds to IRA radiation with upregulation of MMP-1 in the dermis [50]. In parallel, there is a decrease in the skin’s antioxidant content [50]. In vivo, the skin was protected from IRA-induced MMP-1 upregulation with the use of antioxidants [50]. The implication is that the mitochondria ROS signaling that leads to MMP-1 collagen degradation can be prevented by antioxidants.
Select Anti-aging Therapies This section highlights select cellular respiration anti-aging approaches. As mentioned above, one protective approach is with antioxidants. DNA, lipids, and proteins are known to be protected by the application of antioxidants on the skin. Antioxidants can regulate the transfer of electrons or quench the free radicals escaping from the ETC. This can mitigate the effects of photoaging through the prevention of oxidative damage and the related damage to mitochondrial functions. Some examples of antioxidants are glutathione, CoQ10, and N-acetyl cysteine (NAC). At low concentrations of the antioxidant glutathione, UVB-induced mtDNA deletions have been prevented [43], giving further evidence that mtDNA damage by UVR is mediated by ROS. At higher levels of glutathione, when it acts as a reductive antioxidant (electron donor) and hence a deleterious agent, the protective effect ceases and the mtDNA deletions return [43]. CoQ10 is a known antioxidant found in the mitochondria, and serves to carry electrons in the ETC. Its level in the skin declines with age and UV stress [51]. A series of in vitro and in vivo experiments by Hoppe et al. have shown the benefits of CoQ10 in prevention of skin aging. Topical application of CoQ10 reduced wrinkle depth and level of oxidation in vivo. CoQ10 is also effective in protecting the DNA of keratinocytes from UVA-induced oxidative stress. CoQ10 is also able to reduce the expression of collagenase in dermal fibroblasts following UVA irradiation [52]. N-acetyl cysteine (NAC) is an antioxidant which increases the intracellular concentration of glutathione
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(GSH) [53]. Lipoic acid and NAC supplementation of Alzheimer’s patient’s fibroblasts protected the mitochondria from oxidative stress in vitro [54]. Although not performed with skin cells, NAC oral supplementation of mice increases the mitochondrial respiration of senescent liver cells. The liver cells’ ETC complexes had higher activity, while levels of protein carbonyls, a marker of protein oxidation, were reduced [55]. Another therapy for the improvement of skin’s energy metabolism is to provide the skin with energy supplementation. One approach is with the amino acid creatine. Creatine is the precursor to phosphocreatine. Phosphocreatine is synthesized in the mitochondria by creatine kinases. Phosphocreatine can donate a phosphate group to ADP to produce ATP. This provides an additional reserve of ATP that can be used by cells for metabolic activity. Creatine does not offer antioxidant or UV protection, rather its protective effect is from increasing cell energy reserves. Lenz et al. observed the photoprotective effect of creatine on human skin cells in vitro and in vivo [56]. Supplementation of normal human fibroblasts with creatine during repeated UVA exposure showed a mitigation of mtDNA mutations as well as the normalization in oxygen consumption and MMP-1 production [44]. Creatine also prevents the common deletion, and inhibitors of creatine block these effects [44]. These data show that while UVA reduces mitochondrial function, supplementation with creatine can mitigate these effects. The researchers suggest that the prevention of UVA-induced common deletion may be from creatine’s ability to normalize the cell’s energy status. This prevents an upregulation of a deleterious respiratory chain, which generates more ROS [44]. Supplementation with energy precursors also allows for more efficient repair. Maes et al. have shown, in a skin model, that DNA repair from UV stress exposure is enhanced with creatine [57]. In human clinicals, with a formulation containing creatine, acetyl-L-carnitine, and NADH reduced the appearance of aging [58]. The researchers believed that the enhanced repair was due to the increased availability of ATP that the creatine provided. Under the stress of UV, the cells can synthesize the needed repair enzymes using this additional ATP [57, 58]. Combinations of the above therapies are also effective. In vivo, a combination of CoQ10 and a stabilized form of creatine in a topical emulsion improved signs of skin aging, including density of the dermal papillae [59]. Protecting cellular energy metabolism of skin can improve protection from UV stress, provide energy for repair systems during stress, and cause positive changes to the skin morphology.
Conclusion Aging is a complex topic involving all of the functions of the skin, and its underlying mechanism is difficult to attribute to any single biological source. Multiple studies give evidence that mitochondrial damage is either the cause or a marker of age-related dysfunctions in the skin. The damage, and related decline in mitochondrial functions, can create aged skin appearance. The contribution of mitochondrial damage to skin aging may be amplified by the presence of a vicious cycle. Mitigation and prevention of this mitochondrial damage alleviates the signs of skin aging. While by no means the only factor in skin aging, alterations to the skin’s energy metabolism systems lead to a decline in function and hence contribute to skin aging.
Cross-references > Alterations
of Energy Metabolism in Cutaneous Aging
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Cellular Energy Metabolism and Oxidative Stress 13. Barja G. Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity, and relation to aging and longevity. J Bioenerg Biomembr. 1999;31(4):347–366. 14. Harper, et al. Age-related increase in mitochondrial proton leak and decrease in ATP turnover reactions in mouse hepatocytes. Am J Physiol Endocrinol Metab. 1998;275:197–206. 15. Qian T, Nieminen AL, Herman B, Lemasters JJ, Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol. 1997;273:C1783–C1792. 16. Chen Q, Lesnefsky EJ. Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. Free Radic Biol Med. 2006;40:976–982. 17. Chen JJ, Yu BP. Alterations in mitochondrial membrane fluidity by lipid peroxidation products. Free Radic Biol Med. 1994;17: 411–418. 18. Sohal RS, Dubey A. Mitochondrial oxidative damage, hydrogen peroxide release, and aging. Free Radic Biol Med. 1994;16:621–626. 19. Agarwal S, Sohal RS. DNA oxidative damage and life expectancy in houseflies. Proc Natl Acad Sci USA. 1994;91:12332–12335. 20. Mandavilli BS, Santos JH, Van Houten B. Mitochondrial DNA repair and aging. Mutat Res. 2002;509:127–151. 21. Muller F, et al. Trends in oxidative aging theories. Free Radic Biol Med. 2007;43(4):477–503. 22. Kujoth GC, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005;309:481–484. 23. Doonan R, et al. Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 2008;22:3236–3241. 24. Melov S, et al. Extension of life-span with superoxide dismutase/ catalase mimetics. Science. 2000;289(5484):1567–1569. 25. Greco M, et al. Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts. FASEB J. 2003;17: 1706–1708. 26. Laderman KA, et al. Aging-dependent functional alterations of mitochondrial DNA (mtDNA) from human fibroblasts transferred into mtDNA-less cells. J Biol Chem. 1996;271:15891–15897. 27. Declercq L, et al. Age-dependent response of energy metabolism of human skin to UVA exposure: an in vivo study by 31P nuclear magnetic resonance spectroscopy. Skin Res Technol. 2002;8:125–132. 28. Cristofalo VJ, et al. Use of the fibroblast model in the study of cellular senescence. In: Barnett Y, Barnett C (eds) Aging Methods and Protocols. Totowa: Humana Press, 2000, pp. 26. 29. Stockl P, et al. Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts. Exp Gerontol. 2006;41:674–682. 30. Mammone T, Gan D, Foyouzi-Youss R. Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures. Cell Biol Int. 2006;30:903–909. 31. Zwerschke W, et al. Metabolic analysis of senescent human fibroblasts reveals a role for AMP in cellular senescence. Biochem J. 2003;376(Pt 2):403–411. 32. Bulteau AL, Petropoulos I, Friguet B. Age-related alterations of proteasome structure and function in aging epidermis. Exp Gerontol. 2000;35:767–777. 33. Petropoulos I, et al. Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells. J Gerontol Biol Sci Med Sci. 2000;55:B220–B227. 34. Torres CA, Perez VI. Proteasome modulates mitochondrial function during cellular senescence. Free Radic Biol Med. 2008;44: 403–414.
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35. Yang JH, Lee HC, Lin J, Wei YH. A specific 4977-bp deletion of mitochondrial DNA in human ageing skin. Arch Dermatol Res. 1994;286:386–390. 36. Berneburg M, et al. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274(22):15345–15349. 37. Berneburg M, et al. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122(5):1277–1283. 38. Birch-Machin MA, et al. Mitochondrial DNA deletions in human skin reflect photo-rather than chronologic aging. J Invest Dermatol. 1998;111(4):709–710. 39. Ray A, et al. The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J Invest Dermatol. 2000;115:674–679. 40. Koch H, Wittern K-P, Bergemann J. In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet a irradiation. J Invest Dermatol. 2001;117:892–897. 41. Krishnan K, Harbottle A, Birch-Machin MA. The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin. J Invest Dermatol. 2004;123:1020–1024. 42. Eshaghian A, et al. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J Invest Dermatol. 2006;126:336–344. 43. Ji F, et al. Novel mitochondrial deletions in human epithelial cells irradiated with an FS20 ultraviolet light source in vitro. J Photochem Photobiol. 2006;184(3):340–346. 44. Berneburg M, et al. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences. J Invest Dermatol. 2005;125:213–220. 45. Brennan M, et al. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol. 2003;78(1):43–48. 46. Lahmann C, et al. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357(9260):935–936. 47. Varani J, et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114:480–486. 48. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008;43:629–632. 49. Schroeder P, et al. Cellular response to infrared radiation involves retrograde mitochondrial signaling. Free Radic Biol Med. 2007;43(1):128–135. 50. Schroeder P, et al. Infrared radiation-induced matrix metalloproteinase in human skin: implications for protection. J Invest Dermatol. 2008;128:2491–2497. 51. Podda M, et al. UV radiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med. 1998;24:55–65. 52. Hoppe U, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. BioFactors. 1999;9(2–4):371–378. 53. Zafarullah, et al. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci. 2003;60(1):6–20. 54. Moreira P, et al. Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts. J Alzheimer’s Dis. 2007;12(2):195–206. 55. Miquel J, et al. N-Acetylcysteine protects against age-related decline of oxidative phosphorylation in liver mitochondria. Eur J Pharmacol. 1995;292:333–335.
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23 Changes in Nail in the Aged Nelly Rubeiz . Ossama Abbas . Abdul Ghani Kibbi
Introduction Nail complaints are very common among the elderly. These may represent either normal age-related nail alterations or nail dystrophies that tend to be more common in the elderly secondary to several factors including impaired circulation, faulty biomechanics, infections, neoplasms, and dermatological or systemic diseases [1, 2]. Alone or in combination, these factors may affect the matrix, nail bed, hyponychium, or nail folds leading to secondary abnormalities in the nail plate. These nail alterations may be symptomatic and may impair daily activities or may be associated with significant cosmetic problems leading to a negative psychological impact. Knowledge of these age-related nail changes and dystrophies as well as their underlying causes is important in order to effectively reach an accurate diagnosis and thus provide better care for the nail concerns of this large and growing elderly population.
Normal Senile Nail Changes The age-associated nail changes include characteristic changes in morphology, growth, chemical composition, and histology of the nail unit [1, 2]. The mechanisms underlying these changes are not clear, but may be due to a dysfunctional circulation at the distal extremities or to the ultraviolet radiation effects.
Age-Related Morphological Nail Changes These include changes in color, contour, surface, and thickness of the nail plate [1, 2]. Among the most common nail color changes observed in elderly people is a yellow to gray discoloration with dull, pale, or opaque appearance. The normally smooth texture of the nail plate tends to become increasingly friable with advancing age leading to splitting, fissuring, and longitudinal superficial or deep striations [1, 2]. In general, nail plates are thicker in men than women, where the normal average thickness of fingernails and toenails is 0.6 and 1.65 mm in men
and 0.5 and 1.38 mm in women, respectively. With advancing age, the nail plate thickness may become thicker, thinner, or remain the same [1, 2]. ‘‘Neapolitan nails’’ is a peculiar discoloration observed in up to 20% of people above 70 years and manifests as three horizontal bands of white (proximal), pink (middle), and opaque (distally) discoloration in addition to an absent lunula [1]. One study found that this peculiar nail alteration is significantly associated with osteoporosis and thin skin, and suggested an abnormality in collagen leading to these changes in nail bed, bone, and skin [3]. Although usually seen in liver cirrhosis and chronic congestive heart failure, Terry’s nails, a type of apparent leukonychia characterized by a distal transverse pink band and proximal white band, is occasionally seen as a part of the normal aging process [4]. Senile nail contour changes include an increase in the transverse convexity and a decrease in the longitudinal curvature [1, 2].
Age-Related Nail Growth Rate Changes The normal average growth rate of fingernails and toenails is 3.0 and 1.0 mm/month, respectively. In elderly people, there is a decrease in this rate of growth by approximately 0.5%/year after the age of 25 years [1, 2].
Age-Related Changes in the Chemical Composition of the Nail Plate The nail plate is made up of tightly layered cornified cells that are generated by the nail matrix epithelium and consists mainly of intermediate filamentous proteins or keratins (80–90% which are of hard keratins) [5]. The keratins are embedded in a matrix composed of nonkeratin proteins (high-sulfur and high-glycine/tyrosine proteins). Other nail plate constituents include water (7 and 18%), lipids (0.1–5%), and trace elements (mainly iron, zinc, calcium, and magnesium) [1, 2, 5–7]. Normally, the nail plate calcium (Ca) content is low (0.2% by weight), while the sulfur content is high (10% by weight) [4]. It is believed that the relatively higher sulfur content
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to Ca content contributes to nail plate hardness, especially that the former is a reflection of the cysteine disulfide bonds which stabilize fibrous proteins [6]. Even though the Ca content of the nail does not seem to contribute to the hardness of the nail plate, a recent study revealed that both fingernail and toenail Ca concentration decrease with advancing age while magnesium (Mg) concentration tend to increase [6]. Interestingly, this study also showed that measurement of the Ca and Mg contents of the nail plate may be used as osteoporosis predictor. The iron content usually decreases in senile nails [1, 2]. Another constituent of nail plates are membrane-forming integral lipids such as cholesterol and cholesterol sulfate, which are also present in keratinized skin areas and hair. There is an age-related significant decrease in fingernail cholesterol sulfate levels which has been reported in women and may explain the higher incidence of brittle nails in the elderly category [8].
Age-Related Changes in Nail Histology Compared to young individuals, the dermis of the nail bed usually exhibits elastic tissue degeneration and thickening of the blood vessels. In addition, the keratinocytes of the nail plate are commonly enlarged and show increased remnants of keratinocyte nuclei, which are also known as pertinax bodies [1, 2, 9]. To the best of the authors’ knowledge, there are no reports describing the changes that may occur in the nail matrix as a result of aging.
Age-Related Nail Dystrophies Several nail disorders affect the population at large and may appear with advancing age and include, without order of frequency or age-related prevalence, brittle nails, infections (especially onychomycosis), onychauxis, onychocryptosis, onychoclavus, onychogryphosis, onychophosis, splinter hemorrhages, subungual hematoma, and malignancies of the nail apparatus [1, 2, 7–12].
Brittle Nails Brittle nail disorder is characterized by increased nail plate fragility and is considered to be a polymorphic abnormality affecting about 20% of the population with higher incidence in women and the elderly [7]. Clinically, brittle nails manifest with onychoschizia and onychorrhexis, the severity of either may be variable [7].
Onychoschizia, which results from impairment of intercellular adhesion of nail plate corneocytes, usually presents as a lamellar splitting of the distal portion and free edge of the nail plate, and a transverse splitting secondary to breakage of the lateral edges. Underlying causes are usually exogenous and include trauma, repetitive cycles of wetting and drying, the action of fungal proteolytic products, and the use of chemicals or cosmetics such as nail enamel solvents, cuticle removers, and nail hardeners, among others. Onychorrhexis, which results from abnormalities in epithelial growth and keratinization secondary to nail matrix involvement, usually manifests as longitudinal thickening, splitting or ridging of the nail plate, and/or multiple splits leading to triangular fragments at the free edge. Abnormalities of vascularization and oxygenation (such as arteriosclerosis or anemia), dermatological (inflammatory diseases and disorders of cornification), and systemic diseases (endocrine, metabolic, etc.) are among the various factors that may underlie the abnormalities of growth and keratinization responsible for onychorrhexis. Recently, a composite scoring system assessing the severity of nail brittleness based on the degree of ridging, nail thickness, lamellar, longitudinal, and transverse splitting has been proposed [7]. Management of brittle nails may not be easy or simple [7]. The initial therapeutic approach is to determine the predominance of either onychoschizia or onychorrhexis. Underlying factors should then be identified and if possible, be corrected. After that, general and specific measures may be followed. Nail hydration by 15 min daily soaks of the nail and by the application of emollients rich in phospholipids may be useful. Strengthening the nail plate may be accomplished by the application of nail hardeners containing formaldehyde; however, these should be utilized cautiously because they may lead to brittleness, onycholysis, and subungual hyperkeratosis. Although enamel application may mechanically protect the nail and fill the fractures; its removal however, may lead to substantial dehydration. Several studies have shown that daily oral intake of biotin (2.5 mg/d) for 1.5–15 months may be beneficial; however, these studies are small and not double blind placebo-controlled [7, 10, 11].
Infections Infections by different pathogens may affect the nail plate either primarily or through extension from involved adjacent structures such as the nail folds [1, 2, 10–12].
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Onychomycosis, a fungal infection of the fingernails and/or toenails, is the most common infection and represents up to half of all nail diseases [12]. It affects 10–20% of adults, especially the elderly. Multiple factors are associated with an increased risk of onychomycosis including old age, male gender, underlying medical diseases (diabetes, peripheral arterial disease, and immunodeficiency), smoking, and predisposing genetic factors [12]. More than 90% of onychomycosis cases are caused by dermatophytes, among which Trichophyton rubrum and T. mentagrophytes are the most common. Other less commonly encountered causative organisms include yeasts such as Candida and nondermatophyte molds such as Scopulariopsis brevicaulis and Scytalidium hyalinum [12]. Five clinical subtypes of onychomycosis are recognized [12]. Distal and lateral subungual onychomycosis (DLSO), the most common subtype usually caused by T. rubrum, manifests as subungual hyperkeratosis, onycholysis, nail thickening, and discoloration secondary to fungal invasion which starts at the hyponychium and spreads proximally along the nail bed. Superficial onychomycosis usually presents as white (caused by T. mentagrophytes) or black (caused by dematiaceous fungi) patchy nail discoloration due to fungal invasion of the dorsal surface of the nail plate. Proximal subungual onychomycosis (PSO) commonly affects immunocompromised individuals and presents clinically as a white spot under the lunula that progresses distally. It results from fungal invasion (usually T. rubrum), from the proximal nail fold to the nail plate. Endonyx onychomycosis (EO) is an uncommon form caused by T. soudanense; it resembles DLSO; however, the nail thickness is within normal and there is no hyperkeratosis or onycholysis. Total dystrophic onychomycosis (TDO) is an advanced form, characterized by progressive nail plate destruction leaving an exposed abnormally thickened nail bed. TDO may be observed in immunodeficient patients such as those with chronic mucocutaneous candidiasis and is fairly acute or may be progressive representing an end stage of other forms of onychomycosis. Effective treatment of onychomycosis entails making an accurate diagnosis and identifying the causative pathogen [12, 13]. Several diagnostic methods including KOH-based microscopy, fungal cultures, and histopathology with PAS may be used alone or in combination; the latter being the most sensitive [12, 13]. The treatment options include oral and/or topical antifungal agents, mechanical or chemical treatments, or a combination of these. The choice of the therapy should be individualized based on several factors such as the causative agent, the potential for drug interactions and side effects, the
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number of nails involved, the severity of onychomycosis, and the cost. Currently, terbinafine appears to be the most effective oral agent for treating dermatophyte onychomycosis, especially in elderly patients due to its fungicidal action, safety, and low potential for drug interaction [13]. The azoles (ketoconazole, fluconazole, itraconazole) can also be used but are generally considered to be less effective than terbinafine as they are fungistatic rather than fungicidal [13]. Paronychia, seen occasionally in elderly patients, is an acute or chronic infection of the nail folds which may lead to secondary changes in the nail plate [1, 2]. Acute paronychia, usually caused by Staphylococcus aureus, most commonly presents as tender erythematous swelling of only one nail and is typically trauma-induced. Management includes abscess drainage, warm saline soaks, and the use of topical or systemic antibiotics. Chronic paronychia is commonly caused by Candida species or Gramnegative bacteria and presents clinically as red and swollen nail folds with loss of cuticle and multiple secondary transverse ridges in the nail plate. Keeping the nail fold dry coupled with topical antifungal or antiseptic agents are the treatment of choice. Elderly patients, similar to infants and immunosuppressed hosts, are prone to uncommon presentations of Sarcoptes scabiei infestation in which all skin surfaces such as the scalp and face, as well as nails may be affected. The mite may inhabit and persist in subungual hyperkeratotic debris, leading to prolonged infestations and/or epidemics among elderly patients and those caring for them in nursing homes. Cutting the nails as much as possible and brushing their tips with a scabicide is an adjunct modality to the antiscabetic treatment [1, 2].
Onychauxis Onychauxis is a localized hypertrophy of the nail plate which presents clinically as discoloration, hyperkeratosis, loss of nail plate translucency, and often subungual hyperkeratosis [1, 2]. The underlying cause/s may be related to the aging process or to faulty biomechanics that tend to be more common in the geriatric population. Overlapping and underlapping toes, digiti flexi (contracted toes secondary to buckling of toes induced by shortening of the controlling muscles), or foot-to-shoe incompatibility are examples of these faulty mechanics. Onychauxis may be associated with pain, and, with time, may be complicated by distal onycholysis, increased risk for onychomycosis, subungual hemorrhage, and/or subungual ulceration.
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Partial or total debridement of the thickened nail plate should be done periodically. Other treatment options include the use of electric drills, 40% or higher urea paste, or nail avulsion. In complicated cases or those with recurrences, chemical or surgical matricectomy may be done to achieve permanent ablation of the affected plate.
Onychoclavus Onychoclavus, also known as subungual corn, is a hyperkeratotic process most commonly located under the distal nail margins [1, 2]. It results from chronic minor trauma and persistent localized pressure by bony abnormalities such as digiti flexi, rotated fifth toes, foot-to-shoe incompatibility, or hallux valgus (the great toe rotates toward the second toe). It usually presents as a tender dark area under the nail plate (most commonly the great toenail) and may be confused with benign and malignant subungual melanocytic lesions and subungual exostosis [1, 2]. The treatment of this condition is surgical removal of the hyperkeratotic tissue and the correction of the underlying bony abnormality.
Onychogryphosis Onychogryphosis, also known as ram’s-horn nail, is a term used to describe thickening and enlargement of the nail plate, most commonly the great toenails [1, 2]. The affected nail plate is usually brownish to opaque in color, grows faster on one side than the other, may have many grooves and transverse striations, and is commonly associated with nail bed hypertrophy. This nail dystrophy is common among the elderly population, and if untreated, may lead to a walking disability. In patients with diabetes mellitus or peripheral vascular disease, onychogryphosis may be complicated by subungual gangrene due to pressure effects [1]. Infrequent nail cutting, trauma, foot-to-shoe incompatibility, and bony abnormalities such as hallux valgus are responsible for its pathogenesis. Onychogryphosis should be distinguished from hemionychogryphosis, which is characterized by the lateral growth of the nail plate from the onset as a complication of persistent congenital malalignment of the great toenails. Beyond cosmetic considerations, treatment of onychogryphosis in the elderly may be mandatory in order to prevent disability and its complications [1, 2]. Conservative management with the use of an electric drill or burr
to file the involved nail plate is the initial step followed by removal of subungual hyperkeratosis and subsequent periodic nail plate trimming. Other more radical approaches such as nail avulsion, with or without matricectomy may be valuable in selected patients.
Onychophosis Onychophosis is a localized or diffuse hyperkeratosis under the nail plate (subungual), on the lateral or proximal nail folds, or in the space between the nail plate and nail folds [1, 2]. The first and the fifth toes are the digits of predilection. Multiple nail and adjacent soft tissue abnormalities including onychocryptosis, nail fold hypertrophy, and onychomycosis may be the underlying causes of onychophosis. Other external causes such as repeated minor trauma and foot-to-shoe incompatibility may be contributing. Several treatment modalities may be used to treat onychophosis and include keratolytic agents (urea 20% or salicylic acid 6–20%), nail packing, and, if needed, surgical excision. Recurrences may be prevented by wearing appropriate comfortable shoes to minimize pressure effects of the nail plate on surrounding nail folds.
Onychocryptosis (Ingrown Toe Nail) Although more commonly observed in young adults, onychocryptosis may occasionally be encountered in the elderly causing significant pain, difficulty walking, and disability [1, 2, 14]. It occurs when the lateral nail plate penetrates the adjacent nail fold as a result of nail plate over-curvature, subcutaneous in-growing toenail, and/or lateral nail fold hypertrophy. Clinically, patients commonly present with tenderness and inflammation of the lateral nail fold, which at times, may be associated with granulation tissue formation and secondary infection. The most common underlying causative factor/s include improper nail cutting, ill-fitting or high-heeled shoes, hyperhidrosis, long toes, prominent nail folds, and bony abnormalities such as hallux valgus. The management of ingrown toenail consists of treating the acute signs and symptoms and correcting the underlying predisposing factors [1, 2]. There is a evidence that the best chance for complete cure is to excise the lateral nail plate, to curette the granulation tissue, and to perform lateral matricectomy [1, 2, 14]. This procedure may be complicated by postoperative nail bed infection
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or by regrowth of a nail spicule secondary to incomplete matricectomy. In addition this procedure, not uncommonly, may lead to recurrences and poor cosmetic results. Recently, surgical decompression of the ingrown toenail without matricectomy has been proven to be very effective. In this approach, a large volume of soft tissue around the nail plate is removed and the inflammation is relieved [14]. The advantage of this maneuver is complete preservation of the nail anatomy and function with excellent therapeutic and cosmetic results.
Splinter Haemorrhages Splinter hemorrhages are linear discolorations under the nail plate that progress over a period of few days from an initial red color to a dark brown or black color [1, 2]. The location of the splinter hemorrhages may give leads to the underlying pathogenesis. Splinter hemorrhages located in the middle or distal third of the nail plate are usually associated with trauma, while those located proximally are commonly associated with systemic diseases such as infective endocarditis, cholesterol emboli, or connective tissue disorders. Proximal type-splinter hemorrhages especially those associated with systemic diseases are generally more common among young adults, whereas several studies have shown that trauma-associated distal splinter hemorrhages are observed frequently in the elderly population. Trauma-induced splinter hemorrhages commonly resolve on their own, while the proximal-type splinter hemorrhages require treatment of the underlying systemic disorder.
Subungual Hematomas Subungual hematomas are common among the elderly and are most frequently induced by trauma, which may or may not be remembered [1, 2]. The event may result in nail bed laceration and bleeding under the nail plate. Amyloidosis, diabetes mellitus, or anticoagulant therapy may also be less common causes of sunbungual hematomas. Early on, it presents as a tender red subungual discoloration that tends to move forward and becomes bluish and less painful with time. The forward and distal movement of this discoloration under the nail plate is a clinical clue that serves to distinguish this lesion from melanocytic proliferations including melanoma. Occasionally, distal onycholysis with subsequent spontaneous nail plate avulsion may occur.
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Reassurance and observation of the nail is the main management strategy. However, in acute painful cases, pressure may be relieved by drilling a hole through the nail plate. Chronic cases are best left to heal spontaneously after ruling out melanoma.
Bowen’s Disease of the Nail Apparatus Bowen’s disease of the nail unit, also known as in situ epidermoid carcinoma, is a nonaggressive malignancy most commonly originating in the epithelium of the nail folds or grooves [15]. The incidence is highest in patients aged between 50 and 69 years, and usually affects the fingers, particularly the thumb [15]. Classical presentations include subungual or periungual ulcerated hyperkeratotic or papillomatous proliferations with associated onycholysis. Rarely, this condition may present as LM or erythronychia. Ulceration or bleeding is usually indicative of invasion which may be deep and reach the underlying contiguous bone in less than 20% of patients. Distant metastatic rate is usually low. The etiology remains unclear; trauma, X-ray exposure, arsenic, chronic paronychia, and human papilloma virus (HPV) infection have all been implicated. The latter has been implicated because HPV 16, 34, and 35 have been detected in many cases of in situ and invasive Bowen’s disease of the nail apparatus, and this raised speculation about a role for genital-digital transmission of the virus. Mohs’ micrographic surgery is the treatment of choice for this condition [15]. Other less effective modalities have been used and include regular excision, electrosurgery, liquid nitrogen, imiquimod, photodynamic therapy, intra-arterial infusion with methotrexate, and radiation therapy. Amputation of the distal phalanx should be done in case of bone involvement. Regular follow-up is essential in view of the potentially polydactylous nature of this disease.
Nail Apparatus Melanoma Nail apparatus melanoma (NAM) usually occurs in Japanese and African Americans, with a relative incidence of 23 and 25%, respectively [16]. It is rarely observed in the white population. It most commonly occurs in the elderly with a mean age at diagnosis of 60–70 years [16]. The most frequent histogenetic type is acral lentiginous melanoma. The classical presentation is a solitary longitudinal melanonychia (LM) of the thumb, index finger, or big toe. In addition, Hutchinson sign (brown or black pigment
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extension from the matrix and nail bed onto the surrounding tissues) may be present and accounts for the radial growth phase of this melanoma. Rarely, a homogeneous or irregular black spot in the matrix or nail bed may be the only sign of a subungual melanoma. Although NAM appears to have a worse prognosis than its cutaneous counterpart, this may be related to the delay in diagnosis of the former. A strong index of suspicion is required when confronted with an isolated LM, especially in an elderly patient [16]. If a biopsy is obtained and the diagnosis is confirmed, treatment of NAM is then tailored based on the stage of the melanoma. Total excision of the entire nail apparatus or Mohs’ surgery is the treatment of choice for in situ melanoma, while invasive melanomas should be managed with distal phalanx amputation. Adjuvant chemotherapy may also be needed in advanced cases.
Other Nail Conditions Several other conditions should be kept in mind when evaluating nail changes in an elderly patient. These include nail changes associated with cutaneous inflammatory disorders (such as psoriasis or lichen planus) [17], nail cosmetics [18, 19], systemic disorders commonly observed in the elderly (such as renal disease) [20], or medications as the elderly patients are usually on multidrug therapy (such as anticoagulants, anticonvulsants, or beta-blockers) [21].
Conclusion Elderly patients may complain of nail changes and dystrophies that may be of cosmetic concern, cause pain, affect daily activities, or be malignant. Awareness of these conditions is essential to reach the correct diagnosis and provide appropriate management.
References 1. Cohen PR, Scher RK. Geriatric nail disorders: diagnosis and treatment. J Am Acad Dermatol. 1992;26(4):521–531.
2. Singh G, Haneef NS, Uday A. Nail changes and disorders among the elderly. Indian J Dermatol Venereol Leprol. 2005;71(6):386–392. 3. Horan MA, Puxty JA, Fox RA. The white nails of old age (neapolitan nails). J Am Geriatr Soc. 1982;30(12):734–737. 4. Saraya T, Ariga M, Kurai D, et al. Terry’s nails as a part of aging. Intern Med. 2008;47(6):567–568. 5. Lynch MH, O’Guin WM, Hardy C, et al. Acidic and basic hair/nail (‘hard’) keratins: their colocalization in the upper cortical and cuticle cells of the human hair follicle and their relationship to ‘soft’ keratins. J Cell Biol. 1986;103:2593–2606. 6. Ohgitani S, Fujita T, Fujii Y, et al. Nail calcium and magnesium content in relation to age and bone mineral density. J Bone Miner Metab. 2005;23(4):318–322. 7. van de Kerkhof PC, Pasch MC, Scher RK, Kerscher M, Gieler U, Haneke E, Fleckman P. Brittle nail syndrome: a pathogenesis-based approach with a proposed grading system. J Am Acad Dermatol. 2005;53(4):644–651. 8. Brosche T, Dressler S, Platt D. Age-associated changes in integral cholesterol and cholesterol sulfate concentrations in human scalp hair and finger nail clippings. Aging (Milano). 2001;13(2):131–138. 9. Lewis BL, Montgomery H. The senile nail. J Invest Dermatol. 1955;24(1):11–18. 10. Colombo VE, Gerber F, Bronhofer M, Floersheim GL. Treatment of brittle fingernails and onychoschizia with biotin: scanning electron microscopy. J Am Acad Dermatol. 1990;23:1127–1132. 11. Hochman LG, Scher RK, Meyerson MS. Brittle nails: response to daily biotin supplementation. Cutis. 1993;51:303–305. 12. Gupta AK, Ricci MJ. Diagnosing onychomycosis. Dermatol Clin. 2006;24(3):365–369. 13. Gupta AK, Tu LQ. Therapies for onychomycosis: a review. Dermatol Clin. 2006;24(3):375–379. 14. Noe¨l B. Surgical treatment of ingrown toenail without matricectomy. Dermatol Surg. 2008;34(1):79–83. 15. Baran R, Richert B. Common nail tumors. Dermatol Clin. 2006; 24(3):297–311. 16. Andre´ J, Lateur N. Pigmented nail disorders. Dermatol Clin. 2006; 24(3):329–339. 17. Holzberg M. Common nail disorders. Dermatol Clin. 2006; 24(3):349–354. 18. Dahdah MJ, Scher RK. Nail diseases related to nail cosmetics. Dermatol Clin. 2006;24(2):233–239. 19. Rich P. Nail cosmetics. Dermatol Clin. 2006;24(3):393–399. 20. Tosti A, Iorizzo M, Piraccini BM, et al. The nail in systemic diseases. Dermatol Clin. 2006;24(3):341–347. 21. Piraccini BM, Iorizzo M, Starace M, et al. Drug-induced nail diseases. Dermatol Clin. 2006;24(3):387–391.
34 Climacteric Aging and Oral Hormone Replacement Therapy Pascale Quatresooz . Claudine Pie´rard-Franchimont . Ge´rald E. Pie´rard
Introduction In affluent societies of the West, a woman’s appearance is largely appreciated through her skin aspect, which is thought to reflect in part her general health. Inevitably, skin like any other human tissue, undergoes regressive changes with age. Menopause is the time when permanent cessation of menstruation occurs following the loss of ovarian activity. The prefix ‘‘meno,’’ meaning month, is derived from the Greek, and it has been used to refer to the menstrual cycle. ‘‘Pause’’ indicates the cessation of the process. The transition from regular ovulatory cycles to the menopausal state is not an instantaneous event. Rather, a series of progressive hormonal and clinical alterations reflects the decline in the ovarian activity. The period of time between the reproductive period of life and the postmenopausal years is referred to as perimenopause or climacteric. It includes the last years prior to menopause when endocrinologic, clinical, and biologic changes associated with menopause are occurring, as well as the first year following menopause. Postmenopause, on the other hand, is defined as the year of menopausal amenorrhea and the time thereafter. Aging of humans is a physiologic process characterized by a progressive loss in homeostatic capacity of the organism, ultimately increasing the vulnerability to environmental threats and to certain disease status. Obviously, the aging process evolves at different rates among individuals of the same age. In addition, any given subject shows a variable senescence status among his/her organs, and among each of the constituent tissues, cells, and subcellular structures. In addition, intracellular and extracellular molecules are also involved differently in aging. Within each organ system, aging usually manifests as a progressive and almost linear reduction in maximal function and reserve capacity. Menopause is probably an exception to this timetable as it appears as a major turning point of importance in women’s life. In addition, the menopause effects on skin are intermingled with age-associated physiological decrements resulting from acute and chronic
environmental insults. Menopause appears to spot a decline in skin qualities [1]. Any part of the skin is subject to alterations, including the epidermis, dermis, hypodermis, and hair. Life expectancy of women is substantially longer than that of men, but women often experience greater burdens of morbidity and disability. In many societies, the rapid trend in the aging population, combined with the increasing feminization of aging, contributes to the need for a sharp focus on gender issues. As the proportion of older women grows at rapid rates in the global population, the challenges of learning more about the skin condition of this group is welcomed. One of these priorities involves discovering more about the physiology and treatment of menopause and the climacteric period. Both risk factors and health needs are likely to change as women enter the climacteric period. Menopause has been shown to have a potential role in the etiology of some age-related diseases and particular physiological conditions. The continuing increase of woman life expectancy has resulted in a marked increase of women who live years beyond menopause. Indeed, women can nowadays expect to live onethird of their lives in a potential hormonally deficient state. Using age 50 as a proxy for menopause, about 25 million women undergo menopause each year. By 2030, the world population of postmenopausal women is expected to increase to 1.2 billion, with 47 million new entrants each year [1].
Menopause in the Overall Aging Process Menopause appears as a milestone in the women aging process, which is a universal and global evolution showing many different characteristics. It is recognized that the multifacet process of aging is different among organisms. Two distinct classifications of life evolution and aging histories are of major importance when considering aging models. The first classification distinguishes species exhibiting a clear distinction or not exhibiting a clear
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distinction between germ cells and somatic cells. The second classification identifies semelparous species, reproducing only once in their lifetime, and iteroparous species, which reproduce repeatedly. The concept of aging is most clearly defined in iteroparous species, which have a distinct soma separate from the germ line. Aging needs to be clearly qualified when applied to species with other kinds of life history. It is mistaken, for example, to regard the postreproductive end of life of semelparous species, which usually occurs in highly determinate fashion, as being comparable with the more protracted process of senescence in iteroparous species. Physical growth and senescence are both characterized by cumulative progression of interlocking biologic events. They are not always separated in the timetable of life as they may proceed in tandem. There is evidence that depletion in estrogens exert a prominent influence on aging of a series of body systems including bones, the cardiovascular system, and the skin [2]. For years, the rationale for hormone replacement therapy (HRT) in menopausal women appeared straightforward for many physicians. Such replenishment therapy convincingly showed evidence for alleviating the skin atrophy and xerosis in postmenopausal women [1–3].
Skin Aging in Perspective Conceptually, global human aging may be perceived as one single basic process of physiological decline progressing with age. Over the past decades, the understanding of aging skin has considerably expanded, with emphasis on differentiating true chronologic aging changes from photoaging resulting from habitual chronic sun exposure [4]. The action spectrum of photodamages is not fully characterized but it is acknowledged that the cumulative effects from repeated exposures to suberythemal doses of ultraviolet B (UVB) and UVA in human skin are involved in these processes. The role of UVB in elastin promoter activation in photoaging is evident. UVA also contribute significantly to long-term actinic damage, and the spectral dependence for cumulative damages does not parallel the erythema spectrum for acute UV injury on human skin. The near infrared radiations bring additional deleterious effects participating in skin aging. Such a concept based on a duality in skin aging has been challenged because it may appear as an oversimplification in the reality of life. Another more diversified classification of skin aging in seven distinct types was offered [5]. The most important variables include the endocrine and overall metabolic status, the past and
present lifestyle, and several environmental threats including cumulative UV and infrared radiations, and repeated mechanical solicitations by muscles and external forces such as earth gravity (> Table 34.1). In this framework, the climacteric aging is individualized and emphasized in the endocrine type of aging. The global aging is considered to result from the cumulative or synergistic effects of each specific cause. Increased awareness of the distinct age-associated physiologic changes in the skin including the menopause effects allows for more effective skin care regimens, preventive measures, and dermatologic treatment strategies. The immutability of skin aging is challenged by this way. In this context, skin aging appears as a notoriously complex process. In particular, the ideal appearance, structural integrity, and functional capacity of the skin require an adequate balance between many hormonal influences. Any alteration in this controlled system results in significant changes in skin qualities [6]. Among hormones, estrogens and the other sex steroids have profound influences on both skin development and composition [1–3, 6, 7]. The relative hypoestrogenemia associated with menopause contributes to, and probably exacerbates any other deleterious effect of age. Therefore, a gender perspective is required for a full understanding of skin aging. Both from the physiological and psychosocial viewpoints, the determinants of global aging are closely related to the skin aspect and to the gender. Yet until recently the specific gender perspective of aging was rather neglected by investigators. However, the past decade or so has witnessed progresses in understanding the hormonal involvement in the global aging process [6].
. Table 34.1 Types of cutaneous aging Aging type Genetic
Determinant factor Genetic (premature aging syndromes, phototype-related)
Chronologic
Time
Actinic
Ultraviolet and infrared irradiations
Behavioral
Diet, tobacco, alcoholic abuse, drug addiction, facial expressions
Endocrinological Pregnancy, physiological, and hormonal influences (ovaries, testes, thyroid) Catabolic
Chronic intercurrent debilitating disease (infections, cancers)
Gravitational
Earth gravity
Climacteric Aging and Oral Hormone Replacement Therapy
Gender-Linked Aging The effects of estrogen have been studied on several body systems including the skin [1–3]. Since its introduction as a therapeutic agent about 60 years ago, estrogen is acknowledged to exhibit anti-aging effects on women’s skin because several critical functions of the skin are hormonedependent. In particular, estrogen receptors and their associated proteins have been identified [1, 7, 8]. The normal ovarian cycle is the result of a complex interaction between the hypothalamus, pituitary gland, and ovaries. It is further modulated by higher cortical centers, the thyroid gland, the adrenals, and some peripheral hormonal production. The ovulatory cycle starts with the recruitment of a number of follicles from which one becomes dominant and is the source of ovulation. From puberty to menopause, about 200,000 follicles give rise to 500 mature oocytes. Hence, atresia appears as the dominant and continuous process in ovarian physiology. This is a key element, which leads to menopause [1]. During the normal menstrual cycle, estradiol is the dominant estrogen, reaching a peak level at the time of ovulation. Circulating levels of FSH and LH are characterized by a mid-cycle surge. During the climacteric period, the function of the ovaries is progressively failing. The transition from regular ovulatory cycles to the perimenopause and menopause is characterized by variations in cycle length and bleeding pattern. Women who experience menopause at a young age usually have a short transition phase. By contrast, menopause of later occurrence is associated with a variety of long and short intermenstrual bleeding episodes, and an overall increased mean cycle length. During the transition phase of perimenopause, there is large variability in sex steroid production including estrogen release. The amount of circulating estradiol varies from cycle to cycle, probably representing varying degrees of follicular maturation and function. The perimenopausal ovary requires greater amounts of FSH to stimulate estrogen production. Contrary to older belief, estradiol levels do not gradually wane in the years before menopause, but remain in the normal range until follicular growth and development cease [1]. In contrast to estrogens and progestins, androgen levels remain stable during this transitional period. Androstenedione, testosterone, dehydroepiandrostenedione (DHEA), and DHEAsulfate (DHEAS) do not show any change in circulating concentrations prior to menopause. The most sensitive measure for declining ovarian function during perimenopause relies on the assessments of serum gonadotropins, particularly FSH. There is a dramatic rise of FSH during menopause, followed by a
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slow decline over the ensuing decades. LH levels may remain normal in the face of elevated FSH.
Skin Climacteric Aging Estrogens and other sex steroids exert profound influences on both skin biology and composition. Thus, it is believed that adequate hormone levels are required to control the skin structural integrity and functional capacity. In addition to estrogen and androgen receptors in the skin, aromatase activity is present in fibroblasts, adipocytes, and sebocytes in postmenopausal women. As a result, androgens are possibly switched in situ to estrogens. Sex steroids clearly exert a key role in the skin aging process as evidenced by the accelerated decline in skin appearance from the perimenopausal years onwards. These changes have not been studied thoroughly, although histological findings have demonstrated that the estrogen and progesterone receptors in the skin show a relative decline in their expression from the time of the climacteric [7]. Estrogen receptors have been identified in cells of both the epidermis and the dermis [1, 8]. However, their regional distribution within the skin varies considerably in keeping with the concentration seen within the female genital tract. A high estrogen to androgen receptor ratio is present in the vagina, and a reverse ratio with an increase in androgen and a decrease in both estrogen and progesterone receptors is present in the vulva. The menopause and its specific HRT still leave a great many challenges unresolved at the level of the skin. In particular, they address the issues of HRT effects on a series of physiological functions of the epidermis and dermis. In that field, dissension and controversy are rife. Glaring discrepancies are present in the current literature. In a global view, HRT appears to markedly improve climacteric changes in many organs including the skin [9–11]. The bulk of recent studies confirms that estrogens or estro-progestins effectively suppress the climacteric syndrome and genital atrophy, while significantly decreasing the risk of osteoporotic fractures. The influence of menopause on skin and its correction by HRT and specific topical treatments may prove to be difficult to objectivate by clinical inspection alone. Several relevant aspects are, however, conveniently rated in a semiquantitative manner. The visual and tactile perception of skin qualities is a valuable tool in clinical dermatocosmetology. However, it lacks sensitivity and reproducibility when comparative evaluations are made over a prolonged period of time. In addition, the external appearance is sometimes misleading compared to the
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actual changes induced by treatments including HRT. By contrast, noninvasive objective methods of biometrology are well suited for improving the reliability and preciseness of assessments. Some skin changes of climacteric aging are detected as early as in the early postmenopausal years, when HRT might be of benefit to control them. Cutaneous changes observed in the decade following menopause are both age- and hormone-related [9, 10, 12]. Postmenopausal women commonly complain of generalized xerotic, easily bruising and wrinkled skin. Dermal thickness apparently decreases with time after menopause. It has been reported that the decline in dermal collagen occurs at a rapid rate immediately after menopause, and becomes more gradual thereafter [1] Approximately 30% of skin collagen was reported to be lost in the first 5 years after menopause, with an average decline of about 2% per postmenopausal year over a period of 20 years [1]. In this context, it is difficult to differentiate the consequences of menopause from age-associated changes related to a decline in growth hormone [6]. Indeed, both estrogen and growth hormone depletions are combined in aging women. The relative estrogen reduction at the perimenopause contributes to and exacerbates the negative effects of age. As a consequence, the effects of HRT on skin have deservedly attracted much interest [1, 12, 13], although the issue remains controversial [12, 14, 15]. Anyway, the bulk of the literature indicates that hypoestrogenemia has a detrimental effect on skin collagen content, which is partially addressed by HRT. The maximum effect at preventing skin aging appears to occur when HRT is initiated early in the perimenopausal years [1]. However, shortterm treatments fail to bring significant improvements in the skin condition [14, 15]. Some controversial data are also found in the literature [12, 16].
HRT and the Dermal Extracellular Matrix The dermis is a tough connective tissue matrix supporting the various structures embedded in it. It contains highly stable fibers, predominantly made of collagen and elastin. Collagen represents about 80% of the dry weight of the adult skin. It exhibits high tensile strength and prevents the skin from being torn by overstretching. Elastic fibers, which compose about 5% of the dermis serve to recoil the skin to its initial shape after deformation. Fibroblasts and dermal dendrocytes synthesize and control all the components of the extracellular matrix. Light microscopy reveals that the collagen network of sun-protected skin areas are thinner and less compact in aged people.
The interstitial material between the collagen bundles contains hyaluronic acid and other glycosaminoglycans. Interest in molecular biology and morphology have deferred comprehension of more important structural changes in the collagen network. It may become considerably distorted by lifelong mechanical stresses. Moreover, the number of dermal cells declines with age and they exhibit the shape of a shrunken fibrocyte, becoming narrower with a much cytoplasm, suggesting a decreased metabolic activity. A number of studies on the HRT effects on the dermis focused on changes in its thickness, collagen content, and mechanical functions. HRT administration modalities were clearly different among trials. Various estrogens were used in combination or not with cyclic administration of progesterone derivatives to prevent endometrial hyperplasia. Most often, the information has been discussed collectively without distinguishing the effects of estrogens from those of estrogens and progesterone derivatives in combination [1]. Globally, the dermal collagen content and the dermal thickness appear to be maintained in HRT receivers compared to age-matched untreated women [1, 9, 10]. In women with a lowered skin collagen content, estrogen replenishment is believed to be initially of corrective and later of prophylactic value, while in those women with mild reduction of collagen content in the early menopausal years estrogens are of prophylactic value only [1, 16, 17]. Thus, a depletion in skin collagen may be in part corrected but not overcorrected. The replenishment in skin collagen content may show some regional variability with a more pronounced effect on the abdomen than thigh [1]. At present, no consensus has been reached about the value of HRT on climacteric aging of the dermis. Some authors deny any significant effect [14, 15]. Others feel that there are different levels of skin response with good and poor responders [11, 17–19]. The latter poor responsive result may correspond to smokers or to women who have only recently entered the menopausal period and have not yet lost estrogen-replaceable collagen [1]. The water content stored in the dermis is bound to the hydrophilic glycosaminoglycans. Such feature helps protecting the skin against excessive tissue compression while maintaining its suppleness. Estrogens increase dermal hygroscopic properties, probably through enhanced synthesis of dermal hyaluronic acid [1]. A specific role for versican, if any, is not firmly established. The quantitative changes and the decrease in compactness of the collagen bundles in the dermal matrix lead to progressive skin slackness. The resulting aging
Climacteric Aging and Oral Hormone Replacement Therapy
aspect is characterized by a progressive increase in extensibility associated with a loss of elasticity [20, 21]. Some wrinkles are the result of these functional changes [11]. The climacteric period appears to be responsible for wrinkles particularly on the forearms and face. Several controlled trials have shown the benefit of HRT for mitigating these changes [17, 18]. Fine wrinkling, atrophy, and a progressive deepening of facial creases ensue. These skin alterations were reported to be partially reversed in postmenopausal women receiving estrogen or combined HRT [18, 22]. The marked increase in skin extensibility commonly occurring in untreated perimenopausal women, appears limited by HRT, which therefore helps prevent skin slackness [18, 22]. Hence, HRT may exert a beneficial effect on the facial skin by reducing the age-related rheological changes without, however, limiting the number and depth of wrinkles [9, 13, 18, 22]. The maximal effect at preventing skin aging appears to occur when HRT is started early [10].
HRT and the Dermal Microvasculature Flushes at menopause appear to be caused by a prominent vasodilatation particularly in the face, neck, chest, palms, and soles. Their prevalence during the early menopausal years is in part explained by the loss of peripheral vascular control seen in association with estrogen deficiency. This phenomenon is corrected by HRT leading to abolition of the flush. Indeed, estrogens appear to enhance both endothelium-dependent and endothelium-independent vasodilatation in the skin of women [23–25]. A quantitative biometrological study showed a higher red intensity value (parameter a*) in menopausal women receiving HRT for at least 1 year [12]. However, the maximum inducible vasodilation in the forearm skin was reported to be reduced in postmenopausal women receiving estrogen replacement and premenopausal women, compared with untreated postmenopausal women [23]. The beneficial effect of HRT on the skin blood flow has, however, been challenged. HRT users may have fewer chronic leg ulcers and pressure-induced ulcers [26]. Estrogen might increase the wound healing rate in the elderly. This finding warrant confirmation before recommending HRT to improve wound healing.
HRT, Epidermis, and Lips Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses such as those induced by H2O2, other oxygen species, and a variety of chemicals.
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Cells in replicative senescence share common features with cells in SIPS including morphology, senescenceassociated b-galactosidase activity, cell cycle regulation, gene expression, and telomere shortening. Telomere shortening is then attributed to the accumulation of DNA single-strand breaks induced by oxidative stress. Thus, SIPS could be a mechanism of the in vivo accumulation of senescent-like cells in the skin, and DNA damage plays a key role in skin aging and photoaging. The estrogen depletion possibly promotes SIPS. Xerosis is an alteration of the stratum corneum known as dry skin to the laity. This condition results from an altered desquamation process that is often associated with decreased hydration of the upper layers of the stratum corneum and with weakening of the barrier function of the skin. The hydration, the water-holding capacity, and the barrier function of the stratum corneum appear to be increased following HRT [12, 27, 28]. Lip properties are quite distinct from those of the face and other body areas. Lip tissues are subjected to repeated mechanical and other physical and chemical stresses. Age-related changes in geometrical dimensions of lips have been reported with resulting alteration of the levels of extensibility and contractibility [29, 30]. There was also marked differences in the hydration level of the surface as the upper lip appeared more hydrated than the lower one. It was claimed that any hormonal effect was most unlikely in age-related changes in lip surface hydration and lip mechanical properties [30].
HRT and Pilosebaceous Follicles Hair loss, particularly the frontal fibrosing alopecia, is apparently associated with menopause, but it is not corrected by HRT. Tibolone, which is an alternative to HRT may increase the severity of diffuse alopecia and induce facial hypertrichosis [31]. Sebum excretion on facial skin shows large interindividual differences. In nonsupplemented menopausal women sebum excretion has been reported to increase in the perimenopause and later on declines with chronologic aging [9, 30, 32]. HRT-treated women show less prominent variations. However, the benefit differs among women and it remains hardly predictable. Globally HRTmight increase the casual sebum level [11], but there is a lack of consensus about that aspect. Sebaceous glands are privileged targets for sex steroids, particularly 5a-dihydroxytestosterone. Other hormonal controls and neuropeptides are also operative on sebocytes. As a result, the sebum production may be regarded as a marker of some specific hormonal changes.
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The sebaceous gland apparatus is an androgen target exhibiting the highest androgen receptor density in human skin. During climacteric aging, one could expect changes in the sebocyte proliferation, intracellular lipid synthesis, sebum transit time in the follicle, storage in the infundibulum reservoir, rheology and capture at the surface, and inside the stratum corneum. The modifications in the balance in sex hormones at the menopause are often believed to initiate the observed changes in sebum physiology. The decline in estrogen combined with a minimal decrease in androgens results in a relative increase in the androgen–estrogen balance. This should theoretically not hinder the sebocyte activity. These hormonal changes can also affect other segments of the sebaceous follicle, in particular, the size of the sebum reservoir. This is indeed evidenced by the progressive enlargement of the follicular openings. Although sex hormones are tentatively offered as the agents responsible for the objective changes, other hormones and nonhormonal aspects of aging cannot be dismissed. There are quite few objective studies evaluating the amount of sebum released at the skin surface. In addition, in many instances the number of subjects was obviously too low and precluded any sound conclusion. Some studies showed that sebum excretion decreased with aging. In particular, sebum excretion in menopausal women appeared lower than in nonmenopausal women [30]. By contrast, it was claimed to increase in menopausal women under HRT [9, 12, 30]. Contrasting data were reported in another controlled study involving large numbers of women [33]. Data showed that the sebum excretion changes in postmenopausal women were more likely related to hormones than to aging [33]. There was a large diversity among individual values of sebum output at the skin surface. In untreated women, a significant decline in sebum excretion rate accompanied by an increase in both the sebum replacement time and the mean pore size were evidenced during the first decade after menopause. The sebum excretion rate and casual level showed a wide range of interindividual differences early after menopause. These physiological changes were less prominent in women receiving HRT. It was concluded that postmenopausal aging affects the sebum production, but HRT does not significantly control the complex process of seborrhoea. However, HRT mitigates the progressive enlargement of the openings of the sebum follicular reservoir. As a consequence of the diversity of hormonal signals to the sebaceous apparatus, sebum excretion varies according to age, gender, pregnancy, and postmenopause. However, at any given age in men and women, the sebum excretion rate differs between individuals over a wide range. In addition, there is a huge overlap between data
gained in both genders. Hence, it is not the amount of circulating androgens but rather the receptivity of the target tissues that accounts for interindividual differences in sebum excretion. It is clear that additional factors are likely to be operative. The effect of the climacteric and postmenopausal age upon the sebaceous gland function has not been thoroughly and adequately studied using recent biometrological methods. It is generally acknowledged that the sebum dynamics varies throughout adult life. In women, it was reported that the sebum production remained almost stable over about 3 decades and dropped significantly in the age range of 50–59 years. However, those views were challenged. HRT appears to reduce moderately the effects of postmenopausal aging on the sebum rheology. In addition, the follicular pores are kept narrower compared to the skin of untreated women. Indeed, estrogens unquestionably suppress human sebaceous secretion at high pharmacological dosages. It is debatable, however, whether they have any significant effect at physiological levels and whether they play any sizeable part in normal control of the gland. It should be stressed that contraceptives show a moderate sebosuppressive activity in acne-prone young women suffering from increased seborrhea. It is possible that HRT has no effect when seborrhoea is absent or discrete. This does not exclude the possibility of an effect in severe cases. Nevertheless, it should be noted that chronological aging by itself likely mitigates seborrhea.
HRT, Skin, and Bones Menopause has been shown to have a potential role in the etiology of some major age-related diseases including osteoporosis. The one area that has fulfilled the hope of the HRT research has been the changes that occur in the skin and bones. The changes occurring in the dermis and bone, both in the climacteric and with HRT apparently parallel each other [3, 34–36]. There is also a correlation between some skin biomechanical properties and bone density [19, 34, 36]. It is probably the combination of skin thickness, dermal biomechanical functions, and bone mineral density that presents the greater sensitivity and specificity in identifying women vulnerable to osteoporotic fractures after menopause.
Conclusion Most women associate the middle years of life with a negative experience. In particular, it is generally
Climacteric Aging and Oral Hormone Replacement Therapy
acknowledged that a series of skin changes occur when women traverse the menopause and the years beyond. A gender perspective is indispensable for a full understanding of sex-hormone-sensitive cells of the skin. Yet until recently, some of these aspects have rather been neglected by biomedical researchers. However, the concept of climacteric and postmenopausal aging affecting the skin has progressively emerged in recent years. It was particularly studied at the level of the tensile strength of the dermis affected by atrophy and wrinkling. Climacteric xerosis is also recognized and most probably represents the consequence of a defect in the process of desquamation. The administration of HRT appears both safe and effective, provided adequate patient selection is made, and contraindications and appropriate use of hormones (nature, dosages, regimens, routes of administration) are respected. HRT increases the well-being as well as some somatic features in menopausal women. It remains that at present, the pros and cons of HRT make it a complex issue for the physicians taking care of skin changes. All the foregoing findings indicate that chronological aging, the climacteric estrogen deficiency, and HRT exert profound effects on various parts of the skin. In many cases the deleterious effects of low estrogenemia on the skin are reflected in the internal organs. It is acknowledged that skin during the climacteric suffers from some decline in its aspect and physical properties. HRT appears to protect in part the skin from some of the negative changes. HRT acts on the skin at several different sites and thus exhibits a multifactorial effect. The effects can be mediated by a direct hormonal effect on cells enriched in the adequate receptors. These stimulated cells can further produce some paracrine signals to other cells, which are thus indirectly influenced by HRT. It is the interplay between the various skin cell types and their signaling pathways that probably control the skin aspect and healthy look. At least the skin represents the one target organ where the HRT benefits are readily visible to the woman and her relatives.
Cross-references > Aging Genital Skin and Hormone Replacement Therapy
Benefits
References 1. Raine-Fenning NJ, Brincat M, Muscat-Baron Y. Skin ageing and menopause: implications for treatment. Am J Clin Dermatol. 2003;4:371–378. 2. Verdier-Sevrain S, Bonte F, Gilchrest BA. Biology of estrogens in skin: implication for skin aging. Exp Dermatol. 2006;15:83–94.
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3. Dunn LB, et al. Does estrogen prevent skin aging ? Results from the First National Health and Nutrition Examination Survey (NHANES I). Arch Dermatol. 1997;133:339–342. 4. Farage MA, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 5. Pie´rard GE. The quandary of climacteric skin ageing. Dermatology. 1996;193:273–274. 6. Quatresooz P, et al. Skin in maturity. The endocrine and neuroendocrine pathways. Int J Cosmet Sci. 2007;29:1–6. 7. Nelson LR, Bulun SE. Estrogen production and action. J Am Acad Dermatol. 2001;45:S116–S124. 8. Slominski A. Neuroendocrine system of the skin. Dermatology. 2005;211:199–208. 9. Callens A, et al. Does hormonal skin aging exist? A study of influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193:189–291. 10. Sauerbronn AV, et al. The effect of systemic hormonal replacement therapy on the skin of postmenopausal women. Int J Gynaecol Obstet. 2000;68:35–41. 11. Quatresooz P, et al. Skin climacteric aging and hormone replacement therapy. J Cosmet Dermatol. 2006;5:3–8. 12. Guinot C, et al. Effect of hormonal replacement therapy on skin biophysical properties of menopausal women. Skin Res Technol. 2005;11:201–204. 13. Quatresooz P, Pie´rard GE. Downgrading skin climacteric aging by hormone replacement therapy. Exp Rev Dermatol. 2007;2:373–376. 14. Oikarinen A. Systemic estrogens have no conclusive beneficial effect on human skin connective tissue. Acta Obstet Gynecol Scand. 2000;79:250–254. 15. Phillips TJ, et al. Does hormone therapy improve age-related skin changes in postmenopausal women? J Am Acad Dermatol. 2008;59:397–404. 16. Brincat MP, Muscat Baron Y, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123. 17. Castelo-Branco C, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 18. Pie´rard-Franchimont C, et al. Climacteric skin ageing of the face. A prospective longitudinal intent-to-treat trial on the effect of oral hormone replacement therapy. Maturitas. 1999;32:87–93. 19. Pie´rard GE, Vanderplaetsen S, Pie´rard-Franchimont C. Comparative effect of hormone replacement therapy on bone mass density and skin tensile properties. Maturitas. 2001;40:221–227. 20. Pie´rard GE, et al. Effect of hormone replacement therapy for menopause on the mechanical properties of skin. J Am Geriatr Soc. 1995;43:662–665. 21. Hermanns-Leˆ T, et al. Skin tensile properties revisited during ageing. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 22. Henry F, et al. Age-related change facial skin contours and rheology. J Am Geriatr Soc. 1997;45:220–222. 23. Arora S, et al. Estrogen improves endothelial function. J Vasc Surg. 1998;27:1141–1146. 24. Lim SC, et al. The effect of hormonal replacement therapy on the vascular reactivity and endothelial function of healthy individuals and individuals with type 2 diabetes. J Clin Endocrinol Metab. 1999;84:4159–4164. 25. Quatresooz P, et al. Immunohistochemical clues at aging of the skin microvascular unit. J Cutan Pathol. 2008, Epub June 17 (2008). 26. Margolis DJ, Knauss J, Bilker W. Hormone replacement therapy and prevention of pressure ulcers and venous leg ulcers. Lancet. 2002;359:675–677.
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27. Pie´rard-Franchimont C, et al. Skin water-holding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 28. Paquet F, et al. Sensitive skin at menopause; dew point and electrometric properties of the stratum corneum. Maturitas. 1998;28: 221–227. 29. Le´veˆque JL, Gubanova E. Influence of age on the lips and perioral skin. Dermatology. 2004;208:307–313. 30. Caisey L, et al. Influence of age and hormone replacement therapy on the functional properties of the lips. Skin Res Technol. 2008;14: 220–225. 31. Roux C. Randomised, double-masked, 2 years comparison of tibolone with 17b estradiol and norethindrone acetate in preventing postmenopausal bone loss. Osteoporosis Int. 2002;13:241–248.
32. Sator PG, et al. The influence of hormone replacement therapy on skin ageing: a pilot study. Maturitas. 2001;39:43–55. 33. Pie´rard-Franchimont C, Pie´rard GE. Post-menopausal aging of the sebaceous follicle. A comparison between women receiving hormone replacement therapy or not. Dermatology. 2002;204:17–22. 34. Castello-Branco C, et al. Relationship between skin collagen and bone changes during aging. Maturitas. 1994;18:199–206. 35. Shah MG, Maibach HI. Estrogen and skin : an overview. Am J Clin Dermatol. 2001;2:143–150. 36. Pie´rard GE, et al. Relationships between bone mass density and tensile strength of the skin in women. Eur J Clin Invest. 2001;31:731–735.
18 Cluster of Differentiation 1d (CD1d) and Skin Aging Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
Introduction CD1d is a member of CD1 family of transmembrane glycoproteins which represent a third lineage of antigenpresenting molecules. These molecules are distantly related to the classical major histocompatibility complex (MHC) molecules in the immune system [1–4]. However, unlike the first and second lineages of antigen-presenting molecules (the classical MHC class I and class II molecules) that bind peptide antigens, CD1 molecules have evolved to bind lipids and glycolipids [5–7]. CD1 family molecules are closely related to MHC class Ia and Ib proteins by sequence homology, domain organization (a1, a2, a3, and b2m), and association with b2 microglobulin rather than to class II molecules [2, 6]. In contrast to MHC class I molecule which is polymorphic, CD1 molecules are not polymorphic [1–3], and are encoded by linked genes outside the MHC complex; the gene for CD1d is located on chromosome 1 in humans [1–4]. The CD1 family is divided into two groups by sequence homology: group I which consists of CD1a, -b and -c isotypes and groupII which includes CD1d [8]. Only the group II CD1d isotypes are preserved in human, mouse, rat, rabbit, and monkey [4, 9]. Sequence similarity is substantially higher for the same isotype from different species than for different isotypes within the same species [1–3, 10], suggesting that each group of CD1 molecules could have a different function [4]. CD1d binds glycol-and phospholipid antigens, and is essential for the development and activation of a subset of T cells known as natural killer T (NK-T) cells which are characterized by the expression of receptors used by NK cells [1–3, 6, 11] and invariant Va-Ja TCRs, such as Va24JaQ TCR in humans and Va14Ja281 TCR in mice [12]. NK-T cells recognize self or nonself glycolipids presented by CD1d molecule, and respond by secretion of cytokines; most notably IFN-g and IL-4 [1–3, 6, 13]. The synthetic glycolipid molecule a-galactosylceramide (a-GalCer) was shown to stimulate human and mouse NK-T cells in a CD1d-restricted manner [14–16]. CD1d plays,
therefore, via the production of cytokines secreted by NK-T cells, a critical role in performing a number of immunoregulatory functions within the human and mammalian body including protection against autoimmune diseases, microbial infection, and cancer. In mice, it was shown that CD1d regulates UV-induced carcinogenesis by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts [17, 18].
CD1d Expression in the Human Skin Recently, CD1d expression and NK-T cells were demonstrated in the epidermis of acute and chronic psoriatic plaques [19–22]. Not only did CD1d show expression in psoriatic skin, but also in normal sun protected and scalp skin [22–25]. Moreover, it was found that CD1d is expressed on human scalp hair follicle keratinocytes, and that its expression undergoes hair cycle-associated changes, suggesting a role in hair follicle cycle regulation.
CD1d Expression in Human Skin Undergoes Age-Associated Changes The expression pattern of CD1d in different age groups was examined recently and variable profiles found [25]. CD1d had a strong expression in the skin of young (between 6–18 year old), but it declined in the skin of mid (between 30–40 year old) and old (50–81) age groups. In the epidermis, CD1d was expressed in all layers except in the stratum corneum (> Figs. 18.1–18.5). However, its expression had different intensities, with strong immunoreactivity in the stratum basale and stratum spinosum but weak immunoreactivity in the stratum granulare, particularly in old individuals (Table 18.1). The findings of age-related decrease of CD1d protein expression in the human epidermis agree with other groups [26, 27]. Decreased density and function of
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_18, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 18.1 Immunoreactivity of CD1d protein in human skin of 6 (A), 15 (B), 18 (C), 33 (D), 39 (E), 53 (F), 57 (G), 60 (H), 68 (I), 71 (J), 78 (K) and 81 (L) year old individuals, shown in red color with TSA technique. M negative control; N positive control shows CD1d expression in a blood vessel. At the age of 6 years, CD1d immunoreactivity was not only strong, but also seen in all layers of the epidermis except the stratum corneum (A). In the ages between 10 and 30 year old, CD1d immunoreactivity decreased, and was detected in the Malpighian layer, stratum spinosum, and a few layers of stratum granulosum (B–C). In the ages between 30 and 40 year old, CD1d had moderate expression that was mainly seen in the stratum basale (D–E). In the ages between 40 and 60 years, CD1d protein expression was moderate immunoreactivity, and localized to the basal and granular layers (F). In the skin derived from 57 year old donors, the immunoreactivity was however stronger, and the expression was apparent both in the basal and granular layers (G). In the old ages, the expression of CD1d was confined to the stratum basale (H). Sometimes, CD1d protein expression was seen in the stratum spinosum and some cells of the stratum granulosum, in addition to the basal layer (I–L). Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Figure 18.2 Immunoreactivity of CD1d protein in human skin of 6 (A), 18 (B), 33 (C), 53 (D), 60 (E) and 71 (F) year old individuals, shown in red color with ABC technique. At the age of 6 years, CD1d immunoreactivity was not only strong but also seen in all layers of the epidermis except the stratum corneum (A). In the ages between 10 and 30 year old, CD1d immunoreactivity decreased, and was detected in the Malpighian layer, stratum spinosum and a few layers of stratum granulosum (B). In the ages between 30 and 40 year old, CD1d had moderate expression that was mainly seen in the stratum basale (C). In the ages between 40 and 60 years, CD1d protein expression was moderate immunoreactivity and localized to the basal and granular layers (D). Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
Cluster of Differentiation 1d (CD1d) and Skin Aging
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Cluster of Differentiation 1d (CD1d) and Skin Aging
. Figure 18.3 Immunoreactivity of CD1d protein in sweat glands of human skin derived from 6 (A), 33 (B), 39 (C), 64 (D), 71 (E), and 78 (F) years old individuals, shown in red color with TSA technique. CD1d protein expression was strong in sweat glands of all ages. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
Cluster of Differentiation 1d (CD1d) and Skin Aging
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. Figure 18.4 Immunoreactivity of CD1d protein in sebaceous glands of human skin derived from 15 (A), 39 (B) and 71 (C) year old individuals, shown in red color with TSA technique. In sebaceous glands, CD1d protein expression was strong in all ages. Interestingly, CD1d protein was comparable to that in the epidermis, i.e., it was strong in children, moderate in young adults, and weak with aging. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Figure 18.5 Immunoreactivity of CD1d protein in different ages. Reprinted with permission from Adly et al. [23, 25] ß 2005, 2006, Wiley-Blackwell
. Table 18.1 CD1d protein expression in the normal human skin: all the specimens were batch-stained in the same run Age groups (Years)
Stratum basale
Stratum spinposum
Stratum granulare
Stratum corneum
Dermis
6–18
2.6 0.3
2.0 0.5
1.7 0.3
0.0
2.6 0.3
30–40
2.4 0.2
1.6 0.3
1.2 0.2
0.0
2.6 0.3
50–81
1.5 0.3
1.25 0.2
0.75 0.2
0.0
2.7 0.3
The immunostaining experiments were repeated at least three times. The staining results were examined by the authors and were scored as (–) for absent, (1) for weak, (2) for moderate, and (3) for intense staining, following other groups
epidermal dendritic cell populations were found in aged C57BL/6J mice. However, the capacity of the dendritic cells to transport antigen from the skin to the draining lymph nodes was found in vivo to be comparable to that
of young mice. The strong expression of CD1d protein in the skin of children and young individuals may be due to increased recruitment, tissue accessibility, and local proliferatioin of CD1d + cells (> Table 18.1). Molecular
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signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the endoplasmic reticulum stress pathway may be involved in CD1d + cells apoptosis [28–31].
Conclusion CD1d plays, therefore, via the production of cytokines secreted by NK-T cells, a critical role in performing a number of immunoregulatory functions within the human and mammalian body including protection against autoimmune diseases, microbial infection, and cancer. In mice, it was shown that CD1d regulates UV-induced carcinogenesis by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.
References 1. Porcelli SA. The CD1 family: a third lineage of antigen-presenting molecules. Adv Immunol. 1995;59:1–98. 2. Porcelli SA, Segelke BW, Sugita M, Wilson IA, Brenner MB. The CD1 family of lipid antigen-presenting molecules. Immunol Today. 1998;19(8):362–368. 3. Porcelli SA, Modlin RL. The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu Rev Immunol. 1999;17:297–329. 4. Zeng Z, Castano AR, Segelke BW, Stura EA, Peterson PA, Wilson IA. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science. 1997;277 (5324):339–345. 5. Hong S, Scherer DC, Singh N, Mendiratta SK, Serizawa I, Koezuka Y, Van Kaer L. Lipid antigen presentation in the immune system: lessons learned from CD1d knockout mice. Immunol Rev. 1999;169:31–44. 6. Exley M, Garcia J, Balk SP, Porcelli S. Requirements for CD1d recognition by human invariant Valpha24 + CD4-CD8- T cells. J Exp Med. 1997;186(1):109–120. 7. Sidobre S, Kronenberg M. CD1 tetramers: a powerful tool for the analysis of glycolipid-reactive T cells. J Immunol Methods. 2002;268 (1):107–121. 8. Calabi F, Jarvis JM, Martin L, Milstein C. Two classes of CD1 genes. Eur J Immunol. 1989;19(2):285–292. 9. Kashiwase K, Kikuchi A, Ando Y, Nicol A, Porcelli SA, Tokunaga K, Omine M, Satake M, Juji T, Nieda M. Koezuka Y. The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques. Immunogenetics. 2003;54 (11):776–781. 10. McMichael AJ. Lymphocytes. 1. Function. Genetic restrictions in the immune response. J Clin Pathol Suppl (R Coll Pathol). 1979; 13:30–38. 11. Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G, Dellabona P, Kronenberg M. CD1d-mediated recognition of an
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alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J Exp Med. 1998;188 (8):1521–1528. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A. An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4–8- T cells. J Exp Med. 1994;180(3):1171–1176. Fujii S, Shimizu K, Steinman RM, Dhodapkar MV. Detection and activation of human Valpha24 + natural killer T cells using alphagalactosyl ceramide-pulsed dendritic cells. J Immunol Methods. 2003;272(1–2):147–159. Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J Exp Med. 1998;188(8):1529–1534. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E, Koseki H, Taniguchi M. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. 1997;278(5343):1626–1629. Nieda M, Nicol A, Koezuka Y, Kikuchi A, Takahashi T, Nakamura H, Furukawa H, Yabe T, Ishikawa Y, Tadokoro K, Juji T. Activation of human Valpha24NKT cells by alpha-glycosylceramide in a CD1drestricted and Valpha24TCR-mediated manner. Hum Immunol. 1999;60(1):10–19. Matsumura Y, Moodycliffe AM, Nghiem DX, Ullrich SE, Ananthaswamy HN. Resistance of CD1d / mice to ultraviolet-induced skin cancer is associated with increased apoptosis. Am J Pathol. 2004; 165(3):879–887. Matsumura Y, Moodycliffe AM, Nghiem DX, Ullrich SE, Ananthaswamy HN. Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d / mice. Photochem Photobiol. 2005;81(1):46–51. Nickoloff BJ, Wrone-Smith T, Bonish B, Porcelli SA. Response of murine and normal human skin to injection of allogeneic blood-derived psoriatic immunocytes: detection of T cells expressing receptors typically present on natural killer cells, including CD94, CD158, and CD161. Arch Dermatol. 1999;135 (5):546–552. Nickoloff BJ, Wrone-Smith T. Injection of pre-psoriatic skin with CD4 + T cells induces psoriasis. Am J Pathol. 1999; 155(1):145–158. Nickoloff BJ, Bonish B, Huang BB, Porcelli SA. Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system. J Dermatol Sci. 2000;24 (3):212–225. Bonish B, Jullien D, Dutronc Y, Huang BB, Modlin R, Spada FM, Porcelli SA, Nickoloff BJ. Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells. J Immunol. 2000;165(7):4076–4085. Adly MA, Assaf HA, Hussein M. Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations. J Clin Pathol. 2005;58(12):1278–1282. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M. Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression. Br J Dermatol. 2005;153(3):514–520. Adly MA, Assaf HA, Hussein MR, Neuber K. Age-associated decrease of CD1d protein production in normal human skin. Br J Dermatol. 2006;155(1):186–191. Sunderkotter C, Kalden H, Luger TA. Aging and the skin immune system. Arch Dermatol. 1997;133(10):1256–1262.
Cluster of Differentiation 1d (CD1d) and Skin Aging 27. Gilchrest BA, Murphy GF, Soter NA. Effect of chronologic aging and ultraviolet irradiation on Langerhans cells in human epidermis. J Invest Dermatol. 1982;79(2):85–88. 28. Fainboim L, Salamone Mdel C. CD1: a family of glycolypid-presenting molecules or also immunoregulatory proteins? J Biol Regul Homeost Agents. 2002;16(2):125–135. 29. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1986;15(4 Pt 1):571–585.
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30. Fenske NA, Conard CB. Aging skin. Am Fam Physician. 1988;37 (2):219–230. 31. Sprecher E, Becker Y, Kraal G, Hall E, Harrison D, Shultz LD. Effect of aging on epidermal dendritic cell populations in C57BL/6J mice. J Invest Dermatol. 1990;94(2):247–253.
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16 Considerations for Thermal Injury: The Elderly as a Sensitive Population Donald L. Bjerke
Introduction As the ‘‘baby boom population’’ in North America ages, one of the populations at greatest risk of thermal injury continues to expand. According to the US Census Bureau in 2000, the population of US citizens who are 75 years and older was 16,548,000 (6.0%). By 2010 this figure is projected to be 19,101,000 (6.4%), and by 2050 it is expected to exceed 54, 094,000 (13.4%) [1]. Annually, in the United States and Canada, 1.25 million people suffer burn injuries [2]. Populations identified at increased risk of burns include infants and young children, older adults, and people with any type of disability [3–6]. Many of the burns reported are from scalds. Scald injuries are painful, require prolonged treatment, and may result in lifelong scarring and even death. Most burn injuries happen in the home with tap water scalds occurring in the bathroom or kitchen [3, 7, 8]. Burns can also be caused by therapies in medical treatment facilities [9] or from therapeutic use of heat in the home. This premise is supported by a jointly issued public health advisory in 1995 by the United States Food and Drug Administration and the Consumer Products Safety Commission on electric heating pads. This advisory reported approximately 1,600 heating pad burns treated in the emergency room annually and that approximately 45% of those patients were over 65 years of age [10]. The American Burn Association classifies the severity of a burn based on the total body surface area (TBSA) affected and the depth of the injury (> Table 16.1). The elderly are at greater risk of thermal injury, and the outcome of that injury can be devastating. Elderly subjects have a higher mortality than younger subjects who have a similar surface area burn. For example, if half the body surface is burned in a young adult, the mortality is about 50%, whereas a burn of only one fifth of the body surface in the elderly results in a similar mortality [1, 8]. This increased risk in the elderly is due to many factors as a result of both physical and physiologic differences seen in this population. Diminished senses,
impaired mental acuity, slower reaction time, reduced mobility, and bedridden states may lead to the decreased ability of the elderly to identify the severity of the situation, as well as their capacity to escape from harm. Physiologic factors include thinner skin, reduced microcirculation, increased susceptibility to infections in the elderly, and higher incidence of premorbid conditions such as chronic disease, alcoholism, medications, senility, and neurological or psychiatric disorders [3]. This, in turn, may lead to an increased total body surface area burn, deeper burns, and more devastating consequences from thermal injury. This chapter discusses the conditions by which thermal injury occurs and the physiologic factors associated with an increased risk in the elderly population.
. Table 16.1 Classification of Burn Injuries Major: burn injuries Second-degree burns over a body surface area (BSA) greater than 25% in adults or 20% in children; all third-degree burns over a BSA of 10% or greater; all burns involving hands, face, eyes, ears, feet, and perineum; all inhalation injuries; electrical burns; complicated burn injuries involving fractures or other major trauma; and burns on all high-risk patients (i.e., those who are elderly or who have debilitating diseases) Moderate: uncomplicated burn injuries Second-degree burns over a BSA of 15–25% in adults or 10–20% in children; third-degree burns over a BSA of 2–10%; and burns not involving eyes, ears, face hands, feet, or perineum Minor: burn injuries Second-degree burns over a BSA of 15% or less in adults or 10% or less in children; third-degree burns over a BSA of less than 2%; and burns not involving eyes, ears, face, hands, feet, or perineum. Minor burns exclude electrical injuries, inhalation injuries, and burns on all high-risk patients
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_16, # Springer-Verlag Berlin Heidelberg 2010
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Background Information on Thermal Injury The first significant research in the area of thermal injury was conducted by Henriques and Moritz at the Harvard Medical School [11–15] in the 1940s. Prior to this work, remarkably little information existed concerning the mechanism by which hyperthermia leads to irreversible cellular injury, the reciprocal relationships of time and temperature in the production of either cutaneous or systemic injury, the relationship between environmental heat, surface temperature, and the slope of the transcutaneous thermal gradient, the pathogenesis of cutaneous burns, or the physiological mechanisms by which external heat may be responsible for acute disability and death. This research provided information on parameters controlling the flow of heat into the skin and the importance of heat capacity and thermal conductivity and developed an approximate first-order Fourier’s law equation to describe the transient heat flow. In vivo factors that affect skin temperature include site variations in the respective thickness of epidermis, dermis, fat, and muscle; variation of existing temperature gradients within the skin with respect to time and/or position of site; average rate of blood flow through the various skin layers, and variations of the rate of flow with respect to position of site and temperatures within the site; and the appearance of edema fluid in variable quantities. These factors result in site-specific alterations in the density, heat capacity, thickness, and thermal conductivity of the various layers of skin so affected. Using a pig model (skin similar to humans), Henriques and Moritz brought and held constant the skin at various temperatures until the threshold of irreversible injury occurred. From this, they derived the time–temperature relationship in the layer of basal epidermal cells, which are thought to be the most important cell layer for the production of epidermal injury by heat. Cell death (necrosis) is a result of irreversible thermal denaturation of the protein present within the cell [14, 16]. Because second- and third-degree burns involve cell death at the basal epidermal layer, the distance from the surface of the skin to this basal epidermal layer becomes important for the rate of heat transfer. In other words, a thinner epidermis results in more efficient heat transfer from the surface of the skin to the basal epidermal layer, thus increasing the risk of thermal injury. The in vitro work in pigs was extended to in vivo thermal injury in both pigs and humans. Circulating water at various temperatures was brought in contact with skin on the ventral forearms or anterior thoracic region of presumed young healthy military men. Of particular note is that the time–temperature relationship is not linear and the rate at which burning
occurs is almost doubled for each degree rise in temperature between 44 C and 51 C. Discomfort in the form of a stinging sensation occurred between 47.5 C and 48.5 C and was variable between subjects with respect to intensity. For example, severe burns were sustained without discomfort at 47 C while in other cases intense discomfort was noted before irreversible injury at temperatures above 48 C. The lowest temperature resulting in cutaneous burning was 44 C and the time required to cause irreversible damage to epidermal cells at this temperature was approximately 6 h. Alternatively, a surface temperature of 70 C resulted in trans-epidermal necrosis in less than 1 s. The relationship between temperature and duration of exposure to the extent of skin damage was landmark and has served as a guide for all subsequent works. Wu extended the work of Moritz and Henriques by adding the heat transfer reaction for a source of high energy [16]. His treatment, assuming contact between two semi-infinite bodies of finite thermal inertia at different temperatures, showed that sources of low inertia (e.g., wood, insulation, some plastics) cause a slower rise in skin temperature than a source of high thermal inertia (e.g., steel and aluminum) at the same temperature. This is explained by high thermal inertia materials, which can make more energy available at the surface in a given time than those of lesser thermal inertia. ASTM published a document entitled Standard Guide for Heated System Surface Conditions that Produce Contact Burn Injuries in October 2003 [17]. Included in this guide is a summary of the 1947 research of Moritz and Henriques. The review notes that the earlier work neglected the flow of blood to carry heat away and the physiological changes in skin properties as the damaged zone traverses the outer skin layers. Factors that increase the complexity of predicting burns include: (a) site variations with respect to the thickness of different skin layers; (b) variations of initial conditions within the skin with respect to time, position, and physical condition of the subject; (c) the unknown average rate of blood flow through the skin layers and variations within the layers with respect to location and ambient temperatures (warm ambient causes increased flow near surface and cold ambient results in less flow near surface); and (d) the appearance of watery fluids in variable quantities upon exposure that result in alterations of skin density, heat capacity, thickness, and thermal conductivity. The guide is meant to serve as an estimation of the exposure to which an average individual might be subjected and does not assume to be inclusive of unusual conditions of exposure, physical health variations, or nonstandard ambient condition. The guide applies to contact with heated surfaces only. Importantly, > Fig. 16.1 demonstrates the
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. Figure 16.1 Temperature–time relationship for burns (ß ASTM International [17]. Reprinted with permission.)
relationship between skin temperature and time as it relates to thermal injury. The following equations were developed and are reported in the document: TA ¼ 15:005 þ 0:51907 Lnðtime 1000Þ þ 352:97=½Lnðtime 1000Þ TB ¼ 39:468 0:41352 Lnðtime 1000Þ þ 190:60=½Lnðtime 1000Þ where: TA = critical contact temperature for complete transepidermal necrosis ( C) TB = critical contact temperature for reversible epidermal injury ( C) time = elapsed contact time (s) Ln = natural logarithm Exposures below the lower curve should not produce permanent injury in normal humans. Exposure between the curves are described as second-degree burns and have intermediate levels of cell damage. Exposures at levels above the top line are defined as third-degree burns that cause deep, permanent cell damage and scarring.
Reported Burn Injuries in the Elderly Barillo investigated burn injuries in medical treatment facilities [9]. The medical records of 4,510 consecutive admissions to one burn center were reviewed and a cohort
of 54 patients had suffered burn injuries as a result of medical therapy. A number of burns in the home resulted from therapeutic applications of heat, including six patients burned by heating pads, one patient burned by a heat lamp, and four patients burned by contact with hot water bottles or soaks. The average hospital stay for burns resulting from medical therapy (22.9 days) was excessive in comparison with the average total burn size of 3.0% TBSA. In addition to medical therapies, there were two patients, including one fatality, that were scalded while being bathed in nursing homes. The total body surface area (TBSA) burned by the scalding water was 20.3% with third-degree burns on 3% of the body. Contributing factors to thermal injury were advanced age, chronic illness, limited mobility, and altered skin sensation. More recently, Ghods and colleagues published the results of a survey of 36 burn clinics in Germany with regard to hot air sauna burns [18]. In total, 14 patients were treated in the German burn units between 1999 and 2005 and an additional two patients in the author’s clinic. Of note, the average age of the individuals was 67 years and the time spent in the sauna was between 45 and 60 min. In all cases, unconsciousness occurred and was assumed to be a result of orthostatic dysregulation. Deep second- and third-degree burns of the highest exposed body parts were found in all cases (an average of 14% TBSA). In two cases, the involved lower extremities had to be amputated; in four cases, primary amputations of toes or fingers were necessary, and four patients died because
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of multiple system organ failure. Normally, exposure to temperatures up to 120 C in dry, hot air do not cause damage to the skin, because of the low thermal capacity of the air. In the cases presented by the Ghods report, a hypothesis was developed to support a theory for ‘‘apex burns.’’ Unconsciousness leads to a declension of the perfusion of the skin with degraded cardiac achievement and low blood pressure. This effect, in turn results in insufficient cooling of the skin. These two reports clearly demonstrate that conditions that normally do not cause harm in a young healthy population can result in significant thermal injury to older individuals, especially when there are other concomitant factors involved. Because of the medical significance of burns in the elderly, one encouraging trend is the shift from burn treatment to burn prevention. Behavioral changes include testing water temperature before taking a bath or shower, avoidance of wearing loose sleeves while cooking, having carbon monoxide detectors that are tested regularly, and having smoke detectors that are tested regularly [3]. Additional work by Redlick and colleagues has targeted different mass media channels to promote campaigns targeted at the prevention of burns in the elderly. This groundbreaking work by the Burn Centre in Toronto along with Sunnybrook and Women’s College Health Science Centre and the Toronto Western Hospital has led to an effective burn prevention campaign for older adults [19]. Incorporated into the prevention campaign were previous success stories for lowering hot water heater settings [20] and preventing cigarette burns and contact burns with household radiator heaters [21]. While the campaign was effective at improving burn prevention knowledge, whether this results in a change in burn prevention behavior remains unclear.
Risk Factors for Thermal Injury It is well accepted that elderly individuals are at greater risk of thermal injury. Physiological factors that contribute to an increased risk include the thinning of the skin, a compromised ability to dilate the vasculature of the skin, and a reduced thermal sensitivity with advancing age. The significance of these changes are that older adults have thinner skin than their younger counterparts, so contact with a hot surface or liquid can cause deeper burns with even brief exposure. To maintain a safe skin temperature upon thermal challenge, the body mobilizes the blood circulation to the periphery to ‘‘wick away’’ the heat by acting as a convective heat exchanger [22]. With aging comes a reduction in the ability to mobilize the circulation to the periphery. In addition, the compromised ability to feel heat may be decreased with aging due
to certain medical conditions or medications so that the elderly may not realize the thermal insult (e.g., scalding bath or kitchen water) is too hot until injury has occurred. Physical conditions may also contribute to the increased risk of thermal injury. Some older adults have conditions that make them more prone to falls in the bathtub or shower or while carrying hot liquids [7]. The physical factors related to thermal injury are outside the scope of this chapter. The sections that follows will describe in more detail the literature regarding thinning of skin, compromised microcirculation, and reduced thermal sensitivity that accompany aging.
Thinning Skin For additional information on the effects of aging on skin structure, please see the other relevant sections of this book. The skin undergoes several structural and functional changes with advancing age. With regard to thermal injury, the significance of thinning of the skin is that heat applied to the surface can more easily be conducted to the basal layer of the epidermis and beyond because it has less distance to penetrate. Thus, the depth of the thermal injury can be greater in elderly than in younger individuals exposed to the same temperature. While the thinning of the skin with age has been characterized, there remains an opportunity to examine the effects of changes in skin composition that occur with increasing age in relation to thermal capacity, density, and thermal conductance. In addition, elderly skin is also more prone to blistering from mechanical sheer force, and while controversial, this effect may be exacerbated by thermal challenges. The process of aging skin is often divided into two components: intrinsic aging, which is genetically determined and extrinsic aging, which is associated with cumulative damage by UV exposure. Extrinsic aging associated with excess exposure to ultraviolet light is characterized by loss of elasticity, increased roughness and dryness, irregular pigmentation, and deep wrinkling. The epidermis may thin in response to atrophy and may be accompanied with changes in the proportion and/or functionality of the dermal extracellular components [23]. Although there are differences in intrinsic and extrinsic skin aging, it is becoming evident that there are many consistent changes at the molecular level. Changes seen with intrinsic aging such as decreased cellular lifespan, reduced response to growth factors, disruption of matrix synthesis, and elevation of proteolytic activity are all evident in photo-damaged skin. The changes are simply more pronounced [23]. Montagna and Carlisle describe aging skin with the undersurface of the epidermis becoming flattened, with
Considerations for Thermal Injury: The Elderly as a Sensitive Population
little apparent change to the epidermis except for minor alterations in the organization of its cells [24]. The dermis undergoes greater changes with aging as it diminishes in bulk, many of its collagenous elastic fiber are gradually dissolved by enzymes, and the layer of fat becomes thinner. There appears to be a steady decline in the number of fibroblasts and mast cells with advancing age. The upper dermis contains collagenous fiber bundles that are somewhat haphazardly arranged. Cerimele describes the accompanying physiological changes that occur in aging skin [25]. These include impairment of barrier function, decreased turnover of epidermal cells, reduced keratinocytes, and fibroblasts, a reduced vascular network particularly around hair bulbs and glands. These changes result in fibrosis and atrophy, and decreases in hair and nail growth, vitamin D synthesis, and the density of Langerhan cells. Reductions in the immune response, and decreased functioning of Meissner’s and Pacinian corpuscles are noted. Histological alterations of the microvasculature, including thickening of the basement membrane in the exposed areas of alterations in the veil cells in protected zones, combined with the general reduction in vasculature, are probably responsible for the gradual atrophy of the cutaneous appendages that occurs with time. All of these normal changes with aging impact on the risk of thermal injury [25]. There is also a reduction in the number and biosynthetic capacity of fibroblasts and progressive disappearance of elastic tissue in the papillary dermis. Skin collagen content decreases with age and the fine collagen fibers associated with infancy become increasingly dense and tightly packed and far more randomly oriented [23]. Martin conducted a comprehensive cadaver study of body composition with 13 un-embalmed cadavers aged 59–86 years (six male and seven female) [26]. Measurements were made at 12 sites bilaterally and one central site (abdomen). Skin thickness was measured to a precision of 0.05 mm. Skin thickness varied by subject and site with males having thicker skin than females at all individual sites and overall (1.19 mm compared to 0.96 mm). The mean of all 25 sites ranged from 0.81 to 1.43 mm in males and from 0.73 to 1.10 mm in females. The thinnest site was the bicep (0.76 mm in men and 0.49 mm in women). The thickest skin site was at the subscapular site (2.07 mm in men and 1.76 mm in women). The other body sites measured were triceps, forearm, chest, waist, supraspinal, abdominal, front thigh, medial thigh, rear thigh, patellar, and medial calf. Of note, this study determined the entire skin thickness and concluded that skin likely thins with age. Moragas and colleagues studied abdominal skin samples from 96 autopsy cases ranging in age from 3.5 months
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to 86 years [27]. Abdominal skin was chosen as shielded from photoaging and thus, the changes are attributed to intrinsic aging only. Samples, 35 mm by 15 mm, were fixed in 10% buffered formalin, subsampled and embedded in paraffin wax. Sections of 4 mm thickness were stained with hematoxylin and eosin. Four characteristics were evaluated in each case. Three were denoted by linear roughness indices: progressive flattening of the epidermal undersurface related to the rete peg profile, effect of shrinkage on the basal layer, and waviness of the interface between the granular and corneum layers due to shrinkage. The fourth variable corresponded to epidermal thickness in micrometers, measured in zones between the rete pegs. When looking at the extreme age groups (0–20 years and 80–100 years), elderly subjects had a 36% reduction in the roughness index as related to the rete peg profile as compared with younger subjects. In the elderly, the epidermis was 49.5% thinner than in younger individuals. The decrease in shrinkage indices (basal layer and waviness of the interface between the granular and corneum layers) were 6% and 22%, respectively. The average epidermal thickness for age groups 0–20, 41–60, and 81– 90 were 22.6, 17.9, and 11.4 mm, respectively. The reduction in epidermal thickness was not influenced significantly by gender [27]. The progressive decrease in mechanical resistance explains why elderly people complain that they are prone to shear-type skin injuries or show increased blistering [25]. The epidermis makes a major contribution to these changes. The two most striking epidermal features associated with aging are dermo–epidermal junction flattening, with effacement of the so-called rete ridges, and epidermal thinning [23, 27]. Epidermal tissue repair also declines with age, whether measured in terms of wound closure time or blister roof regeneration. The flattening of the dermo–epidermal junction in elderly may result in greater separation of the layers and blistering in response to shear force as compared to younger individuals. The effect of heat on pressure ulcers was investigated by Kokate and colleagues [28] in a swine model (considered to have skin similar to humans). Higher skin temperature causes an increase in tissue metabolism and oxygen consumption (about 10% for a 1 C rise). The heightened need for nutrients and oxygen cannot be fulfilled because of tissue compression resulting in ischemia and tissue damage. Kokate applied 100 mmHg of pressure and temperatures of 25 C, 35 C, 40 C, and 45 C for 5 h in the swine model. This model produced both pressure ulcers and injuries consistent with burns. At 35 C there was deep tissue damage (i.e., pressure damage), while 40 C resulted in both dermal and deep tissue damage, and 45 C caused full-thickness cutaneous and deep tissue injuries. In contrast, 25 C
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resulted in the absence of damage and was considered protective of pressure ulcers. Thus, the interplay of pressure and temperature should be considered in elderly individuals who develop injuries consistent with pressure ulcers and/or thermal injury, especially in the areas of bony prominences.
Decreased Ability for Peripheral Vasodilatation With advancing age, structural and functional changes take place in the peripheral blood vessels that result in a reduced ability to mobilize blood to the cutaneous vessels when challenged with a heat stimulus. Without the optimal convective heat exchange capacity, the skin temperature can rise more rapidly and thus shorten the time to a skin burn relative to younger individuals. This section will describe the structural and functional changes to the peripheral vasculature with age and the impact on maintaining skin temperature. In addition, studies using pharmacological actives affecting vascular tone to better understand the mechanism by which aging affects peripheral vasodilation in response to thermal stimuli will be presented. These studies will demonstrate that resting blood flow and vascular conductance decrease with age due to increased sympathetic nerve activity but during exertional heat stress, the diminished blood flow is independent of sympathetic vasoconstriction. With advancing age, there is decreased nitric oxide-mediated vasodilation and a decreased response to histamine. Thus, the reduced ability to increase blood flow to the peripheral dermal vasculature is multifactorial with effects on structure and function. Histological changes in cutaneous microvessels with age was investigated by Braverman [29]. The veil cells around normal, diabetic, and aged vessels were reconstructed in three dimensions by a computer graphics system from 120 to 140 serial ultrathin sections. While there did not appear to be any differences in the metabolic capability of the veil cells in the different groups, there were structural differences. The normal vessel was surrounded by a single layer of veil cells, which had a wrinkled and pleated surface. The veil cells around aged vessels appeared to have the same length as young veil cells but were underdeveloped in their lateral extensions so that they did not cover the vessel circumferentially as well as did the normal veil cells. Mortiz and Henriques [12] were the first to investigate the importance of blood flow with regard to the protection of the epidermis from reversible injury. Using a pig model, and applying a constant flow of water at 49 C
and 51 C for durations subthreshold to injury, increasing the pressure from 0 to 80 mmHg (thought to compress the most distal blood vessels) did not result in irreversible trans-epidermal injury. Thus, the thinness of the epidermis was considered more important than the protective effect of removing heat via the blood circulation. These early conclusions later came under scrutiny. The ASTM review notes that the increased pressure in the Moritz and Henriques study was not sufficient to collapse the blood vessels [17]. The impact of vasodilatation on protecting the surface skin temperature was subsequently demonstrated by Lipkin and Hardy [30] on the human forearm. These authors evaluated thermal inertia, which is a product of thermal conductivity, density, and thermal capacity. They noted that as heating of intact skin progressed, the influence of the increased blood flow became more pronounced, finally causing a decrease in skin temperature in spite of continued irradiation. This phenomenon did not occur when blood flow was occluded as skin temperature continued to increase. Therefore, the values of thermal conductivity, density, and thermal capacity for living skin are not constant and will depend upon blood flow, thickness of the stratum corneum, and possibly upon the state of hydration. To investigate the effects of age on the response of skin blood flow in the forearm to direct heat [31], three groups of 20 male subjects each – young (20–39 years); middleaged (40–59 years), and older (60–79 years) had blood flow measured by Doppler flowmeter. The forearm was in a water bath at 30 C that was elevated to 35 C and then 40 C. The older group demonstrated a significantly lower volume in response to 35 C and 40 C, and there was a significant reduction in blood flow for both middle-aged and older men at 40 C. Thus, aging decreases the response of cutaneous blood flow in the forearm to the direct effects of heat. The reduced blood flow suggests that this is mediated by a reduced flow response of individual microvessels in middle-aged and older men. The reduced blood volume data suggest that, in addition, vessel recruitment is depressed in older individuals. To understand the underlying physiological changes to the peripheral vasculature that occur with aging, several authors have used pharmacologic tools to investigate the sympathetic and parasympathetic nervous system. Dinenno [32] examined hemodynamic changes related to aging. Resting limb blood flow and vascular conductance are reduced with age in adult humans and these changes are related to elevations in sympathetic vasoconstrictor nerve activity and reduction in limb oxygen demands. Sixteen young males (28 1 years; mean SEM)
Considerations for Thermal Injury: The Elderly as a Sensitive Population
and 15 older males (63 1 years) were compared for femoral artery blood flow (Doppler ultrasound), vascular conductance, femoral artery resistance, and muscle sympathetic activity. Whole-limb blood flow represents the sum of flow to skeletal muscle, skin, subcutaneous tissue, and bone. Flow to subcutaneous tissue and bone is thought to be negligible at rest. Data on young adult humans in which relative measurements of whole-forearm blood flow were performed before and after skin flow was abolished with epinephrine iontophoresis suggest that skin blood flow represents 30–35% of the total flow under these conditions [33]. Femoral artery blood flow was 26% lower in the older men, despite similar levels of cardiac output. Femoral artery vascular conductance (femoral blood flow/mean arterial pressure) was 32% lower and femoral vascular resistance (mean arterial pressure/femoral blood flow) was 45% higher in older men. Muscle sympathetic nerve activity was 74% higher in the older men and correlated with femoral artery blood flow, vascular conductance, and vascular resistance. Thus, basal whole-leg arterial blood flow and vascular conductance are reduced with age in healthy adult men under resting conditions, these changes are associated with elevations in sympathetic vasoconstrictor nerve activity; and the lower wholelimb blood flow is related to a lower oxygen demand that is independent of tissue mass. The authors raise other possibilities for the findings, including a reduced bioavailability of nitric oxide with age or elevations in locally released (e.g., endothelin) or systemically circulating (e.g., vasopressin) levels of vasoconstrictor agents may have played a role. While the reduction in whole-limb blood flow of older adults at rest is thought primarily to involve differences in skeletal muscle blood flow, it raises the question as to how well skin blood flow can respond in terms of a thermal challenge. Evidence is accumulating that older adults are limited in their capacity to augment blood flow and vascular conductance in response to acute increases in functional demand imposed by large-muscle dynamic exercise, energy intake, and ambient heat stress. Kenney tested the hypothesis that an attenuated increase in cutaneous vascular conductance in elderly in response to local or reflex-mediated heat stress is due to an augmented or sustained noradrenergic vasoconstriction [34]. Forearm skin perfusion was measured by laser Doppler flowmetry in 15 young (22 þ 1 years) and 15 older (66 þ 1 years) men who exercised at 50% peak oxygen uptake in a 36 C environment. Blood flow was monitored in two sites, one of which was pretreated with bretylium tosylate (BT) to block the local release of norepinephrine and thus vasoconstriction. Forearm vascular
16
conductance was 40–50% lower in the older adults. Decreased active vasodilator sensitivity to increasing core temperature, coupled with structural limitations to vasodilation, appears to limit the cutaneous vascular response to exertional heat stress in older subjects. At rest in a thermoneutral environment, human cutaneous vascular conductance is under the tonic influence of noradrenergic vasoconstrictor activity. During dynamic exercise in a warm environment, during which cutaneous vascular conductance can increase more than tenfold, vasoconstriction is withdrawn and the active vasodilator system is activated. For a given mean arterial pressure, under any given set of exercise and environmental conditions, the balance between vasoconstriction and vasodilation activity determines cutaneous vascular conductance. The conclusions of the study were that the diminished cutaneous vascular response in the skin of older subjects during exertional heat stress occurred independent of noradrenergic vasoconstriction. The mechanistic alterations that could explain this diminished vascular response were: (a) a relatively greater vasoconstrictor activity, (b) decreased vasodilator activity, or (c) end-organ response differences, which could be independent of efferent neural activity. The study of Kenney eliminates the first possibility and suggests that a combination of the latter two may be involved [34]. Minson extended the work on the effects of aging on the cutaneous microvasculature by investigating the role of nitric oxide (NO) and the axon reflexes in the skin blood flow response to local heating with advanced age [35]. Two microdialysis fibers were placed in the forearm skin of ten young (22 2 years) and ten older (77 5 years) men and women. Skin blood flow was measured by laser Doppler flowmeter. Both sites were heated to 42 C for 60 min while 10 mM NG-nitroL-arginine methyl ester (L-NAME) was infused throughout the protocol to inhibit NO synthase (NOS) in one site and 10 mM L-NAME was infused after 40 min of local heating in the second site. Local heating before L-NAME infusion resulted in a significantly reduced initial peak and plateau maximum vasodilation in elderly subjects. This finding suggests that healthy aging impacts the nerves that mediate the axon reflex or vascular responsiveness to the neurotransmitters released from these nerves. When NOS was inhibited after 40 min of heating, vasodilation declined to the same value in the young and older subjects. The initial peak response was significantly lower in the older subjects in both microdialysis sites. These data suggest that age-related changes in both axon reflex-mediated and NO-mediated vasodilation contribute to attenuated cutaneous vasodilator responses
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Considerations for Thermal Injury: The Elderly as a Sensitive Population
in the elderly. A diminished ability to rapidly increase skin blood flow in response to directly applied heat may make the elderly more susceptible to local tissue damage. Research by Tur has demonstrated age-related differences in skin blood flow in response to histamine administration [36]. In this study, the cutaneous microvascular response of older individuals (64–74 years) as compared to younger individuals (25–35 years) was slower to peak blood flow and took longer to decay. There were also regional differences such that peak blood flow was greater in the back of the young as compared to the forearm, while both sites were similar in the older cohort. While heat challenge should produce a vasodilatory response, Khan investigated the effect of aging on the vasculature during a cooling challenge and concluded that elderly subjects have diminished sympathetic vasoconstrictor responses [37]. This may be a significant factor contributing to thermoregulatory impairment in the elderly, thereby rendering them more susceptible to the harmful effects of cold weather. Using laser Doppler flowmetry sympathetic vascular responses in fingertip skin was evaluated. Indirect body heating was employed to minimize variability. The change in fingertip blood flow produced by inspiratory gasp and contralateral arm cold challenge was determined. The normal response is a rapid vasoconstriction with a subsequent decrease in fingertip blood flow, which returns to its pre-stimulus value. The study evaluated 28 elderly (mean age 68 years with SD of 4 years) and 20 younger subjects (mean age 26 years with SD of 5 years). Experiments were conducted in a room set at 25 C (55% relative humidity) and the subjects’ right arm was placed up to the elbow in a water bath maintained at 43 C. The increase in blood flow is directed mainly through arteriovenous anastomoses, but capillary blood flow also increases owing to raised local tissue temperature as a consequence of high shunt flow. The inspiratory gasp consisted of a sudden deep breath with the right arm in 43 C water throughout followed by a transfer of the right arm into a cold water bath at 15 C. A second experiment took place where subject bodies were placed in a temperature-controlled chamber in a room set at 25 C. The chamber was heated to 40 C to induce central dilation. The chamber was subsequently cooled to 12 C, which took approximately 10 min and was maintained for another 20 min. The time of blood flow to fall to 75%, 50%, and 25% from steady state was determined. Vasoconstrictor responses were significantly reduced in the elderly group in response to inspiratory gasp and cold challenge, although individual responses varied from normal to absent. The authors concluded that there is a 65% probability that an otherwise normal
elderly person will have a vasoconstrictor response considered abnormal for healthy young subjects. Whole-body cooling yielded similar results in that some elderly subjects demonstrated rapid vasoconstriction, whereas others responded with poor vasoconstrictor ability. Time to minimum blood flow after vasoconstriction was longer in the elderly, but only the time to 25% blood flow was statistically significant. While specific details on the location and nature of the effect is not clear, diminished vasoconstrictor responses most likely also result form general changes in sympathetic nervous function with age, since fingertip vasoconstriction produced by inspirator gasp and cold challenge is dependent upon sympathetic nervous activity. There was wide variability among elderly subjects in their response. The authors did not comment on the changes in blood flow with regard to heat challenge. The significance of the findings are that since thermal equilibrium is protected by reflex adjustments of cutaneous blood flow in the extremities, diminished vasoconstrictor response would promote significant heat loss in the elderly during cold exposure. The reduced vasoconstrictor response most probably occurs in the thermoregulatory shunts because a major proportion of the laser Doppler finger blood flow signal arise from flow through arteriovenous anastomoses.
Effects of Aging on Thermoregulation While increased morbidity and mortality in the elderly population during heat waves has been well documented in several medical reports, relatively few scientific studies have focused on the physiological basis of the aging process in thermoregulation, and those that have produced conflicting results. This section examines research on the effects of age on thermal regulation during exertion or exposure to increased ambient temperatures. Changes in the basic physiological mechanisms of thermoregulation may contribute to a decreased ability to avoid hyperthermia in the elderly. These changes may involve the ability to sweat and the vasomotor response to heat and could result from structural changes in the skin as well as less-effective neural regulation of blood flow and sweating. Weiss in 1992 examined the capillary blood flow velocity in the feet of ten young (ages 28–43) and 12 elderly (ages 72–84) men at skin temperatures of 32 C and 44 C [38]. The mean peak capillary blood flow in the elderly (102 mV measured by laser Doppler flowmetry) was lower than in the young population (163 mV). Considering that the magnitude, but not the pattern of skin perfusion varied between the groups, the authors concluded that aging is associated with the loss of capillary
Considerations for Thermal Injury: The Elderly as a Sensitive Population
plexus functional units, and therefore skin perfusion is lower in aged people [38]. Martin measured the maximal forearm skin vasodilatory capacity across a group of 74 subjects ranging in age from 5 to 85 years [39]. Maximal forearm skin vascular conductance was the endpoint of choice and represents the maximum forearm skin blood flow divided by the mean arterial blood pressure. The results demonstrated a progressive decrease in maximum forearm skin vascular conductance with age from young adulthood through old age. The authors note substantial histological and scanning electron micrographic evidence in the underside of the epidermis including the collapse, disorganization, and even total disappearance of vessels comprising the microcirculation. Others report a decrease in the number of superficial capillary loops in the skin as it ages. Such changes are consistent with the attenuated maximal skin blood flow response in older adults. However, a reduced blood flow to existing vessels cannot be ruled out as well. To evaluate the effect of age on thermoregulation Sagawa exposed six older (61–73 years of age) and ten younger (21–39 years of age) Japanese men to 40 C and 40% relative humidity (while sitting) for up to 130 min and examined sweat responses, esophageal, and skin temperatures, non-evaporative heat exchange, heart rate, cardiac output, blood pressure, forearm blood flow, and metabolic heat production [40]. There was no significant difference in sweat rate or in onset of sweating between the groups (> Table 16.2). Changes in skin temperature, non-evaporative heat exchange, metabolic heat production, heart rate, and cardiac output were the same during heat exposure in both groups. However, forearm blood flow before and after exposure to heat was significantly lower in the elderly group. These data suggest that the greater health risk posed to resting, yet healthy, aged
. Table 16.2 Effect of heat exposure (40 C and 40% relative humidity) on select hemodynamic parameters from Sagawa et al., 1988 study Age group
Control
Onset of sweating
At 95 min
Forearm blood flow (mL/100 mL/min) Elderly
1.3 0.2
6.0 1.7
4.6 0.7
Young
2.6 0.4*
7.2 0.9
8.7 1.4*
Forearm vascular conductance (mL/100 mL/min/Torr ¥ 100) Elderly
1.3 0.2
7.2 2.4
5.2 0.7
Young
3.0 0.5*
9.3 1.0*
12.2 1.9*
*Significant difference between age groups (p < 0.05)
16
men by hot environments is not a consequence of inadequate sweating but could be associated with retardation of the cutaneous vasodilatation reflex, which can prevent effective transfer of the body heat to its shell, thus resulting in greater heat storage. The impairment of vasomotor function in aged persons is not related to inadequate cardiac response, but is perhaps associated with insufficient vasoconstriction of the blood supply to the viscera, resulting in less blood being shunted to the skin. This suggestion is probable because, in old age, the responsiveness of the circulatory system to adrenergic nerve control is known to decrease [41]. Therefore, in elderly individuals, a decreased response to beta stimulation could explain the impaired vasodilatory responses, and a lowered response to alpha stimulation could be the underlying mechanism for diminished vasoconstriction in the viscera. The effects of age and acclimation on responses to passive heat were studied by Armstrong and Kenney [42]. Six older men (61 1 years) were compared to six young men (26 2 years) in an environmental chamber during a systematic increase in dry-bulb temperature from 28 C to 46 C followed by 30 min in a constant 46 C environment. If older and younger subjects are matched for VO2max, anthropometry (height, weight, and surface area-to-mass ration), and body composition (skinfold thickness and adiposity), no temperature differences are seen during a passive thermal stress of this magnitude. The authors note that previous studies concluded that older subjects respond to passive heat stress with greater elevations in core temperature than young subjects of the same gender, although this is not a universal finding. Much of the discrepancy can be attributed to subject selection criteria, i.e., such factors as body surface area-to-mass ratio, adiposity, and especially VO2max. The control of heat-induced cutaneous vasodilatation in relation to age was determined in subjects of 55–68 years of age as compared to subjects of 19–30 years of age [43]. Subjects performed 75 min cycle exercises in a hot environment (37 C, 60% relative humidity). Core body temperature and skin temperature rose to the same level in both groups and there were no differences in rate of sweating. However, older subjects responded with lower arm blood flow by about 40% as compared to the younger counterparts. These results suggest an altered control of skin vasodilatation during exercise in the heat in older individuals. Havenith extended this work by examining the relative influence of age (ranging from 20 to 73 years of age) on cardiovascular and thermoregulatory responses to low intensity cycle exercise (60 W for 1 h) in a warm humid environment (35 C, 80% relative humidity) [44]. The results suggest that age is an important contributory
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factor in cardiovascular effector responses to a humid heat stress test, in particular, for heart rate and skin blood flow (forearm blood flow and forearm vascular resistance), both lower with advancing age. Again, there were no age-related effects on sweating rate. In the warm humid climate chosen for the experiment, in which both dry and evaporative heat loss are minimal, the effect of a reduced skin blood flow on core temperature is likewise minimal. During exercise in warm environments, as core temperature rises, skin blood flow increases to facilitate the convective transfer of heat from core to skin. Both the slope of the skin blood flow–core temperature relationship and the steady-state skin blood flow achieved are attenuated in older subjects. It has been hypothesized that this decreased cutaneous vasodilatory response with aging is due to structural changes in the cutaneous vasculature [26]. Rooke examined maximum blood flow in elderly men and noted that blood flow does not increase as much when participants were subjected to total body heating and exercise as compared to younger adults [45]. Local heating of the forearm of seven young men (average age 31 years) and seven elderly men (average age 71 years) showed differences between the two age groups. Skin temperature was raised from 32–35 C to 42 C for 60 min. At baseline, skin blood flow in the two age groups (26–37 years and 66–82 years of age) were comparable. During the last 10 min of heating, blood flows as measured by venous plethysmography were much lower in the elderly than in the young subjects (11.1 2.7 vs 19.9 5.2 mL/min/100 mL, respectively). Thus, aging results in a reduction of the maximal conductance of the cutaneous vasculature. The authors suggest that the major limitation of skin blood flow in the elderly is intrinsic to the structure and function of the skin and not due to autonomic dysfunction. Changes in the number, size, and tortuosity of blood vessels in aged skin could be the cause of limited skin blood flow in the elderly. With age, the dermal thickness decreases by 20% and becomes relatively avascular. The implications are that the elderly appear to be less effective at maintaining normal body temperature than young adults. This applies to both cold and heat exposure, as suggested by a disproportionately high occurrence of hypothermia and heat stroke in the elderly.
Decreased Ability to Feel Heat Insensate skin and chronic illness such as diabetes mellitus are common risk factors in patients burned by therapeutic heat application [9]. The scald potential from hydrotherapy in patients with diabetic or other neuropathy is well
documented [46, 47]. A second group at risk are patients requiring cutaneous, fasciocutaneous, or myocutaneous flap procedures for surgical reconstruction. Transposed flap tissue may be insensate and may also have compromised circulation, which interferes with heat dispersal [48]. Burns have been accidentally produced in anesthetized, unconscious, or immobilized patients by the use of hydrotherapy, heating blankets, hot water bottles, or other warming devices both within the hospital and in the field by Emergency Medical Services. Similarly, the elderly have been identified as a population at greater risk of thermal injury secondary to decreased sensory perception and having a higher threshold for pain [25]. This demonstrates the importance of the ability to sense noxious thermal stimuli so as to remove one self from the burn hazard before extensive damage occurs. Pacinian and Meissner’s corpuscles, which are responsible for pressure and superficial tactile perception, respectively, undergo progressive disorganization and histological degeneration, possibly accompanied by functional loss, with advancing age. Free nerve endings do not seem to be substantially modified. While Buettner [49] reports on the effects of radiant and direct contact heat being similar with a pain threshold at 44.8 C, this work was done with a small group (N ¼ 5) presumably in the general population. Lautenbacher and Strian [50] studied the thresholds for heat pain in 64 healthy persons from 17 to 63 years of age (32 women and 32 men). The stimuli were applied to the thenar and the dorsum pedis with a contact thermode. The thresholds increased significantly with age for the foot, but not the hand. The length of the afferent pathways seems to influence the degree of agerelated changes both in heat-pain perception and in thermal sensitivity, resulting in a distal-proximal pattern of age-dependent decline (> Table 16.3). While the pain threshold for the hand did not show statistical significance with regard to age, there was a positive trend for an elevation in pain threshold with advancing age. Estimated threshold elevations based on quadratic regression curves between 15 and 65 years of age are 0.6 C on the hand and 2.2 C on the foot. The increase of threshold for determining warmth and cool on the foot (but not hand) increased significantly with advancing age. The authors note that it is unlikely that the findings of reduced heat pain and thermal sensitivity on the foot are produced only by age changes of the skin: the free nerve endings of the nociceptive and thermoceptive afferents are mainly located near the epidermal–dermal junction. The flattening of the junction and the thinning of the epidermis and dermis with increasing age may indeed result in more frequent damage to the free nerve endings, and also
16
Considerations for Thermal Injury: The Elderly as a Sensitive Population
. Table 16.3 Thresholds for detecting cool, warm, and heat pain when the stimulus is applied to the hand or foot of subjects 17 to 63 years of age, as reported by Lautenbacher and Strain, 1991 Modality
Site
Cooling sensitivity
Hand
Warming sensitivity
Pain threshold
Modality
Age 17–29 Age 30–44 Age 45–63 years years years 0.8 0.3 C
1.0 0.4 C
0.9 0.4 C
1.4 1.0 C
1.7 1.0 C
2.2 1.6 C
Handb 1.3 0.7 C
1.9 1.0 C
1.6 0.9 C
Foota
. Table 16.4 Thresholds for detecting cool, warm, and heat pain when the stimulus is applied to the chin or lip of subjects 20 to 89 years of age, as reported by Heft et al., 1996
Foota
4.5 1.9 C
6.1 2.8 C
6.2 3.2 C
Hand
45.6 2.5 C
45.2 2.5 C
45.7 1.8 C
Foota
44.9 1.5 C
44.8 1.7 C
45.7 1.2 C
Significant age effect by linear trend analysis, p < 0.05 Significant age effect by quadratic trend analysis, p < 0.05
a
b
in a decrease in the thermal resistance, which would tend to appear as heightened sensitivity. Chakour investigated the effects of age on pain perception mediated by Ad-fibers and C-fibers [51]. During preand post-nerve block periods, older adults (over 65 years of age) exhibited a significant elevation in thermal pain threshold relative to younger adults (20–40 years of age) in response to a noxious CO2 laser thermal stimulus. However, when Ad-fiber function was impaired and only C-fiber information was available, both groups responded similarly. These findings support the notion of a differential age-related change in Ad-fiber-mediated epicritic pain (phasic pain, sharp and pricking in nature) perception versus C-fiber-mediated protopathic pain (tonic pain, dull, burning, or aching in nature) with older adults have an increased thermal pain threshold (i.e., decreased pain sensitivity) as compared to younger adults. The magnitude of loss in sensitivity to mechanical stimuli is greater than to thermal stimulation and several authors have suggested that myelinated fiber function may be more prone to the effects of advancing age. Kenshalo investigated absolute thresholds for six modes of cutaneous stimulation applied to two sites in 27 young (ages 19–31) and elderly (ages 55–84) humans at the thenar eminence (hand) and the plantar foot [52]. The modes were tactile, vibration at 40 and 250 Hz, temperature increases or decreases, and noxious
Cool Warm Pain
Site Chin
Age 30 yearsa 31.5 C
Age 80 yearsa 30.8 C
Change/year ( C) 0.01
Lip
32.4 C
31.9 C
0.01
Chin
33.8 C
36.1 C
0.05
Lip
33.8 C
34.3 C
0.01
Chin
43.9 C
47.1 C
0.06
Lip
43.2 C
45.6 C
0.05
a
Predicted by linear regression
heat (via conductive heat application). For heat-pain threshold, the temperature applied was 40 C and increased at a rate of 0.3 C/s until the participant pushed a spring loaded button signifying the detection of heat pain. In this study, no statistically significant differences were found between young (mean 44.60 C and 46.46 C) and elderly (mean 44.95 C and 46.69 C) hands or feet, respectively, in their sensitivity to heat-pain stimulation. In both populations, the hands were more sensitive to a noxious heat stimulus than the feet. A larger study was conducted by Heft that evaluated 179 healthy adults aged 20–89 years who rated threshold and suprathreshold warming, cooling, and painful stimuli applied to the upper lip and chin of the face [53]. The results agree with those of Kenshalo in that while there were slight elevations in detection thresholds for cool, warm, and painful stimuli in older subjects, under suprathreshold conditions there were no statistically significant age differences for the painful stimuli (> Table 16.4). The observed threshold changes and the less consistent changes in suprathreshold performance for the non-noxious stimuli may be related to changes in peripheral nerve function, skin composition, or central nervous system function. Peripheral changes of note would include changes in the underlying innervation of the tissue, changes in the thermal conductivity of the supporting tissues, or both. Free nerve endings, which are associated with the thermal and pain sensations, remain intact into old age [54]. It can be concluded that there is a slight diminution in threshold and supra-threshold thermal performance with increasing age, and they speculate that these changes are best explained at this time by alterations in the skin thermal conductivity. Using linear regression of the data, Heft summarized the thresholds for warming, cooling, and pain conditions at the lip and chin sites in 179 subjects [53].
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Considerations for Thermal Injury: The Elderly as a Sensitive Population
Burn Prevention
Acknowledgments
One example of burn prevention is that associated with water heaters in the home. Feldman reported on unsafe bath water temperatures in the Seattle area where 80% of homes tested had bathtub water temperatures of 54 C or greater, exposing the occupants to the risk of fullthickness scalds within 30 s exposure [55]. Such burns can be prevented by limiting household water temperatures to less than 52 C. This work led to educational campaigns and legislation to lower water heater temperatures to 49–54 C [56, 57]. This work, which reduced hot water heater set points from 60–65 C down to 49 C was effective in reducing the frequency, morbidity, and mortality of tap water burn injuries. The average bath temperature of a group of 20 subjects after lowering the water heater set point was 40.5 C; with a range from 36 C to 42.5 C. Average shower temperatures (seven subjects) were slightly lower than for baths, 40 C (range 38.5– 41.0 C) [58]. These and other easy interventions in the home can reduce the risk of injury to elderly individuals.
The author expresses his appreciation and gratitude to Dr. Karen Blackburn, Dr. Rob Rapaport, and Dr. Jim McCarthy for their valuable scientific comments and suggestions.
Conclusion Thermal injuries in the general population are not uncommon. One particular population at increased risk, the elderly, continues to expand. This increased risk can be explained by several physical and physiological changes that occur with aging. These changes include thinning of the skin, reduced ability to vasodilate the peripheral vasculature in a protective response to a heat stimulus (to remove the heat from the area and maintain a safe skin temperature), and a reduced sensitivity to noxious heat stimuli with advancing age. While the landmark work of Henriques and Moritz has provided data that demonstrates the temporal relationship between skin temperature and thermal injury, additional work is needed to understand this relationship in elderly individuals. Whether advancing age shifts the time–skin–temperature curve relative to a younger population is not known. Regardless, more and more evidence demonstrate that the elderly are less able to defend against a thermal challenge relative to their younger counterparts. Because thermal injury in an elderly population can have more significant consequences with respect to morbidity and mortality, the prevention of these injuries has utmost significance. Burn prevention campaigns thus become very important and effective tools in communicating that older adults are at greater risk of thermal injury and simple changes in behavior in the home environment can prevent these injuries [3, 7, 19, 56].
References 1. Lionelli GT, Pickus EJ, Beckum OK, et al. A three decade analysis of factors affecting burn mortality in the elderly. Burns. 2005;31: 958–963. 2. Burn Foundation. Burn incidence and treatment in the United States 1999 fact sheet. Philadelphia, PA, 1999. 3. Redlick F, Cooke A, Gomez M, et al. A survey of risk factors for burns in the elderly and prevention strategies. J Burn Care Rehabil. 2002;23:351–356. 4. Baptiste MS, Feck G. Preventing tap water burns. Am J Public Health. 1980;70:727–729. 5. Petro JA, Belger D, Salzberg CA, et al. Burn accidents and the elderly: what is happening and how to prevent it. Geriatrics. 1989;44 (3):25–48. 6. Stassen NA, Lukan JK, Mizuguchi NN, et al. Thermal injury in the elderly: when is comfort the right choice? Am Surg. 2001;67: 704–708. 7. American Burn Association. Scalds: a burning issue. A campaign kit for burn awareness week, 2000. 8. Bull JP, Lawrence JC. Thermal conditions to produce skin burns. Fire Mater. 1979;3(2):100–105. 9. Barillo DJ, Coffey EC, Shirani KZ, et al. Burns caused by medical therapy. J Burn Care Rehabil. 2000;21:269–273. 10. Burlington DB, Brown A. FDA/CPSC public health advisory: hazards associated with the use of electric heating pads, 1995. http://www.fda.gov/downloads/MedicalDevices/Safety/AlertsandNotices/PublicHealthNotifications/UCM062624.pdf. 11. Henriques FC, Moritz AR. Studies of thermal injury. I: the conduction of heat to and through skin and the temperatures attained therein. A theoretical and experimental investigation. Am J Pathol. 1947;23:531–549. 12. Moritz AR, Henriques FC. Studies of thermal injury. II: the relative importance of time and surface temperature in the causation of cutaneous burns. Am J Pathol. 1947;23:695–720. 13. Moritz AR, Henriques FC, Dutra FR, et al. Studies of thermal injury. IV: an exploration of casualty-producing attributes of conflagrations; local and systemic effects of generalized cutaneous exposure to excessive circumambient (air) and circumradiant heat of varying duration and intensity. Arch Pathol. 1947;43:466–488. 14. Henriques FC. Studies in thermal injury. V: the predictability and the significance of thermally induced rate processes leading to irreversible epidermal injury. Arch Pathol. 1947;43:489–502. 15. Henriques FC. Studies of thermal injury VII. Automatic recording calorie applicator and skin tissue and skin surface thermocouples. Rev Sci Instr. 1947;18:673–680. 16. Wu Y-C. Material properties criteria for thermal safety. J Mater. 1972;1(4):573–579. 17. ASTM Designation. C 1055–03: Standard guide for heated system surface conditions that produce contact burn injuries. Published October 2003, pp. 1–8.
Considerations for Thermal Injury: The Elderly as a Sensitive Population 18. Ghods M, Corterier C, Zindel K, et al. Case report. Hot air sauna burns. Burns. 2008;34:122–124. 19. Tan J, Banez C, Cheung Y, et al. Effectiveness of a burn prevention campaign for older adults. J Burn Care Rehabil. 2004;25:445–451. 20. Katcher ML. Prevention of tap water scald burns: evaluation of a multi-media injury control program. Am J Public Health. 1987;77: 1195–1197. 21. Harper RD, Dickson WA. Reducing the burn risk to elderly persons living in residential care. Burns. 1995;21:205–208. 22. Diller KR. Analysis of burns caused by long-term exposure to a heating pad. J Burn Care Rehabil. 1991;12:214–217. 23. Jenkins G. Molecular mechanisms of skin ageing. Mech Ageing Dev. 2002;123:801–810. 24. Montagna W, Carlisle K. Structural changes in ageing skin. Br J Dermatol. 1990;122(Suppl 35):61–70. 25. Cerimele D, Celleno L, Serri F. Physiological changes in aging skin. Br J Dermatol. 1990;122(Suppl 35):13–20. 26. Martin AD. Skin thickness: caliper measurement and typical values. CRC Press, Boca Raton, 1995, pp. 293–296. 27. Moragas A, Castells C, Sans M. Mathematical morphologic analysis of aging-related epidermal changes. Anal Quant Cytol Histol. 1993;15:75–82. 28. Kokate JY, Leland KJ, Held AM, et al. Temperature-modulated pressure ulcers: a porcine model. Arch Phys Med Rehabil. 1995;76: 666–673. 29. Braverman IM, Sibley J, Keh-Yen A. A study of the veil cells around normal, diabetic, and aged cutaneous microvessels. J Invest Dermatol. 1986;86:57–62. 30. Lipkin M, Hardy JD. Measurement of some thermal properties of human tissues. J Appl Physiol. 1954;7:212–217. 31. Richardson D. Effects of age on cutaneous circulatory response to direct heat on the forearm. J Gerontol. 1989;44:M189–M194. 32. Dinenno FA, Jones PP, Seals DR, et al. Limb blood flow and vascular conductance are reduced with age in healthy humans. Relation to elevations in sympathetic nerve activity and declines in oxygen demand. Circulation. 1999;100:164–170. 33. Detry JMR, Brengelmann GL, Rowell LB, et al. Skin and muscle components of forearm blood flow in directly heated resting man. J Appl Physiol. 1972;32:506–511. 34. Kenney WL, Morgan AL, Farquahar WB, et al. Decreased active vasodilator sensitivity in aged skin. Am J Physiol. 1997;272: H1609–H1614. 35. Minson CT, Holowatz LA, Wong BJ, et al. Decreased nitric oxideand axon reflex-mediated cutaneous vasodilation with age during local heating. J Appl Physiol. 2002;93:1644–1649. 36. Tur E. Age-related regional variations of human skin blood flow response to histamine. Acta Derm Venereol (Stockh.) 1995;75: 451–454. 37. Khan F, Spence VA, Belch JJF. Cutaneous vascular responses and thermoregulation in relation to age. Clin Sci. 1992;82:521–528.
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38. Weiss M, Milman B, Rosen B, et al. Analysis of the diminished skin perfusion in elderly people by laser Doppler flowmetry. Age Ageing. 1992;21:237–241. 39. Martin HL, Loomis JL, Kenney WL. Maximal skin vascular conductance in subjects aged 8–85 yr. J Appl Physiol. 1995;79(1):297–301. 40. Sagawa S, Shiraki K, Yousef MK, et al. Sweating and cardiovascular responses of aged men to heat exposure. J Gerontol. 1988;43: M1–M8. 41. Roberts J, Steinberg GM. Effects of aging on adrenergic receptors: introduction. Fed Proc. 1986;45:40–41. 42. Armstrong CG, Kenney WL. Effects of age and acclimation on responses to passive heat exposure. J Appl Physiol. 1993;75 (5):2162–2167. 43. Kenney WL. Control of heat-induced cutaneous vasodilatation in relation to age. Eur J Appl Physiol. 1988;57:120–125. 44. Havenith G, Inoue Y, Luttikholt V, et al. Age predicts cardiovascular, but not thermoregulatory, responses to humid heat stress. Eur J Appl Physiol. 1995;70:88–96. 45. Rooke GA, Savage MV, Brengelmann GL. Maximal skin blood flow is decreased in elderly men. J Appl Physiol. 1994;77(1):11–14. 46. Katcher ML, Shapiro MM. Lower extremity burns related to sensory loss in diabetes mellitus. J Fam Pract. 1987;24(2):149–151. 47. Balakrishnan C, Rak TP, Meininger MS. Burns of the neuropathic foot following use of therapeutic footbaths. Burns. 1995;21:622–623. 48. Cavadas PC, Bonanad E. Unusual complications in a gracilis myocutaneous free flap. Plast Recontstr Surg. 1996;97:683. 49. Buettner K. Effects of extreme heat and cold on human skin. II. Surface temperature, pain and heat conductivity in experiments with radiant heat. J Appl Physiol. 1951;3:703–713. 50. Lautenbacher S, Strin F. Similarities in age differences in heat pain perception and thermal sensitivity. Funct Neurol. 1991;6:129–135. 51. Chakour MC, Gibson SJ, Bradbeer M, et al. The effect of age on Adand C-fibre thermal pain perception. Pain. 1996;64:143–152. 52. Kenshalo DR. Somesthetic sensitivity in young and elderly humans. J Gerontol. 1986;41:732–742. 53. Heft MW, Cooper BY, O’Brien KK, et al. Aging effects on the perception of noxious and non-noxious thermal stimuli applied to the face. Aging Clin Exp Res. 1996;8:35–41. 54. Montagna W, Carlisle K. Structural changes in aging human skin. J Invest Dermatol. 1979;73:15–20. 55. Feldman KW, Schaller RT, Feldman JA, et al. Tap water scald burns in children. Pediatrics. 1978;62(1):1–7. 56. Liao C-C, Rossignol AM. Landmarks in burn prevention. Burns. 2000;26:422–434. 57. Erdman TC, Feldman KW, Rivara FP, et al. Tap water burn prevention: the effect of legislation. Pediatrics. 1991;88:572–577. 58. Lawrence JC, Bull JP. Thermal conditions which cause skin burns. Inst Mech Engineers Eng Med. 1976;5:61–63.
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36 Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones Enzo Berardesca . Joachim Fluhr
Introduction The stratum corneum is viewed currently as a layer of protein-enriched corneocytes embedded in a lipidenriched, intercellular matrix [1], the so-called bricks and mortar model. The ‘‘bricks’’ are corneocytes surrounded by a cornified cell envelope made up of proteins, mainly loricrin, filaggrin, and involucrin, and covalently bound to the hydroxyceramide molecules of a lipid envelope. These ‘‘bricks’’ are embedded in a ‘‘mortar’’ of lipid bilayers [2–4]. The so-called mortar contains a variety of intercellular lipids including, ceramides, free sterols and sterolesters, cholesterolsulfate, and free fatty acids. The stratum corneum continually renews itself, and there is a steady state between the proliferation and differentiation process of keratinocytes and desquamation of corneocytes. Two important forces are responsible for the adherence of corneocytes and build the functional barrier of the skin: the corneodesmosomes as a morphomechanical force and the intercellular lipids as a functional force. Entering the process of differentiation, keratinosomes containing lamellar structured lipid bilayers reach to the apical cell pole from the center of the cytosol in the stratum spinosum and are extruded at the border between the stratum granulosum and stratum corneum. In the intercellular space, lipids form bi- and multilamellar structures, adhering to the corneocytes [1, 2]. Apart from an intact barrier function, the water content of the epidermis depends on the so-called natural moisturizing factors (NMF). These are amino acids, lactic acid, pyrrolidone carboxylic acid, and urea which are released after the breakdown of filaggrin in the mid-portion of the stratum corneum corneocytes, exhibiting an osmotic force and thus binding water. The effect of age on the thickness of skin strata is one of the more controversial topics among dermatological researchers. Comparing measures of skin layer thickness between individuals (and among studies) is especially challenging due to the significant variation in the measurements between individuals and between sites within each individual. Light and electron microscopic studies
have provided important evidence for morphological changes in skin strata with age, even though there is a general agreement that skin thickness (in terms of epidermis, dermis, and also stratum corneum) decreases with age (> Fig. 36.1).
Changes in Stratum Corneum with Age There have been few attempts to measure the rate of corneocyte loss and desquamation in relation to the aging process. This is odd because desquamation is a very important process. Corneocyte size and renewal (or turnover) depend not only on the rate of input into the system (epidermopoiesis), but also on the rate at which cells are lost (desquamation). The epidermis shows a linear decrease in thickness with age, both in absolute terms and in cell number. The reduction in epidermal population size suggests that there may also be a decrease in the rate of production of epidermal cells, and the apparent lengthening of the stratum corneum renewal time seems to confirm it. In addition, there is some evidence that the rate of reepithelization of wounds decreases with age. Using tritiated thymidine and an autoradiographic labeling method, Kligman [5] reported a reduced value for an elderly cohort compared to a younger group; in a study comparing the effects of aging between sun-exposed and non-exposed sites, this has not been detected [6]. A more sensitive but complicated assay using the FACS fluorescent assay demonstrated an age-related decrease in the DNA synthesis and therefore in a longer cell cycle through the stratum corneum [7]. Stratum corneum cell turnover and replacement time have been evaluated using also the dansyl chloride staining technique. Dansyl chloride is a fluorescent dye, which penetrates the full thickness of the stratum corneum, and when applied topically to the skin in vivo, becomes florescent under Wood’s light [8]. The time the fluorescence takes to disappear corresponds to the turnover cycle of the stratum corneum; these studies have shown a progressive increase in the turnover time of the stratum corneum
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. Figure 36.1 Correlation between thickness of the stratum corneum and age (Marks R [10])
. Figure 36.2 Corneocyte surface area and age. There is a significantly positive correlation (Plewig G [11])
associated with increasing age [9]. The lengthening of the turnover implies a reduction in the desquamation rate, but this is not as large as thought. The reason is the increase of corneocyte size during aging. Thus, there are fewer corneocytes in an old individual’s stratum corneum compared to a young one, per unit volume (> Fig. 36.2). Studies measuring the release of corneocytes from the skin also showed that there is a decrease of corneocyte loss at least when measured under these experimental conditions [10]. The evolution of corneocyte size during the aging process has been studied by several authors; there is a consensus that the size progressively increases with age, even though there are body site and seasonal variations (changes due to hormonal status will be discussed later in this document). The more investigated sites are the arm and the forearm, and data show a progressive increase of corneocyte size from birth to age (> Fig. 36.3) [11–14]. Some differences have been reported between sunexposed and non-exposed areas (> Fig. 36.4) [15] where in general UV irradiation increases epidermal turnover leading to smaller corneocytes compared to a similar photo-protected site. Indeed, seasonal variations in corneocyte size have been reported with smaller corneocytes in summer as a consequence of prolonged solar irradiation [16]. In a study on professional cyclists, it was found that the size of corneocytes from the area of the
arm protected by the shirt was ‘‘normal,’’ while in the adjacent exposed site the area of the cells was significantly smaller [17]. In conclusion there is a correlation and an inverse relationship between stratum corneum turnover and dimensions of corneocytes (> Fig. 36.5).
Influence of Sex Hormones The influences of sex hormones on morphologic and functional parameters of the epidermis are of increasing interest. The effects of hormones and aging on stratum corneum structure, function, and composition are not yet known in detail. Although age-dependent factors have been studied, few data are available concerning changes in perimenopausal women [18] with a significantly decreased sebum content of the forehead in menopausal women and higher stratum corneum hydration of the forehead in late menopausal women. Influences of female hormones on the composition of stratum corneum sphingolipids have been described, as well as the negative impact of age on the biosynthesis of sphingolipids [19]. With age a decline occurs in hormone levels, especially in sex hormones like estrogen, testosterone, dehydroepiandrosterone, and growth hormones [20, 21]. Hormone replacement therapy (HRT) leads to an increase in
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
. Figure 36.3 Evolution of the corneocyte size versus age on the forearm. Data of different groups from (Plewig G) [11] (solid squares), 12 (solid circles), 13 (open circles), 14 (open squares)
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collagen content [22]. Under basal conditions the physiologic functions of stratum corneum seems to remain unchanged with age. Under stressed conditions, however, aged skin is more susceptible to barrier disruption than younger skin, i.e. an aged epidermal permeability barrier shows decreased cohesion as well as delayed barrier repair with age under stress conditions [23, 24]. In a recent study [25], corneocyte size in pre- and postmenopausal women of the same age group (40–50 years) was investigated and compared to men of the same decade using a videomicroscopic technique: despite the close age range, the significantly smaller corneocytes in premenopausal women versus postmenopausal women or men are likely to be attributed to the different levels of female sex hormones (> Fig. 36.6). The detected differences support the hypothesis that sexual hormones have an impact on corneocyte surface area. Female sex hormone levels of premenopausal women are supposed to be higher than those of nonhormonal substituted postmenopausal women or men, and thus the smaller corneocyte surface area could be explained by the influence of female sex hormones. The barrier function and the stratum corneum hydration parameters are not involved in this mechanism as no correlation between these parameters and corneocyte surface area was detectable. In this study, no other major differences in barrier function or stratum
. Figure 36.4 Comparative evolution of corneocyte size at different body sites (From Corcuff P, Leveque JL [15])
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. Figure 36.5 Epidermal turnover and corneocyte size as influenced by age. Increasing size of corneocytes derives from slowing down of turnover related to the aging process
. Figure 36.6 Corneocyte size pre- and postmenopause as compared to men of the same age. Menopause induces a fast increase in corneocyte size comparable to men of the same age (Fluhr et al. [25])
corneum water-holding properties have been detected (maybe for the close chronological age range of the groups investigated), even though there some reports in the literature on the positive impact of hormone replacement therapy on cutaneous mechanical properties and water-holding capacity [26, 27]. Further investigation is necessary to study the physiology of perimenopausal skin especially under stress conditions. The role of hormonal replacement has been documented in a study [28] where menopausal women who had been treated for at least 5 or 10 years, the biophysical measurements were significantly higher for the parameters evaluating hydration and sebum secretion, which generally decrease after the menopause, associated with higher values for the yellow
intensity parameter and the skin relief parameters on the forehead. The skin relief parameters on the forehead were significantly higher in menopausal women since at least 5 years and taking HRT. This is one of the few studies that have demonstrated an effect of exposure to HRT on skin color assessed by colorimetry, and on skin relief with an increase of the roughness parameters on the forehead. An investigation assessed the effect of HRT on the skin, using high frequency diagnostic ultrasound combined with computerized image analysis. The study was a crosssectional observational study carried out on 84 women (comprising 34 HRT users, 25 postmenopausal controls, and 25 premenopausal controls). The time that volunteers had been taking for HRT varied from 6 months to 6 years. The skin was shown to be thicker in the HRT group than in the postmenopausal control group [29]. An additional study evaluating the severity of facial wrinkling by an eight-point photographic scale in a sample of Korean women, estimated the HRT exposition impact among 85 postmenopausal women, comprising 15 taking HRT. HRT was found to be associated with a lower risk for facial wrinkling in the postmenopausal women group [30]. These results support the subjective impression and the clinical evaluation concerning the impact of HRT on the development and the severity of some properties associated with skin aging after menopause.
Conclusion The aging process, associated with hormonal changes in women during menopause has a significant impact on the
Corneocyte Size and Cell Renewal: Effects of Aging and Sex Hormones
physiology of the skin and the stratum corneum. In particular, at this level, corneocytes are larger due to the slowing down of the metabolic processes and to the keratinocyte turnover; this can cause changes in the physical properties of the upper layers causing some ‘‘cosmetic’’ effects such as decreased brightness and reduction of transcutaneous penetration; cell renewal is slower, even though desquamation rate seems to be constant. Hydration of the stratum corneum seems not to change too much during aging, despite contradictory reports: probably this is related to an uneven distribution of the water profile on the skin surface, which can be investigated today by new imaging techniques [31].
Cross-references > Stratum > The
Corneum Cell Layers Stratum Corneum and Aging
References 1. Landmann L. The epidermal permeability barrier. Anat Embryol (Berl). 1988;178:1–10. 2. Swartzenruber DC, Wertz PW, Madison KC, et al. Evidence that the corneocyte has a chemically bound lipid envelope. J Invest Dermatol. 1987;88:709–713. 3. Wertz PW, Downing DT. Covalently bound o-hydroxyacylsphingosine in the stratum corneum. Biochim Biophys Acta. 1987;917:108–111. 4. Steven AC, Steinert PM. Protein composition of cornified cell envelopes of epidermal keratinocytes. J Cell Sci. 1994;107:693–700. 5. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–56. 6. Marks R, et al. The effects of phoageing and intrinsic ageing on epidermal structure and function. G Ital Chir Dermatol Oncol. 1987;2:252–263. 7. Marks R. The epidermal engine. A commentary on epidermopoiesis, desquamation and their interrelationships. J Cosmet Sci. 1986;8:135–144. 8. Jansen LH, Hojyo-Tomoko MT, Kligman AM. Improved fluorescence staining technique for estimating turnover of the human stratum corneum. Br J Dermatol. 1974;90:9–14. 9. Roberts D, Marks R. Determination of age variations in the rate of desquamation. A comparison of four techniques. J Invest Dermatol. 1979;74:13–16. 10. Marks R. Measurement of biological ageing in human epidermis. Br J Dermatol. 1981;104:627–633. 11. Plewig G. Regional differences in cell sizes in the human stratum corneum II. Effect of sex and age. J Invest Dermatol. 1970;54:19–23. 12. Marks R, Nicholls S, King CS. Studies on isolated cornocytes. Int J Cosmet Sci. 1981;3:251–258. 13. Grove GL, Lavker RM, Hoelzle E, Kligman AM. Use of noon intrusive tests to monitor age associated changes in human skin. J Soc Cosmet Chem. 1981;32:15–26.
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14. Leveque JL, Corcuff P, DeRigal J, Agache P. In vivo studies on the evolution of physical properties of the human skin with age. Int J Dermatol. 1984;23:322–329. 15. Corcuff P, Leveque JL. Corneocyte changes after acute UV irradiation and chronic solar exposure. Photodermatology. 1988;5:110–115. 16. Hermann S, Scheuber E, Plewig G. Exfoliative cytology: effects of seasons. In: Marks R, Plewing G (eds) Stratum Corneum. Beriln: Springer-Verlag, 1983, pp. 181–185. 17. Leveque JL, Porte G, DeRgal J, Corcuff P, Francois AM, Saint-Leger D. Influence of chronic sun exposure on some biophysical parameters of the human skin; an in vivo study. J Cutan Aging Cosmet Dermatol. 1988;1:123–127. 18. Ohta H, Makita K, Kawashima T, Kinoshita S, Takenouchi M, Nozawa S. Relationship between dermato-physiological changes and hormonal status in pre-, peri-, and postmenopausal women. Maturitas. 1998;30:55–62. 19. Denda M, Koyama J, Hori J, Horii I, Takahashi M, Hara M, Tagami H. Age- and sex-dependent change in stratum corneum sphingolipids. Arch Dermatol Res. 1993;285:415–417. 20. Tazuke S, Khaw KT, Barrett-Connor E. Exogenous estrogen and endogenous sex hormones. Medicine (Baltimore). 1992;71:44–51. 21. Roshan S, Nader S, Orlander P. Review: ageing and hormones. Eur J Clin Invest. 1999;29:210–213. 22. Sauerbronn AVD, Fonseca AM, Bagnoli VR, Saldiva PH, Pinotti JA. The effects of systemic hormonal replacement therapy on the skin of postmenopausal women. Int J Gynaecol Obstet. 2000;68:35–41. 23. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95:2281–2290. 24. Reed JT, Ghadially R, Elias PM. Skin type, but neither race nor gender influence epidermal permeability barrier function. Arch Dermatol. 1995;131:1134–1138. 25. Fluhr JW, Pelosi A, Lazzerini S, Dikstein S, Berardesca E. Differences in corneocyte surface area in pre- and post-menopausal women: assessment with the noninvasive videomicroscopic imaging of corneocytes method (VIC) under basal conditions. Skin Pharmacol Appl Skin Physiol. 2001;14(Suppl 1):10–16. 26. Pierard-Franchimont C, Letawe C, Goffin V, Pierard GE. Skin waterholding capacity and transdermal estrogen therapy for menopause: a pilot study. Maturitas. 1995;22:151–154. 27. Pierard GE, Letawe C, Dowlati A, Pierard-Franchimont C. Effect of hormone replacement therapy for menopause on the mechanical properties of skin. J Am Geriatr Soc. 1995;43:662–665. 28. Guinot C, et al. Effect of hormonal replacement therapy on skin biophysical properties of menopausal women. Skin Res Technol. 2005;11:201–204. 29. Chen L, Dyson M, Rymer J, et al. The use of high frequency diagnostic ultrasound to investigate the effect of hormone replacement therapy on skin thickness. Skin Res Technol. 2001;7:95–97. 30. Youn CS, Kwon OS, Won CH, et al. Effect of pregnancy and menopause on facial wrinkling in women. Acta Dermatol Venereol. 2003;83:419–424. 31. Batisse D, Giron F, Leveque JL. Capacitance imaging of skin surface. Skin Res Technol. 2006;12:99–104.
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39 Cyanoacrylate Skin Surface Strippings Claudine Pie´rard-Franchimont . Jorge Arrese-Estrada . Pascale Quatresooz . Ge´rald E. Pie´rard
Introduction In its most widely appreciated context, the stratum corneum (SC) exerts a major barrier function extending to protection from ultraviolet light, microorganisms, oxidants, and other toxic xenobiotics. In addition, it protects from loss of water and electrolytes from the body. Despite limited metabolic activity, the SC is viewed as a highly specialized structure showing continuous renewal keeping ideally a steady state in its structure and thickness. However, it is structurally and biochemically heterogeneous. In addition, it acts as a unique sophisticated biosensor that signals the underlying epidermis to respond to a series of external stresses. On most body sites, the SC typically consists of 12–16 layers of flattened corneocytes. These cells are about 1 mm thick and have a mean area of approximately 1,000 mm2. However, the surface area depends on age, anatomical location, and conditions that influence the epidermal renewal such as chemical irritation and UV irradiation. In particular, the average corneocyte size increases with age. This is sometimes assumed to be related to the increased transit time within the SC. Each corneocyte contains a water-insoluble protein complex made predominantly of a highly organized keratin microfibrillar matrix. Such a structure is encapsulated in a protein- and lipid-enriched shell. This cornified cell envelope shows differences in maturation among corneocytes. Two distinct types of cornified cell envelopes were distinguished as ‘‘fragile’’ and ‘‘rigid,’’ or ‘‘immature’’ and ‘‘mature’’ [1, 2]. In some instances, the SC homeostasis is altered. Indeed, the SC is the repository of many biological events that occurred below it in previous days. The SC structure is further altered by diverse and repeated external threats. The genetic background, nutritional status, and some physical agents, as well as drugs, cosmetics, toiletries, and other chemical xenobiotics represent other major modulators of the SC structure. Knowledge about fine SC structure is crucial in many aspects of the dermocosmetic science, particularly when dealing with age-related xerosis and effects of surfactants, emollients, and squamolytic agents [3].
Critical Factors for Clinical Practicability of CSSS Cyanoacrylate skin surface stripping (CSSS) is a timehonored method [4]. After its clever discovery, it was soon applied for diagnostic purposes in dermatology. The CSSS method consists of depositing a drop of cyanoacrylate liquid adhesive onto a supple transparent sheet of terephthalate polyethylene, 175 mm thick, cut to the size of a conventional coverslip (1.5 6 cm). The material (3S-biokit, C + K electronic, Germany) is pressed firmly on the lesion. After 15–30 s, a sheet of SC of uniform thickness can be conveniently harvested (> Fig. 39.1). As the adhesion mechanism of cyanoacrylate relies on a chemical reaction, the depth of the removed SC is determined by the depth of penetration of the adhesive before it hardens. The cleavage level is exclusively located inside the SC. Oozing and eroded lesions are not adequately studied using CSSS. The sampling procedure is often painless and bloodless. Anesthesia and antiseptic procedures are unnecessary. The cost is minimal. The following laboratory procedure is simple and not timeconsuming. CSSS are conveniently harvested from any part of the body, with two main provisos. On the one hand, sampling from a hairy area is painful because of pulling out hairs. In addition, the CSSS quality may be inadequate owing to the poor contact with the SC. It is, therefore, advisable to shave these areas before sampling a CSSS. On the other hand, intercorneocyte cohesion on the palms and soles is frequently stronger than the cyanoacrylate bond, thus impairing the collection of an unbroken sheet of corneocytes. However, a CSSS sampling from these sites is possible in certain physiopathological conditions associated with a compromised texture and cohesion of the SC.
Overall Microscopic Aspect of Normal Skin on CSSS CSSS of normal skin reveals a regular network of highpeaked crests related to the skin surface hollow depressions corresponding to the so-called first- and second-order lines.
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Their pattern is typical for specific parts of the body and is subject to variations with age [5]. The primary lines of the skin surface correspond to grooves in the latticework papillary relief at the dermo–epidermal junction [3]. In young individuals, regular intersections of primary and secondary lines delimit regularly shaped polyhedral plateaus (> Fig. 39.2). With aging, this network progressively loses its configuration, being preferentially oriented along the skin tension lines. The process ends with the disappearance of the shallow wrinkles [6]. It is, therefore, possible to indirectly assess the texture of the superficial dermis on CSSS. Accordingly, dermal aging, corticosteroidinduced atrophy, sclerosis, striae distensae, scars, and many other changes in the connective tissue are readily visible in a noninvasive way using CSSS. This morphological assessment of the skin microrelief is conveniently quantified by computerized image analysis using any regular profilometric method [3]. . Figure 39.1 CSSS sampling
. Figure 39.2 CSSS: regular crisscross pattern of primary and secondary order lines of the skin surface
Velus hairs are commonly captured within the CSSS. In addition, CSSS collects follicular casts corresponding to the horny material present at the opening of the pilosebaceous follicles near the skin surface [7]. It is therefore possible to assess the density of the follicles per unit surface area, and to observe the presence of follicular hyperkeratosis (kerosis) as well as comedones, trichostasis spinulosa, intrafollicular bacteria, and mites [3, 5, 7–9].
Cytological Aspects of Normal Skin on CSSS Cytological characteristics of corneocytes are hardly visible on CSSS unless histological dyes are used [5]. A number of stains are suitable. The most useful and simplest one is a mixture of toluidine blue and basic fuschin in 30% ethanol. Normal skin shows a regular cohesive pattern of adjacent anucleated corneocytes. Their boundaries are clearly identified as a thin polyhedral rim (> Fig. 39.3). Parakeratotic cells are rare and dispersed singly on normal skin. They are recognized by the presence of a nucleus central to the polyhedral cell. Saprophytic microorganisms are present at the skin surface forming the biocene of resident bacteria. Thus, most of them are encased within the cyanoacrylate bond during sampling, and they are not accessible to the staining procedure. As a result, only a portion of the surface microflora is seen on CSSS [5]. By contrast, microorganisms present inside the follicular casts are collected distinctly from the skin surface biocene by scraping out these horny spiky structures appending to the CSSS. Viability of the intrafollicular bacteria can be assessed using flow cytometry [10].
. Figure 39.3 CSSS: regular corneocyte paving
Cyanoacrylate Skin Surface Strippings
Corneomelametry Melanin is present in normal corneocytes of phototype V and VI individuals. The dusty load can be specifically revealed using argentaffin-staining procedures. The relative darkness of these CSSS can be assessed using corneomelametry [11, 12]. This method consists of measuring the reduction of light transmission through the CSSS using a photomicroscope equipped with an internal photodensitometer device. On a cytological viewpoint, it is important to distinguish melanin-laden anucleated corneocytes from neoplastic dendritic melanocytes after their migration into the SC covering a malignant melanoma.
Diagnostic CSSS in Inflammatory Conditions Obviously the diagnostic indications for CSSS only apply to disorders characterized by changes taking place in the SC. Many common dermatoses are conveniently diagnosed using CSSS [3, 5, 13]. Straightforward diagnoses can be established in superficial, infectious, and parasitic skin diseases. Morphological examination, possibly combined with fungal cultures, can be carried out to identify these dermatoses. By essence, infectious agents that are made visible on CSSS are not those adhering on top of the skin surface (see above), but rather those invading the SC. Fungi, including yeasts and dermatophytes, exhibit typical morphology (> Fig. 39.4), forming clusters or a network of globular or filamentous structures. In the group of parasitic disorders, scabies may pose problem at the time of sampling. In fact, this diagnosis . Figure 39.4 CSSS of a dermatophytosis: fungal hyphae are clearly identified
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can be established only if the mite, its eggs, or its dejecta are present in the sample. Duplicate CSSS should therefore be sampled from a typical scabies burrow. The first one removes the roof of the burrow and the second one may collect the parasite. Any sample taken outside such lesion, for instance, from nonspecific prurigo, will be unhelpful because the diagnosis will only suggest the presence of a spongiotic dermatitis [5, 14]. Demodex mites are conveniently recognized [5, 14] and highlighted in the follicular casts using the Fite stain. Noninfectious erythemato-squamous disorders conveniently assessed using CSSS include spongiotic and parakeratotic dermatoses and xeroses [3, 5, 14]. Spongiotic dermatitides represent superficial inflammatory reactions responsible for spongiosis, microvesiculation, and serosity leakage inside the SC. Contact dermatitis, atopic dermatitis, and pityriasis rosea are examples that belong to the spongiotic group. Parakeratotic dermatoses encompass id reactions, chronic eczema, and stable psoriasis. The parakeratotic cells are clustered in sheets or thicker bulks (> Fig. 39.5). Seborrheic dermatitis also comes within this parakeratotic category particularly in cases when Malassezia yeasts are rare. In active psoriasis, clusters of neutrophils are found in the center of parakeratotsic foci [14].
Diagnostic CSSS in Cutaneous Neoplasms Some epithelial neoplasms display typical aspects on CSSS. Seborrhoeic keratoses show spotty lenticular foci of soft hyperkeratosis. Widening of shallow furrows with hyperkeratosis is often present [5]. Samples of actinic
. Figure 39.5 CSSS: sheet of parakeratoic cells
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Cyanoacrylate Skin Surface Strippings
keratosis often exhibit irregular thickness with interfollicular parakeratosis and xerosis. The perifollicular rim is, by contrast, featureless. Basal cell and squamous cell carcinomas do not exhibit specific features or suggestive clues on CSSS. Actinic porokeratosis is revealed by the rim of cornoid lamellation and loss of the normal microrelief inside the lesion [5]. Verrucous surfaces overlying melanocytic nevi and dermatofibromas are less pathognomonic, but sharp circumscription with normal surrounding skin and uniformity of the changes in the texture of the SC usually are seen in such benign neoplasms. In melanocytic neoplasms, melanin is found inside corneocytes and eventually in atypical melanocytes. Melanin restricted only inside corneocytes is a feature of benign neoplasms such as lentigines and melanocytic nevi. By contrast, the presence of atypical melanocytes inside the SC is strongly suggestive of malignant melanoma, but also, in rare instances, of a benign melanoacanthoma [5, 13–15]. Thus, CSSS proves to be sensitive and specific for distinguishing malignant melanoma from benign melanocytic tumors such as common melanocytic nevi, dysplastic nevi, or pigmented seborrheic keratoses [13]. For research purposes, karyometry of neoplastic melanocytes is conveniently performed on CSSS [15].
CSSS Assessment of Disease Severity and Therapeutic Activity Disease severity and therapeutic improvement are possibly assessed noninvasively on CSSS exhibiting specific features in the SC. An example is given by xeroses, which correspond to various forms of predominantly orthokeratotic hyperkeratosis [3]. This condition encompasses what is commonly referred to as sensitive skin or dry skin, but this appearance is also found to a more severe degree in ichthyoses [3, 5, 14]. Several types and grades of orthokeratotic hyperkeratosis are distinguished on CSSS [3, 5, 14]. Type 0 is the absence of hyperkeratosis, except for some discrete focal accumulation of corneocytes in the primary order lines of the skin. Type 1a corresponds to a continuous linear hyperkeratosis of the primary lines. Type 1b is characterized by hyperkeratosis predominant at the site of adnexal openings either at hair follicles or at acrosyringia. Type 2 corresponds to focal hyperkeratosis of the skin surface plateaus covering less than 30% of the surface of the sampling. Type 3 resembles type 2, but with a xerotic area over 30% of the CSSS. Type 4 is defined by a homogeneous and diffuse hyperkeratosis with persistence of the trace of primary order lines. Type 5a resembles Type 4, but with
loss of recognizable primary lines. Type 5b corresponds to the most heterogeneous and diffuse hyperkeratosis with loss of or marked remodeling of the primary line network.
Corneofungimetry In superficial dermatomycoses, fungal cells are readily visible on CSSS. In experimental settings, some assessments of disease severity and therapeutic activity on dermatomycoses can be performed on CSSS using computerized image analysis. In an in vitro procedure, fungi are conveniently cultured using corneocytes [16], and particularly CSSS as growth substrates [17, 18]. Quantifications of the restricted fungal growth after the application of antifungals in experimental dermatomycosis are conveniently performed using corneofungimetry [17, 19–21]. The oral or topical antifungals are administered to healthy volunteers for a given period of time (usually a couple of days). CSSS are sampled afterward. A controlled amount of fungal cells collected from a primary culture is deposited onto the CSSS supposedly impregnated by the antifungal test. After a given time (usually 7–10 days) of culture in a clean environment, the CSSS are stained for revealing fungi. Computerized image analysis is used to fine-tune the quantification of the mycelium growing on CSSS. The comparison with control untreated CSSS allows to derive the percentage of inhibition of the fungal growth. Corneofungimetry has several advantages over conventional in vitro evaluation of antifungals: (a) the treatment is applied in vivo in conditions normally encountered by patients, (b) the initial fungal load is controlled, (c) the growth medium is only composed of keratinocytes without any artificial compounds, and (d) any influence of keratinocytes including natural antimicrobial peptides is respected.
Comedometry Comedometry allows the computerized quantification of the number and size of follicular casts present on CSSS. This method finds application in the assessment of comedogenesis-related disorders and in their treatments [9, 10, 22]. Acne is the major indication. In vivo comedometry on human skin appears more relevant than animal models of comedogenesis. The sensitivity of the method is such that microcomedolysis is possibly objectivated by computerized image analysis after a few days or weeks of treatment.
Cyanoacrylate Skin Surface Strippings
Corneosurfametry and Corneoxenometry The interaction between the SC and various chemical xenobiotics is conveniently assessed on CSSS. Corneosurfametry (CSM) refers to the effects of surfactants and wash solutions [3, 23–25]; CSSS are harvested from healthy volunteers. A solution of the test product is sprayed on the CSSS which are placed in covered plastic trays. After a given time of incubation at controlled temperature, the samples are thoroughly rinsed in tap water, dried and stained for 3 min in a toluidine blue-basic fuschin solution. The samples are then copiously rinsed with water and dried prior to color determination using reflectance colorimetry. Indeed, surfactants remove lipids and denaturate corneocyte proteins, thus revealing sites available for staining deposition. A combined dotted and rimmed pattern is visible at the microscopic examination (> Fig. 39.6). Using quantitative reflectance colorimetry, mean luminancy (L*) and Chroma C* are calculated from measurements made at three sites on each sample placed on a white reference plate. It is known that mild surfactants with little effect on corneocytes give high L* values and low Chroma C* values. L* decreases and Chroma C* increases with the irritancy potential of the product. The differences between L* and Chroma C* values of each sample give colorimetric indices of mildness (CIM). The CSM index (CSMI) of the test product, corresponds to the difference in color between water-treated control . Figure 39.6 CSM bioassay: corneocyte alterations due to surfactants
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samples and those exposed to the test product. It is conveniently calculated according to the following formula: CSMI = [(DL*)2 + (DC*)2]0.5. Microwave CSM is a more rapid procedure [26]. CSSS are immersed in a flask containing the test surfactant solution. Samples are then placed in a microwave oven with a 500-mL water load. Microwave CSM is typically run at 750 W for 30 s. The next steps are identical to the standard CSM procedure. Responsive CSM is a variant of the method where skin has been pretreated before CSSS sampling [27]. The method is based on repeat subclinical injuries by surfactants monitored in a controlled forearm immersion test. At completion of the in vivo procedure, CSSS are harvested for a regular or microwave CSM bioassay using the same surfactant as in the preliminary in vivo procedure. Preconditioning the skin in this way increases CSM sensitivity to discriminate among mild surfactants [27]. Shielded CSM is used for testing skin protective products (SPP) [28]. SPP claiming for being barrier creams should be shields against noxious agents. In shielded CSM, the CSSS are first covered by the test SPP before performing regular CSM using a reference surfactant. Comparative screenings of SPP are conveniently performed using shielded CSM without exposing volunteers to hazards linked to in vivo testing. Animal CSM can be performed [29] in a way similar to human CSM. The method is available for safety testing of cleansing products specifically designed for some
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Cyanoacrylate Skin Surface Strippings
animal species. In addition, interspecies differences in surfactant reactivity of the skin to surfactants are conveniently assessed [29]. The corneoxenometry (CXM) bioassay is used for testing any chemical xenobiotic other than surfactants [28, 30, 31]. The basic procedure is similar to CSM and its variants. One main indication is found in the field of skin irritation while avoiding the in vivo hazards. Another indication concerns the comparative assessment of penetration enhancers commonly used in topical formulations [30].
Conclusion Besides conventional biopsies and cytology of exudates, imprints, and scrapings, CSSS provide useful information in the field of dermatopathology. This simple, rapid, cheap, and noninvasive method allows the clinician to avoid a conventional biopsy within the limits of welldefined indications. Less than 3 min are necessary between sampling and examination. There are evident features and subtle characteristics discernible in the structure of the SC that enable a diagnosis to be made in a variety of skin diseases. It is important to stress that no single criterion should usually be relied upon for a definitive diagnosis, but rather a constellation of clues should be sought. Quantifications are made possible on CSSS using computerassisted image analysis. A series of derived methods have been designed for investigative purposes. When performed under controlled procedures, the information appears reproducible and sensitive. In many instances, the procedures help to bypass animal testing and to avoid a number of hazards bound to in vivo human trials.
References 1. Hirao T, Denda M, Takahashi M. Identification of immature cornified envelopes in the barrier-impaired epidermis by characterization of their hydrophobicity and antigenicities of the components. Exp Dermatol. 2001;10:35–44. 2. Harding CR, et al. The cornified cell envelope: an important marker of stratum corneum maturation in healthy and dry skin. Int J Cosmet Sci. 2003;25:157–167. 3. Pie´rard GE. EEMCO guidance for the assessment of dry skin (xerosis) and ichthyosis: evaluation by stratum corneum strippings. Skin Res Technol. 1996;2:3–11. 4. Marks R, Dawber RPR. Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Dermatol. 1971;84:117–123.
5. Pie´rard-Franchimont C, Pie´rard GE. Assessment of aging and actinic damages by cyanoacrylate skin surface stripping. Am J Dermatopathol. 1987;9:500–509. 6. Quatresooz P, et al. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet. 2006;28:389–395. 7. Uhoda E, et al. The conundrum of skin pores in dermocosmetology. Dermatology. 2005;210:3–7. 8. Pagnoni A, et al. Determination of density of follicles on various regions of the face by cyanoacrylate biopsy: correlation with sebum output. Br J Dermatol. 1994;131:862–865. 9. Letawe C, Boone M, Pie´rard GE. Digital image analysis of the effect of topically applied linoleic acid on acne microcomedones. Clin Exp Dermatol. 1998;23:56–58. 10. Pie´rard-Franchimont C, et al. Lymecycline and minocycline in inflammatory acne. A randomized, double-blind study on clinical and in vivo antibacterial efficacy. Skin pharmacol. Appl Skin Physiol. 2002;15:112–119. 11. Thirion L, Pie´rard-Franchimont C, Pie´rard GE. Whitening effect of a dermocosmetic formulation. A randomized double-blind controlled study on melasma. Int J Cosmet Sci. 2006;28:263–267. 12. Pie´rard-Franchimont C, et al. Analytic quantification of the bleaching effect of 4-hydroxyanisole-tretinoin combination on actinic lentigines. J Drugs Dermatol. 2008;7:873–878. 13. Pie´rard GE, et al. Cyanoacrylate skin surface strippings as an improved approach for distinguishing dysplastic nevi from malignant melanomas. J Cutan Pathol. 1989;16:180–182. 14. Pie´rard-Franchimont C, Pie´rard, GE. Skin surface stripping in diagnosing and monitoring inflamnatory, xerotic and neoplastic diseases. Pediatr Dermatol. 1985;2:180–184. 15. Pie´rard GE, et al. Karyometry of malignant melanoma cells present in skin strippings. Skin Res Technol. 1995;1:177–179. 16. Faergemann J. A new model for growth and filament production of Pityrosporum ovale (orbiculare) on human stratum corneum in vitro. J Invest Dermatol. 1989;92:117–119. 17. Rurangirwa A, Pie´rard-Franchimont C, Pie´rard GE. Culture of fungi on cyanoacrylate skin surface strippings: a quantitative bioassay for evaluating antifungal drugs. Clin Exp Dermatol. 1989;59:425–428. 18. Aljabre SHM, et al. Germination of Trichophyton mentagrophytes on human stratum corneum in vitro. J Med Vet Mycol. 1992; 30:145–152. 19. Pie´rard GE, Pie´rard-Franchimont C, Arrese Estrada J. Comparative study of the activity and lingering effect of topical antifungals. Skin Pharmacol. 1993;6:208–214. 20. Arrese JE, et al. Euclidean and fractal computer-assisted corneofungimetry. A comparison of 2% ketoconazole and 1% terbinafine topical formulations. Dermatology. 2002;204:222. 21. Pie´rard-Franchimont C, et al. Activity of the triazole antifungal R12663 as assessed by corneofungimetry. Skin Pharmacol Physiol. 2006;19:49–56. 22. Uhoda E, Pie´rard-Franchimont C, Pie´rard GE. Comedolysis by a lipohydroxyacid formulation in acne prone subjects. Eur J Dermatol. 2003;13:65–68. 23. Pie´rard GE, et al. Surfactant induced dermatitis. A comparison of corneosurfametry with predictive testing on human and reconstructed skin. J Am Acad Dermatol. 1995;33:462–469. 24. Henry F, et al. Regional differences in stratum corneum reactivity to surfactants: quantitative assessment using the corneosurfametry bioassay. Contact Dermatitis. 1997;37:271–275.
Cyanoacrylate Skin Surface Strippings 25. Xhauflaire-Uhoda E, et al. Skin capacitance imaging and corneosurfametry. A comparative assessment of the impact of surfactants on stratum corneum. Contact Dermatitis. 2006;54:249–253. 26. Goffin V, Pie´rard GE. Microwave corneosurfametry and the shortduration dansyl chloride extraction test for rating concentrated irritant surfactants. Dermatology. 2001;202:46–48. 27. Uhoda E, Goffin V, Pie´rard GE. Responsive corneosurfametry following in vivo preconditioning. Contact Dermatitis. 2003;49: 292–296. 28. Xhauflaire-Uhoda E, et al. Skin protection creams in medical settings: successful or evil? J Occup Med Toxicol. 2008;3:15–20.
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29. Goffin V, Fontaine J, Pie´rard GE, et al. Comparative surfactant reactivity of canine and human stratum corneum. A plea for the use of the corneosurfametry bioassay. Altern Lab Anim. 1999;27: 103–109. 30. Goffin V, et al. Penetration enhancers assessed by corneoxenometry. Skin Pharmacol Appl Skin Physiol. 2000;13:280–284. 31. Xhauflaire-UHoda E, Pie´rard-Franchimont C, Pie´rard GE. Effect of various concentrations of glycolic acid at the corneoxenometry and collaxenometry bioassays. J Cosmet Dermatol. 2008;7:198–294.
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Physiology
4 Degenerative Changes in Aging Skin Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
Introduction Over the last 2 centuries, medical progress has dramatically extended the human lifespan, more than doubling life expectancy across the world. Average global life expectancy has risen from about 25 (for both sexes) to 65 for men and 70 for women [1]. Women, whose average life expectancies exceed those of men, can now expect to spend more than one-third of their lifetimes in post menopause [2]. More than 40 million postmenopausal women live in the USA today, comprising 17% of the total population [3]. Although the human skin is incredibly durable, like all other organ systems it is affected by aging [3–5]. A sophisticated and dynamic organ comprising 17% of the body’s weight, the skin primarily acts as the barrier between the internal environment and the world outside. Yet it performs numerous functions beyond simply acting as a barrier [6]: homeostatic regulation, prevention of percutaneous loss of fluid, electrolytes, and proteins; temperature maintenance; sensory perception; and immune surveillance [7]. Aging involves both intrinsic and extrinsic processes occurring in parallel [8]. Intrinsic aging proceeds at different rates in all organisms at a genetically determined pace. It is caused primarily by the build-up of reactive oxygen species (ROS) as a by-product of cellular metabolism and by ROS-induced damage to critical cellular components like membranes, enzymes, and DNA. Skin cells become increasingly senescent as they age: [8] the rate of cell proliferation in the epidermis drops, which contributes to deterioration of skin structure and function [9]. Extrinsic aging is accelerated aging that is superimposed on intrinsic effects of age that result from environmental insults to the skin, often controllable exposures such as solar radiation [10]. As a human being ages, the skin thins, dries, wrinkles, and becomes unevenly pigmented [11]. A loss of subcutaneous fat, as well as underlying bone and cartilage, manifests as sagging skin and fallen nasal tips [12]. Skin complaints by older adults, particularly women, are largely esthetic – plastic surgery has become the fastest growing medical specialty [11] – but aging of the skin also can
produce significant morbidity. In fact, most people over 65 have at least one skin disorder, and many have two or more [13]. Chronic dryness and itching are particularly prevalent; in one study of healthy Japanese over 60 years of age, 95% suffered dry skin at least part of the year [14]. Irritant contact dermatitis associated with incontinence also rises among older adults [15, 16]. Various inflammatory, infectious, and vascular disorders become more common [17]. The prevalence of cutaneous malignancy also rises with age [17, 18]. Distinguishing the precludable aspects of cutaneous aging (primarily hormonal and lifestyle influences) from the inexorable (primarily intrinsic aging) is essential to preventing and treating the ailments of the aging skin. As the population ages, medical care of older skin must shift in focus from cosmetic improvements to reducing morbidity and mortality from dermatological disorders. This will improve the quality of life for the growing population of elderly adults [19].
Structure and Function of Normal Skin The skin is composed of three layers: epidermis, dermis, and hypodermis (> Fig. 4.1).
Epidermis The outer layer of the skin, the epidermis, contains primarily keratinocytes with smaller populations of melanocytes and immune cells (Langerhans cells) [19]. Epidermal thickness, which varies according to anatomic site and individual, averages from 50 to 100 mm [20]. The epidermis is a dynamic system whose structure and metabolism fulfill two main functions: to protect the skin from external insult and to maintain hydration of internal tissues [21]. Both functions are accomplished primarily by the stratum corneum, the outermost layer of the epidermis [22]. Epidermal keratinocytes originate in a single layer of cells at the basement membrane (the layer between the dermis and the epidermis). Cells produced at this layer move upwards; as they ascend, they
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_4, # Springer-Verlag Berlin Heidelberg 2010
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Degenerative Changes in Aging Skin
. Figure 4.1 Normal skin structure showing layers of epidermis, dermis and hypodermis
produce the definitive skin-cell protein, keratin, as well as a variety of lipids. As the keratinocytes continue to move upward toward the skin surface, they change shape as they mature. The stratum corneum (SC), the surface layer of skin, is composed of the flattened cell bodies of dead keratinocytes, now called corneocytes [23]. The SC averages 15 layers over most of the body, but ranges from as little as 3 layers in the very thin skin under the eye [29] to more than 50 layers on the palms and soles of the feet [24]. A dynamic and metabolically interactive tissue [22], the SC, comprises about 60% structural proteins, 20% water, and 20% lipids [19, 25]. The corneocytes of the SC are covered by a highly cross-linked and cornified envelope. The extracellular lipid lamellae consist of ceramides, long-chain free fatty acids, and cholesterol. The ceramides strongly adhere to the cornified envelope of the corneocytes, yielding a barrier membrane which, in healthy adults, maintains the water content of the viable portion of the epidermis at about 70% [26]. The strength of the water barrier also depends on its specific lipid composition [22] and relative
proportions of cholesterol, ceramides, and free fatty acids [19, 22]. These intercellular lipids, as well as sebum, natural moisturizing factor (NMF), organic acids and inorganic ions, impart the water-holding capacity of the SC [23]. Several minor components also contribute to maintaining skin hydration. Hyaluronic acid, a major waterbinding component of the dermis, has been recently shown to play a role in the barrier function and hydration of the SC [27]. Glycerol, which acts as an endogenous humectant, recently has been identified as another component of the SC [28]. In addition, a water-transporting protein named aquaporin-3, expressed from the basal layer up to one cell layer below the SC, acts to facilitate the movement of water between the basal layer of the epidermis and the SC in order to maintain a constant level of hydration in the viable epidermis [29]. The water content of the SC (about 20%) contrasts dramatically with that of the epidermis (about 70%), a sharp drop observable at the juncture between the stratum granulosum (SG) and the SC [26]. Tight junction structures, recently identified at the corneo-epidermal
Degenerative Changes in Aging Skin
junction, are protein aggregates that both control paracellular permeability and act as another way to prevent water in the epidermis from escaping into the stratum corneum [30]. When the barrier function and water water-retaining capacity of the SC is compromised [19], pathologic skin dryness can develop, at which point the stratum corneum becomes less flexible and begins to crack or fissure [21]. Skin is considered clinically dry when moisture content of the stratum corneum falls below 10%. Skin dehydration and cracking may facilitate entry of pathogenic microbes [19].
Dermis The dermis is a dense and irregular layer of connective tissue, 2 to 3 mm thick, that comprises most of the skin thickness (> Fig. 4.1) [2]. Dermal connective tissue contains elastin and collagen; collagen fibers contribute most of the mass of the skin and the bulk of its tensile strength [2]; elastin fibers provide elasticity and resilience [2]. The dermis also contains much of the skin’s vasculature, its nerve fibers and sensory receptors, and its
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primary water-holding components, i.e. hyaluronic acid (responsible for normal turgor of dermis because of its extraordinary water-holding capacity) and supportive glycosaminoglycans [2]. The dermis also serves as underpinning to the epidermis and binds it to the hypodermis [31].
Hypodermis The hypodermis is a layer of loose connective tissue below the dermis (> Fig. 4.1). It contains the larger blood vessels of the skin, subcutaneous fat (for energy storage and cushioning), and areolar connective tissue. The hypodermis provides cushioning, insulation, and thermoregulation, and it stabilizes the skin by connecting the dermis to the internal organs (> Fig. 4.1) [32].
Structural Changes in Aged Skin Changes in the thickness and other characteristics of the epidermis and dermis as skin ages are detailed below (> Fig 4.2 and > Table 4.1).
. Figure 4.2 Differences in skin structure between young and aged skin. With permission from Informa HealthCare- MA Farage, KW Miller, P Elsner and HI Maibach. 2007. Structural Characteristics of the Aging Skin: A Review. Journal of Cutaneous and Ocular Toxicology 26:343–357
27
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Degenerative Changes in Aging Skin
. Table 4.1 Changes in the structure of aged skin Observed effect of aging Lower lipid content Epidermis
Dermis
Hypodermis Appendages
Reference [77]
Dermal–epidermal junction flattens
[55]
Number of enzymatically active melanocytes decreases by 8% to 20% per decade
[20]
Number of Langerhans cells decreases
[41]
Capacity for re-epithelization diminishes
[78]
Number of pores increases
[79]
Thickness reduced (atrophy)
[36]
Vascularity and cellularity decrease
[12]
Collagen synthesis decreases
[44]
Pacinian and Meissner’s corpuscles degenerate
[44]
Structure of sweat glands becomes distorted, number of functional sweat glands decreases
[47]
Elastic fibers degrade
[21]
Number of blood vessels decreases
[12]
Number of nerve endings reduced
[9]
Distribution of subcutaneous fat changes
[44]
Overall volume decreases
[12]
Hair loses normal pigments
[44]
Hair thins
[44]
Number of sweat glands decreases
[44]
Nail plates become abnormal
[44]
Sebum production reduced
[79]
Skin Thickness Skin thickness rises over the first 20 years of life; subsequently, even though the number of cell layers remains stable, [33] adult skin thins progressively at a rate that accelerates with age [37]. This phenomenon occurs in all layers of the skin. The epidermis decreases in thickness with age [18]. The unexposed epidermis thins by up to 50% between the ages of 30 and 80 [34], but changes in epidermal thickness are most pronounced in exposed areas, such as the face, neck, upper part of the chest and the extensor surface of the hands and forearms [35]. Overall, epidermal thickness decreases at about 6.4% per decade [36, 37], decreasing faster in women than in men. Dermal thickness, [36] vascularity and cellularity also decrease with age. [12] The loss of dermal collagen and elastin makes up most of the reduction in total skin thickness in elderly adults: for example, in postmenopausal women, a decrease in skin thickness of 1.13% per year parallels a 2% decrease per year in collagen content [38].
Dermal thickness decreases at the same rate in both genders [21]. The hypodermis loses much of its fatty cushion with age. The basement membrane, a very small fraction of the total skin thickness, actually increases in thickness with age [39].
Changes in Composition of Aging Skin Epidermis As skin ages, epidermal cell numbers [40] and the epidermal turnover rate decrease [32, 41]. Characteristic changes occur in each of the cell types in the epidermis. Cells of the basal layer become less uniform in size, although average cellular size rises [42]. Keratinocytes change shape as skin ages, becoming shorter and fatter [40]; corneocytes become bigger due to decreased epidermal turnover [33, 43]. Enzymatically active melanocytes
Degenerative Changes in Aging Skin
decrease at a rate of 8% to 20% per decade, resulting in uneven pigmentation in elderly skin [44]. Langerhans cells, like other epidermal cells, display more heterogeneous appearance and function [45]. The number of Langerhan’s cells in the epidermis also decreases with age, leading to impairment of cutaneous immunity [44]. Langerhans cells that are produced have been observed to have fewer dendrites and therefore less antigen-trapping capability [45]. Although the number of sebaceous glands in the epidermis does not change, sebum production decreases [44]; the evolutionary and biologic significance of this remains unclear. The water content of aged skin, particularly that of the stratum corneum, is lower than that of younger skin [18, 19, 21]. Age-related changes in the amino acid composition [21] reduce the amount of cutaneous NMF, thereby decreasing the skin’s water- binding capacity [19]. The water content of the SC decreases progressively with age and eventually falls below the level necessary for effective desquamation; this causes corneocytes to pile up and adhere to the skin surface, which accounts for the roughness, scaliness, flaking that accompanies xerosis in aged skin. The integrity of the SC barrier is dependent on an orderly arrangement of critical lipids [3]. However, total lipid content of the aged skin decreases by as much as 65% [40]. Ceramide levels, particularly ceramide 1 linoleate [46] and ceramide 3 [47], are particularly depleted in older skin. Triglycerides are also reduced, as is the sterol ester fraction of stratum corneum lipids [18]. Although the levels of NMF in the SC are higher in aged skin than in younger (a consequence of the slower rate of epidermal turnover in older individuals [23]), amino acid levels are lower [28]. Corneocytes are fewer but much larger [28], with higher intercorneal cohesiveness [48]. Because permeability does not appear to be significantly increased in the skin of the aged individual, it has been generally assumed that barrier function does not alter significantly with aging [49]. Some differences in barrier function parameters, however, have been noted: Baseline transepidermal water loss (TEWL), a measure of the functional capacity of the stratum corneum to maintain the moisture content of the skin, however, is lower in older patients as compared to younger [18, 50], an observation believed to be due to the reduction of the water content of aged skin. (The elderly have less water to lose [50].) Recovery of baseline TEWL values after occlusion is also impaired in older skin [18]. In addition, it has been demonstrated that the permeability barrier of aged skin is also more vulnerable to disruption. In a study which used tape-stripping to effect loss of barrier integrity, adults over 80 required only 18
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stripping as compared to 31 strippings in young and middle-aged adults. (Tape-stripping is a common method of abrogating the SC by removing one layer of skin at a time by applying and then removing a strip of tape.) Recovery of barrier function in the aged subjects was also dramatically different [49]. Only 15% of those older than 80 had recovered barrier function at 24 h (as assessed by return to baseline TEWL), compared to 50% of the younger group [49]. Artificially-induced water gradients (such as produced by occlusion) were shown, in addition, to dissipate more slowly in older skin than in younger, again indicating reduced recovery capacity in aged skin [51]. The findings reveal that aging may have a profound impact on barrier integrity even though barrier function appears normal. A profound abrogation of functional capacity is exposed when the epidermal permeability barrier is under stress, and the barrier function is more easily disturbed and less able to recover. Interestingly, one study found that as skin dries as an inevitable aspect of intrinsic aging, TEWL and the water content of the stratum corneum drop in parallel, while in pathological conditions, TEWL increases even though stratum corneum water content stays low. In stripped skin both values increase, confirming a derangement of actual barrier function as skin ages [52]. The most widely observed structural change in aged skin is a flattening of the dermal–epidermal junction, which occurs as a result of the decreasing numbers and size of dermal papillae [53]. Histological studies reveal that the number of papillae per unit of area decreases dramatically [54], from an average of 40 papillae/mm2 in young skin, down to 14 papillae/mm2 in those aged over 65 [53]. The flattening of the dermal–epidermal junction, observed by about the sixth decade [36], creates a thinner epidermis primarily because of retraction of rete pegs [36], decreasing the thickness of the dermal– epidermal junction by 35% [32, 55]. As a consequence of the reduced interdigitation between dermis and epidermis and the flattened dermal– epidermal junction, the skin becomes less resistant to shearing forces and more vulnerable to insult [33]. Furthermore, flattening of the dermal–epidermal junction results in a smaller contiguous surface between the two layers and reduces communication between the dermis and epidermis; consequently the supply of nutrients and oxygen to the epidermis diminishes [32, 53]. This flattening also may limit basal cell proliferation and may affect percutaneous absorption [36]. The flattening of the dermal–epidermal junction may also contribute to wrinkle formation [33] by increasing the potential for dermal– epidermal separation [32, 53].
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Degenerative Changes in Aging Skin
Dermis
Hypodermis
The three major extracellular components of the dermis are collagen, elastin, and hyaluronic acid. All three are depleted in older skin. Collagen content decreases at about 2% per year [41], primarily because the production of matrix metalloproteinases, which degrade collagen, increases with age [56]. Degradation of dermal collagen by matrix metalloproteinases impairs the structural integrity of the dermis [57]. Mechanical tension or stress on dermal fibroblasts, created by a healthy collagen matrix, is critical for the maintenance of a proper balance between the synthesis of collagen and the synthesis of collagendegrading enzymes [58]. Fibroblast collapse, due to the accumulation of degraded collagen fibers that prohibit construction of a healthy collagen matrix, causes the ratio of collagen synthesis to collagen degradation to become deranged in a self-perpetuating cycle [57]. Aging is also associated with a decrease in collagen turnover (due to a decrease in fibroblasts and their collagen synthesis) [12] The relative proportions of collagen types are also disrupted over the lifespan. The proportion of Type I collagen to Type III collagen in young skin is approximately 6:1, a ratio which drops significantly over the lifespan as Type I collagen is selectively lost [59], although some increase in collagen Type III synthesis occurs as well [60]. In the aged dermis, collagen fibers become thicker and collagen bundles more disorganized than in younger skin [41]. Collagen cross-links stabilize, reducing elasticity in aged skin. Functional elastin also declines in the dermis with age, as elastin becomes calcified in aged skin and elastin fibers degrade [35]. Elastin turnover also declines [12]. The amount of glycosaminoglycans (GAGs), an important contributor to the structure and water-holding capacity of the dermis, declines with age [32, 53], as does the amount of hyaluronic acid produced by fibroblasts [32, 53] and the amount of interfibrillary ground substance, also a component of a healthy dermal matrix [61]. The loss of structural integrity of the dermis leads to increased rigidity, decreased torsion extensibility [32, 41] and diminished elasticity [2, 21], these properties eroding faster in women than in men [21], with a concomitant increase in vulnerability to shear force injuries [32, 41] The impact of these changes is dramatic: for example, when skin is mechanically depressed, recovery occurs in minutes in young skin, but takes over 24 h in skin of aged individuals [32, 41]. Perception of pressure and light touch also decrease in aged skin as pacinian and Meissner’s corpuscles degenerate. The number of mast cells and fibroblasts in the dermis also decreases [12].
The overall volume of subcutaneous fat typically diminishes with age, although the overall proportion of subcutaneous fat throughout the body increases until approximately age 70. Fat distribution changes as well; that in the face, hands, and feet decreases while a relative increase is observed in the thighs, waist, and abdomen. The physiological significance may be to increase thermoregulatory function by further insulating internal organs.
Physiological Changes Physiological changes in aged skin include changes in (i) biochemistry, (ii) neurosensory perception, (iii) permeability, (iv) vascularization, (v) response to injury, (vi) repair capacity, and (vii) increased incidence of some skin diseases as discussed below (> Table 4.2).
Biochemical Changes Vitamin D content of aged skin declines: synthesis of this compound slows because the dermis and epidermis lack its immediate biosynthetic precursor (7-dehydrocholesterol), which limits formation of the final product [41]. The surface pH of normal adult skin averages pH 5.5. This cutaneous acidity discourages bacterial colonization; it also contributes to the skin’s moisture barrier as amino acids, salts, and other substances in the acid mantle absorb water [54]. The pH of the skin is relatively constant from childhood to approximately age 70 [36], then rises significantly. This rise is especially pronounced in lower limbs, possibly due to impaired circulation [36].
Permeability The penetration and transit of permeants through the skin involves (i) absorption to the stratum corneum; (ii) diffusion through the stratum corneum, epidermis, and papillary dermis; and (iii) the removal by microcirculation [18]. The first two steps depend on the integrity and hydration of the stratum corneum, which in turn is a function of the level and composition of intracellular lipids [18]. The final step depends on the integrity of the microcirculation [18]. Heightened interest exists in transdermal administration of medications for long-term drug delivery in chronic disease, as this results in fewer side effects and promotes
Degenerative Changes in Aging Skin
4
. Table 4.2 Changes in the function of aging skin Function Barrier function Sensory and pain perception Thermoregulation Response to injury
Permeability
Immune Function Miscellaneous
Change
Reference
Renewal time of stratum corneum increased by 50%
[41]
Baseline TEWL lower in elderly skin
[51]
Loss in sensitivity, especially after age 50
[33]
Increased itching
[80]
Decreased sweat production
[44]
Lower inflammatory response (erythema and edema)
[38]
Decreased wound healing
[41]
Reduced re-epithelization
[41]
Increased vulnerability to mechanical trauma
[41]
Decreased percutaneous absorption
[44]
Decreased sebum production
[41]
Decreased vascularization
[65]
Decreased chemical clearance
[41]
Decreased number of circulating thymus-derived lymphocytes
[33]
Decreased risk and intensity of delayed hypersensitivity reactions
[70]
Decreased Vitamin D production
[44]
Reduced elasticity
[2]
TEWL = transepidermal water loss
compliance. Consequently, data on percutaneous drug absorption in older adults have gained importance [62]. In general, older adults seem to absorb topical substances more slowly than younger subjects [63]. However, studies on percutaneous absorption in the aged have produced conflicting results. In people over 65, tetrachlorosalicylanilide was absorbed more slowly, but ammonium hydroxide was absorbed more rapidly, than in younger adults [33]. Increased permeability of aged skin to fluorescein and testosterone was observed in vitro [36, 41]. Absorption of radiolabeled testosterone was demonstrated to be three times that of younger subjects [29]. However, in a separate in vivo study, no difference between estradiol and testosterone absorption was observed in aged skin, while hydrocortisone and benzoic acid were both absorbed far less readily in aged skin as compared to younger [50]. These conflicting results may reflect compound- and body-site differences in the rates of percutaneous absorption [62]. Epidermal penetration of a substance is strongly associated with its hydrophobicity relative to the lipid content of the skin: consequently, hydrophobic compounds penetrate more readily in areas of the body areas that have high percentage of skin lipids. For example on the face, where the weight percentage of skin lipids is 12–15%,
hydrophobic compounds (lipophiles) penetrate more readily than hydrophilic ones, whereas on the soles of the feet, where the weight percentage of skin lipids is 1% to 2%, hydrophilic compounds penetrate more readily than hydrophobic ones [32, 50]. Using topically applied radiolabeled penetrants, excretion of lipophilic compounds, testosterone and estradiol, was compared to excretion of the more hydrophilic hydrocortisone and benzoic acids. Percutaneous absorption was quantified from urinary excretion profiles of radiolabel. No difference in percutaneous absorption of testosterone and estradiol was noted between younger and older skin, but absorption of both hydrocortisone and benzoic acid were nearly doubled in younger skin [62]. Because aged skin is drier and has a lower lipid content than younger skin, it may be less amenable to penetration by hydrophilic moieties [62] (> Table 4.3).
Vascularization and Thermoregulation In older skin, capillaries and small blood vessels regress and become more disorganized [33], blood vessel density diminishes [36], and a 30% reduction in the number of
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Degenerative Changes in Aging Skin
. Table 4.3 Percutaneous absorption of testosterone, estradiol, hydrocortisone, and benzoic acid in young and elderly people Cumulative % dose excreted in 5 days Compound
Molecular weighta
Log K (O/W)a
Testosterone
288.4
3.31
Estradiol
272.4
Hydrocortisone Benzoic acid
Aqueous solubility
Young
Elderly
Insoluble
13.2 3.0 n = 17
2.7
Almost insoluble
10.6 4.9 n=3
362.5
1.93
0.28 g/L
1.87 1.6 n = 15
15.2 8.4 n=7 9.0 5.6 n=6 0.86 0.5b n=9
122.1
1.87
3.4 g/L
42.6 16.5 n=6
23.1 7.0c n=9
Source: Ref. [62] a Values taken from the Merck Index; O/W = octanol–water partition b Significantly different from young control group at p < 0.01 c Significantly different from young control group, p < 0.05
venular cross sections per unit area of the skin surface occurs in non-exposed areas of the skin [33]. Capillaroscopy measurements using fluorescein angiography and native microscopy suggest a decrease in dermal papillary loops, which house the capillary network [36]. Although the pattern of blood flow through individual capillaries remains unchanged, [22] the maximum level of blood flow diminishes as functional capillary plexi are lost. A significant time delay in autonomic vasoconstriction in the aged (e.g., after postural changes, cold arm challenge, inspiratory gasp, body cooling) [18, 36] is well documented; this phenomenon is due primarily to declining function of the autonomic nervous system [18]. Eccrine sweating is markedly impaired with age. Spontaneous sweating in response to dry heat was 70% lower in healthy older subjects compared to young controls, due primarily to decreased output per gland [64]. Vascularity is also lost. Cross-sections of photodamaged skin reveal a 35% reduction in vascularity in the papillary dermis of aged skin [65], as well as reduced blood flow, depleted nutrient exchange, dysfunctional thermoregulation, reduced skin surface temperature, and increased skin pallor [66]. Facial skin temperatures were lower in aged subjects [38], and older people exhibited a wider temperature difference between groin and toes [33]. The elderly are predisposed to both hypothermia and heat stroke, as reduced eccrine sweating rates, lower vasodilation or vasoconstriction of dermal arterioles, and the loss of subcutaneous fat impair thermoregulation [64].
Irritant Response Inflammatory response to an exogenous agent declines in people over 70 years old [18, 32, 50]. The inflammatory response is slower and less intense, and some clinical signs of skin damage are absent [18, 38] (> Fig. 4.3). Diagnosis of common dermatological problems becomes difficult, and allergic sensitization tests may be meaningless [33]. Sunburn response also is attenuated and delayed [33]. Fewer inflammatory cells are seen in cantharidin blisters in older subjects [33]. The manifestation of skin irritation is blunted. Patch testing found less erythema, vesicles, pustules, and wheals in aged skin, as well as a decrease in TEWL [32, 55] in response to a range of skin irritants, including toilet soap [33], kerosene [33], dimethyl sulfoxide [DMSO], ethyl nicotinate, chloroform-methanol, lactic acid [18], chemicals which elicit inflammation by clearly different mechanisms [18]. In some cases, the response is also delayed. Analysis of changes in TEWL after sodium lauryl sulfate (SLS) application to the skin confirmed that in aged skin, the irritation reaction is slower and less frequent in postmenopausal then in premenopausal women [67]. Moreover, although blistering caused by ammonium hydroxide exposure is elicited more rapidly in older people, the time required to attain a full response is much longer than in younger ones [18]. The characteristics of the irritant response may be compound dependent in ways specific to older skin, as
Degenerative Changes in Aging Skin
. Figure 4.3 The inflammatory response is slower and less intense, and some clinical signs of skin damage are absent
chemical irritants induce their effects though different mechanisms. SLS as well as nonanoic acid disrupted keratinocyte metabolism and differentiation, while dithranol induced marked swelling of keratinocytes in the upper epidermis [32, 50]. In a study of croton oil, thymoquinone, and crotonaldehyde on older skin, decreased responsiveness was observed only to croton oil [18, 68].
Immune Response The immune response of aged skin is generally diminished. Numbers of Langerhans cells in the epidermis decrease by about 50% between the age of 25 and the age of 70 [41]. The total number of circulating lymphocytes decreases, as does the number of T-cells [32, 41] and B-cells [32, 41], both of which lose functional capacity with age [69]. Delayed hypersensitivity reactions decrease with age: numerous reports have demonstrated a decrease in the capacity for allergic response [32, 41, 70]. For example, healthy older subjects did not develop sensitivity to some known sensitizers and exhibited a lower frequency of positive reactions to standard test antigens compared to young adult controls [32, 41]. The frequency of IgE-mediated, positive prick tests to common allergens declined with age: peak reactivity was observed among people in their twenties, with 52% of subjects reacting to at least one test allergen; positive response rates dropped steadily with age, declining to 16% frequency among subjects older than 75 years [18]. Levels of circulating autoantibodies increase with age; this occurs in parallel with a decrease in useful antibodies as the aged individual’s existing immunity to specific allergens erodes [32, 46].
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Regenerative Capacity and Response to Injury In healthy skin, about one layer of corneoyctes desquamates every day, so that the whole stratum corneum replaces itself about every 2 weeks [23]. In contrast, elderly stratum corneum may take twice as long [71]. Repair of an impaired barrier requires the presence or the three main lipids in appropriate proportions [72] as well as stratum corneum turnover, both of which are suboptimal in older subjects. Injury repair diminishes with age. Wound-healing events begin later and proceed more slowly. For example, a wound area of 40 cm2, which in 20-year old subjects took 40 days to heal, required almost twice as long – 76 days – in those over 80 [32, 41]. The risk of post-operative wound reopening increased 600% in people in their mid-80s compared to those in their mid-thirties [32, 41]. The tensile strength of healing wounds decreased after the age of 70 [32, 41]. Repair processes like collagen remodeling, cellular proliferation, and wound metabolism are all delayed in the aged [32, 41]. The rate at which fibroblasts initiated migration in vitro following wound initiation was closely related to the age of the cell lines [32, 41, 73]. Barrier function requires twice as long to restore in the aged as compared to younger controls [74]; stratum corneum renewal times were much longer in the aged (about 30 days compared to 20 days in normal skin) [74]. Re-epithelialization of the stratum corneum after blistering is also diminished [75], being twice as long for people over 75 than for those aged 25 [32, 41]. The production of messenger RNA (mRNA) and IL-1 protein is also decreased in the aged, contributing to sluggish barrier recovery [75].
Neurosensory Perception Itching is reported more frequently by older adults. However, pain perception declines and pain perception is delayed after age 50 [33]. Consequently, the risk of tissue injury rises, as the most obvious warning signals – pain, erythema, and edema – appear more slowly [33]. This, coupled with longer wound repair times, results in higher morbidity in the aged.
Conclusion Humans now live to twice their reproductive age, an achievement that is unique [76]. Although many
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Degenerative Changes in Aging Skin
profound changes occur over a skin’s lifetime, the human integument remains relatively functional when protected from excessive environmenpal insult. However, the skin of older adults is compromised in many ways [18]. Structural changes lead to undesirable visible characteristics, as well as a decreased elasticity and resilience. Decreases in neurosensory capacity increase the risk of unrecognized injury. The intrinsic drying of the skin makes the skin itchy and increasingly uncomfortable. The decrease in the skin’s ability to repair itself slows wound repair and re-epithelization dramatically and increases the risk of surgical dehiscence. As the proportion of older adults in the industrialized world increases, caring for the problems of aged skin will improve the quality of life in those later years of life gained by medical advances.
Cross-references > Skin
Aging: A Brief Summary of Characteristic Changes
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60. Savvas M, Bishop J, Laurent G, et al. Type iii collagen content in the skin of postmenopausal women receiving oestradiol and testosterone implants. Br J Obstet Gynaecol. 1993;100:154–156. 61. Castelo-Branco C, Figueras F, Martı´nez de Osaba MJ, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 62. Roskos KV, Guy RH, Maibach HI. Percutaneous absorption in the aged. Dermatol Clin. 1986;4:455–465. 63. Kligman AM. The treatment of photoaged human skin by topical tretinoin. Drugs. 1989;38:1–8. 64. Ohta H, Makita K, Kawashima T, et al. Relationship between dermato-physiological changes and hormonal status in pre-, peri-, and postmenopausal women. Maturitas. 1998;30:55–62. 65. Gilchrest BA, Stoff JS, Soter NA. Chronologic aging alters the response to ultraviolet-induced inflammation in human skin. J Invest Dermatol. 1982;79:11–15. 66. Baumann L. Skin ageing and its treatment. J Pathol. 2007;211: 241–251. 67. Elsner P, Wilhelm D, Maibach HI. Sodium lauryl sulfateinduced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal women. J Am Acad Dermatol. 1990;23:648–652. 68. Coenraads PJ, Bleumink E, Nater JP. Susceptibility to primary irritants: age dependence and relation to contact allergic reactions. Contact Derm. 1975;1:377–381. 69. Szewczuk MR, Campbell RJ. Loss of immune competence with age may be due to auto-anti-idiotypic antibody regulation. Nature. 1980;286:164–166. 70. Robinson MK. Population differences in skin structure and physiology and the susceptibility to irritant and allergic contact dermatitis: implications for skin safety testing and risk assessment. Contact Derm. 1999;41:65–79. 71. Baker H, Blair CP. Cell replacement in the human stratum corneum in old age. Br J Dermatol. 1968;80:367–372. 72. Man MQM, Feingold KR, Thornfeldt CR, et al. Optimization of physiological lipid mixtures for barrier repair. J Invest Dermatol. 1996;106:1096–1101. 73. Muggleton-Harris AL, Reisert PS, Burghoff RL. In vitro characterization of response to stimulus (wounding) with regard to ageing in human skin fibroblasts. Mech Ageing Dev. 1982;19:37–43. 74. Grove GL, Kligman AM. Age-associated changes in human epidermal cell renewal. J Gerontol. 1983;38:137–142. 75. Barland CO, Zettersten E, Brown BS, et al. Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis. J Invest Dermatol. 2004;122:330–336. 76. Naftolin F. Prevention during the menopause is critical for good health: skin studies support protracted hormone therapy. Fertil Steril. 2005;84:293–294, discussion 295. 77. Saint Le´ger D, Franc¸ois AM, Le´veˆque JL, et al. Age-associated changes in stratum corneum lipids and their relation to dryness. Dermatologica. 1988;177:159–164. 78. Holt DR, Kirk SJ, Regan MC, et al. Effect of age on wound healing in healthy human beings. Surgery. 1992;112:293–297; discussion 297–298. 79. Rawlings AV. Ethnic skin types: are there differences in skin structure and function? Int J Cosmet Sci. 2006;28:79–93. 80. Buckley C, Rustin MH. Management of irritable skin disorders in the elderly. Br J Hosp Med. 1990;44:24–26, 28, 30–32.
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Biomarkers
47 DNA Biomarkers in Aging Skin Kimberly G. Norman . Alex Eshaghian . James E. Sligh
Introduction Aging of the skin is the result of both the intrinsic chronological aging process and extrinsic damage caused by environmental factors. A major role of the skin is that of protection from external environmental factors. Ultraviolet radiation (UVR) is the most significant environmental insult to the skin. UVR comprises the spectrum of electromagnetic radiation between the wavelengths of 200 and 400 nm. UVR is subdivided into three categories, each of which has distinct biological effects: UVA (320– 400 nm), UVB (280–320 nm), and UVC (200–280 nm). The stratospheric ozone blocks the radiation whose wavelength is below 290 nm, effectively preventing the entire UVC spectrum and part of the UVB spectrum from reaching human skin. The UVR that does reach the human skin can cause molecular defects including DNA damage, lipid peroxidation, and protein cross-linking, which can lead to premature skin aging or photoaging. Photoaging is a term used to describe the clinical and histological features of chronically UV-exposed skin [1]. Photoaging occurs more frequently in people with fair skin and tends to be located in sun-exposed areas such as the head, neck, hands, and forearms. Sun-exposed areas of the skin exhibit characteristic features of aging in common with sun-protected, chronologically aged skin as well as with other chronologically aged tissues. However, certain features of sun-exposed skin are exclusive to these tissues. Hence, the term photoaging refers to the physiologic and pathological changes that occur specifically in aged tissue that has experienced chronic sun exposure over time. Clinical symptoms of photoaging include dry skin, formation of lentigines and nevi, hyperpigmentation, telangiectasia, leathery appearance, increased wrinkle formation, reduced recoil capacity, increased skin fragility, blister formation, and impaired wound healing ability [1, 2]. UVR also causes histologic changes in the skin including hyperkeratosis, thickening of the basement membrane, irregular melanocyte distribution, elastosis, dermal intercellular and perivascular edema, and perivascular infiltration [1]. Further changes include deposition of glycosaminoglycans, fragmented elastic fibers, and interstitial collagen.
Of the UV lights that reach the skin, UVB is mostly absorbed in the epidermis, whereas UVA penetrates through the epidermis and into the dermis. Therefore, UVB affects keratinocytes in the epidermis and UVA affects keratinocytes in the epidermis and fibroblasts in the dermis. UVB most commonly causes damage in the form of cyclobutane pyrimidine dimers (> Fig. 47.1). The characteristic hallmarks of UVB damage are C to T and CC to TT DNA changes. These occur in semiconservative DNA replication due to the A rule, which states that when DNA polymerase comes across un-interpretable changes, it inserts A residues by default. Thus, two A residues are inserted into DNA on strands opposite to cyclobutane-type cytosine-cytosine dimmers, leading to two TTresidues on the template strand. UVA, on the other hand, primarily causes DNA damage indirectly by the production of short-lived reactive oxygen species (ROS) such as singlet oxygen (O ), O2 , and H2O2 via endogenous photosensitizers. ROS leads to single-stranded DNA breaks, nucleotide changes, and DNA-protein cross-links. Potential sites of ROS-induced DNA damage are shown in > Fig. 47.2, and the formation of 8-hydroxyguanine, the most common altered base due to ROS, is shown in > Fig. 47.3. 8-Hydroxyguanine lesions are employed as a DNA marker of overall oxidative stress in the cell and UV damage in the skin. Singlet oxygen produced by UVA light has been shown to cause strand breaks in the mitochondrial DNA (mtDNA), which has resulted in mtDNA deletions [3]. mtDNA deletions are thought to be involved in the photoaging phenotype and serve as biomarkers of aging in the skin, based upon the observation that chronically UV-exposed skin with clinical signs of photoaging has a high frequency of mtDNA deletions as compared to UV-protected skin [4, 5].
Mitochondrial Implications in Photoaging Mitochondria have well-recognized roles both in the generation of cellular energy and as mediators of cellular events such as apoptosis. Mitochondria contain their own genome, a maternally inherited, circular, double-stranded DNA of 16,569 bp encoding 22 tRNAs, two rRNAs,
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. Figure 47.1 Cyclobutane-type pyrimidine dimer formation. The formation of cyclobutane-type pyrimidine dimers as a result of UVB is shown between thymidine (T) and cytosine (C) nucleotides
. Figure 47.2 Potential sites of oxidative DNA damage. The sites of potential DNA damage due to reactive oxygen species (ROS) are shown in red on the four deoxyribonucleotides. A: adenosine, G: guanosine, T: thymidine, and C: cytosine
. Figure 47.3 8-Hydroxyguanine formation. The formation of 8-hydroxyguanine is the most common DNA defect as a result of reactive oxygen species (ROS). 8-Hydroxyguanine can be formed when a hydroxide radical (OH ·) or H2O2 reacts with guanosine (G)
DNA Biomarkers in Aging Skin
and 13 polypeptides, all of which are subunits in the electron transport chain [6-8]. The remainder of the proteins that function in the mitochondrion are encoded in the nucleus and imported into the mitochondrion. Because the mitochondria contain multiple copies of the mtDNA and cells may contain thousands of mitochondria, newly acquired somatic mutations are heteroplasmic, or mixed with wild type mutations, in nature. However, replicative segregation may allow mutant mtDNA molecules in some cells to become prominent or even to become the exclusive mtDNA in the cell, a condition known as homoplasmy. The mitochondrion serves as the major site for the production of ATP through the process of oxidative phosphorylation (OXPHOS). ROS, natural by-products of this pathway, can damage lipids, proteins, and DNA [9, 10]. mtDNA has a high mutation rate due to its lack of histones, decreased capacity for repair, and its close proximity to the site of ROS formation [11]. Imbalances between oxidative stress and free radical scavenging enzymes have been suggested as the underlying causes of most of the mtDNA damage [12, 13]. There is emerging evidence for mtDNA changes in the complex processes of cellular aging and neoplasia [14–16]. The mitochondrial theories of aging hold as their basic principles that OXPHOS produces a main sources of cellular energy in the form of ATP and that there is an age-related decline in OXPHOS. The mtDNA may be particularly important in this energetic decline because its mutation rate is thought to be at least tenfold higher that that of the nuclear genome [17]. A multitude of factors are likely responsible for this increased mutation rate including the lack of protective histones and a less sophisticated system of proofreading than that present in the nucleus [18]. Also, mtDNA is exposed to higher concentrations of oxygen-free radicals as a consequence of their liberation from natural event occurring in the electron transport chain. Continued exposure of the mtDNA to oxidative damage results in an accumulation of somatic mtDNA mutations over time. These mutations may further decrease the efficiency of OXPHOS and increase the likelihood of additional oxygen-free radical production with further subsequent mtDNA damage. This cycle results in a progressive decline in the energy-generating capacity of the cell. Disease ensues when energy output falls below the minimum energetic threshold for normal tissue function. mtDNA changes have been associated with a variety of inherited and acquired human neurodegenerative disorders, myopathies, and endocrinopathies. Common characteristics of these clinical phenotypes are both delayed onset and age-related progression [16]. Age-related onset and a history of sun
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exposure are typical for acquired cases of non-melanoma skin cancer, which is the most common malignancy of older individuals and is one of the most frequently occurring health problems requiring surgical procedures in the elderly. Although there is substantial data correlating mtDNA changes with aging, in general, and with photoaging in the skin, there is no evidence of mtDNA mutations directly causing these phenotypes.
Mitochondrial DNA Mutations and Photoaging In contrast to other tissues, the skin is subject to both chronological aging and environmental insult in the form of UVR. The resulting genetic changes may lead to specific phenotypes of aging skin: photodamage and neoplasia. Changes in the mtDNA in the skin are well recognized in association with photoaging [19–21]. The most common mutation found in aging tissues is the 4,977 bp ‘‘common’’ deletion [22–28]. In autopsy specimens, the common deletion has been found primarily in sun-exposed areas and not in sun-protected areas [29]. Imbalances between oxidative stress and free radical scavenging enzymes have been suggested as the underlying causes of most of the mtDNA damage [12, 13]. The common deletion has been shown to be inducible both in vitro and in vivo in human skin and is thought to occur as a result of mtDNA damage mediated through singlet oxygen [3, 30]. Additionally, the 4,977 bp ‘‘common’’ deletion has been proposed to be a biomarker of photoaged skin because the level of heteroplasmy in the sun-exposed skin increases with age while such levels are not increased in sun-protected skin [29]. In a study by Ray et al. many mtDNA deletions in addition to the common deletion were identified in the epidermis of skin from older individuals and these deletions were strongly associated with UVR [4]. A report of 200 and 260 bp duplications in the non-coding D loop adds another class of mtDNA rearrangements that have been observed in aged human skin [31]. Additionally, an mtDNA deletion of 3,895 bp was identified as a quantitative marker for sunlight exposure in the human skin [32, 33], and the agingdependent T414G mutation within the control region of mtDNA was found to be accumulated in UVR-damaged skin [34]. It was also discovered that the T414G mutation, which may serve as both a marker for chronological aging and photoaging, was commonly identified within a 3,895 bp deleted mtDNA population. In a study in the authors’ lab to examine mtDNA mutations in photoaging, mtDNA from photodamaged skin was screened for the presence of deletions using long
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extension PCR. mtDNA deletions were found to be abundant in photoaged skin specimens from older patients and their number correlated with the patient age, supporting the use of mtDNA mutations as biomarkers of photoaging in the skin [35]. These mtDNA deletions were typically absent in the paired non-melanoma skin cancers. The observed DNA deletions from skin were often unreported (19 of 21 deletions), but usually shared structural features with mtDNA deletions reported in other tissues in that they generally contain short direct or indirect repeats at the breakpoints, and a single copy of the repeat is left behind in the deleted molecule. The structural similarities between the mtDNA deletions observed in the skin and those seen in tissues not exposed to UVR [36] imply a potential common factor in their generation or resolution. Some of the identified deletions were detected from the numerous skin samples, including 3,715 and 6,278 bp deletions. Interestingly, the frequency of these newly identified deletions approached that of the well-characterized 4,977 bp deletion. A consistently higher level of heteroplasmy of the 4,977 bp ‘‘common’’ deletion was found in the dermis as compared to the epidermis in split samples used, consistent with previous studies [29]. Furthermore, the number of novel deletions in photoaged skin not reported in other tissues suggests that skin may be more vulnerable to such mutations via direct exposure to UVR. The mechanism of formation of such mtDNA deletions has been proposed to be a slip mispairing of the repeats during replication [3, 13, 37, 38]. In order for such a mutation to occur, however, both breakpoints must be single stranded simultaneously, which does not normally occur. However, the sequences flanking the repeats or the sequences within the repeats may render the DNA susceptible to structural conformations allowing mispairing [39]. The findings show that most deletions identified contain such repeats, supporting the slip replication model. The novel 3,715 and 6,278 bp deletions contain 10 and 11 bp direct repeats, respectively, similar in size to that of the 4,977 bp ‘‘common’’ deletion (13 bp). Additionally, the novel 6,278 bp deletion contains a homopolymeric track of seven consecutive cytosine residues interrupted by a single adenosine residue within its repeated breakpoint sequence. Such sequences have been proposed to take on structural characteristics allowing for mispairing to occur [40]. These features may explain the reason why the frequencies of detection of the novel 3,715 and 6,278 bp deletions approached that of the 4,977 bp ‘‘common’’ deletion. It has been proposed that ROS, which are normally generated in response to UVR [41], play a role in the generation of the 4,977 bp
‘‘common’’ deletion [3]. Thus, it is not surprising that so many unreported mtDNA deletions were identified in photoaged skin given its role as a barrier to UVR. As mtDNA deletions accrue in photoaged skin, they may be useful as biomarkers of the combined effects of chronological aging and UV exposure. Additionally, specific mtDNA deletions may complement other deletions and the potential level of a single mtDNA deletion may plateau with time as it adversely affects the bioenergetic properties of the cell. Measurement of a panel of deletions may be a more useful assay of photodamage than heteroplasmy levels of any single deletion. Quantitative analysis of other deletions, in addition to the 4,977 bp ‘‘common’’ deletion may be useful for this purpose.
Conclusion While the 4,977 bp ‘‘common’’ deletion has been proposed to be a biomarker for photoaging, it may reflect only a small portion of total mtDNA damage. The 3,715, 6,278, and 3,895 bp deletions and T414G mutation may prove valuable as additional markers of photoaging in the skin. Furthermore, in this study, the photoaged skin contained an abundance of various deletions beyond the aforementioned mtDNA changes. Thus, the use of the 4,977 bp ‘‘common’’ deletion as a biomarker for photoaged skin may indeed be the tip of the iceberg.
References 1. Berneburg M, Plettenberg H, Krutmann J. Photoaging of human skin. Photodermatol Photoimmunol Photomed. 2000;16:239–244. 2. Scharffetter-Kochanek K. Photoaging of the connective tissue of skin: its prevention and therapy. Adv Pharmacol. 1997;38:639–655. 3. Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274:15345–15349. 4. Ray AJ, Turner R, Nikaido O, Rees JL, Birch-Machin MA. The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J Invest Dermatol. 2000;115:674–679. 5. Berneburg M, Krutmann J. Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J Invest Dermatol. 1998;111:709–710. 6. Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457–465. 7. Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet. 1999;23:147.
DNA Biomarkers in Aging Skin 8. Giles RE, Blanc H, Cann HM, Wallace DC. Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA. 1980;77:6715–6719. 9. Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA. 1994;91: 10771–10778. 10. Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995;27:647–653. 11. Wallace DC, Lott MT, Shoffner JM, Brown MD. Diseases resulting from mitochondrial DNA point mutations. J Inherit Metab Dis. 1992;15:472–479. 12. Lu CY, Lee HC, Fahn HJ, Wei YH. Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin. Mutat Res. 1999;423:11–21. 13. Shoffner JM, Lott MT, Voljavec AS, Soueidan SA, Costigan DA, Wallace DC. Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy. Proc Natl Acad Sci USA. 1989;86:7952–7956. 14. Copeland WC, Wachsman JT, Johnson FM, Penta JS. Mitochondrial DNA alterations in cancer. Cancer Invest. 2002;20:557–569. 15. Eng C, Kiuru M, Fernandez MJ, Aaltonen LA. A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev Cancer. 2003;3:193–202. 16. Wallace DC. Mitochondrial diseases in man and mouse. Science. 1999;283:1482–1488. 17. Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, Morrow JD, Van Remmen H, Sedivy JM, Yamasoba T, Tanokura M, Weindruch R, Leeuwenburgh C, Prolla TA. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005;309:481–484. 18. Croteau DL, Bohr VA. Repair of oxidative damage to nuclear and mitochondrial DNA in mammalian cells. J Biol Chem. 1997;272:25409–25412. 19. Pang CY, Lee HC, Yang JH, Wei YH. Human skin mitochondrial DNA deletions associated with light exposure. Arch Biochem Biophys. 1994;312:534–538. 20. Yang JH, Lee HC, Lin KJ, Wei YH. A specific 4977-bp deletion of mitochondrial DNA in human ageing skin. Arch Dermatol Res. 1994;286:386–390. 21. Yang JH, Lee HC, Wei YH. Photoageing-associated mitochondrial DNA length mutations in human skin. Arch Dermatol Res. 1995;287:641–648. 22. Brierley EJ, Johnson MA, Lightowlers RN, James OF, Turnbull DM. Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle. Ann Neurol. 1998;43:217–223. 23. Cortopassi GA, Shibata D, Soong NW, Arnheim N. A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues. Proc Natl Acad Sci USA. 1992;89:7370–7374. 24. Cortopassi GA, Arnheim N. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res. 1990;18:6927–6933. 25. Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992;256:628–632.
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26. Ikebe S, Tanaka M, Ohno K, Sato W, Hattori K, Kondo T, Mizuno Y, Ozawa T. Increase of deleted mitochondrial DNA in the striatum in Parkinson’s disease and senescence. Biochem Biophys Res Commun. 1990;170:1044–1048. 27. Nagley P, Wei YH. Ageing and mammalian mitochondrial genetics. Trends Genet. 1998;14:513–517. 28. Sciacco M, Bonilla E, Schon EA, DiMauro S, Moraes CT. Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from patients with mitochondrial myopathy. Hum Mol Genet. 1994;3:13–19. 29. Birch-Machin MA, Tindall M, Turner R, Haldane F, Rees JL. Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J Invest Dermatol. 1998;110:149–152. 30. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, GersBarlag H, Gefeller O, Krutmann J. Induction of the photoagingassociated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. 31. Durham SE, Krishnan KJ, Betts J, Birch-Machin MA. Mitochondrial DNA damage in non-melanoma skin cancer. Br J Cancer. 2003;88:90–95. 32. Harbottle A, Birch-Machin MA. Real-time PCR analysis of a 3895 bp mitochondrial DNA deletion in nonmelanoma skin cancer and its use as a quantitative marker for sunlight exposure in human skin. Br J Cancer. 2006;94:1887–1893. 33. Krishnan KJ, Harbottle A, Birch-Machin MA. The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin. J Invest Dermatol. 2004;123:1020–1024. 34. Birket MJ, Birch-Machin MA. Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin. Aging Cell. 2007;6:557–564. 35. Eshaghian A, Vleugels RA, Canter JA, McDonald MA, Stasko T, Sligh JE. Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J Invest Dermatol. 2006;126:336–344. 36. Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. MITOMAP: a human mitochondrial genome database. Nucleic Acids Res. 1996;24:177–179. 37. Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi GM, Koga Y, DiMauro S, Schon EA. Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucleic Acids Res. 1990;18:561–567. 38. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science. 1989;244:346–349. 39. Hou JH, Wei YH. The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA. Biochem J. 1996;318(Pt 3):1065–1070. 40. Fullerton SM, Bernardo Carvalho A, Clark AG. Local rates of recombination are positively correlated with GC content in the human genome. Mol Biol Evol. 2001;18:1139–42. 41. Scharffetter-Kochanek K, Wlaschek M, Brenneisen P, Schauen M, Blaudschun R, Wenk J. UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol Chem. 1997;378: 1247–1257.
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31 DNA Damage and Repair in Skin Aging Daniel B. Yarosh
Introduction DNA has many roles in skin cell function, including directing metabolism, storing the information of heredity, and sensing cell danger. Damage to DNA is a major cause of the chronic conditions of aging and photoaging. The natural repair system offers significant protection, and new compounds offer the promise of augmenting DNA repair. This chapter focuses to a large extent on Ultraviolet (UV) damage to DNA because solar UV is by far the greatest danger to DNA. Sun exposure is a major public health concern, and has been directly linked to most of the (more than one million) new types of skin cancer that arise in the USA each year [1]. From 1990 to 1994, premalignant and malignant skin cancers accounted for 19% of visits to the dermatologist [2]. DNA damage caused by solar UV has been directly linked to these skin cancers, as the cancers contain suppressor genes mutations that are characteristic of UV in their inactivated tumor [3].
Sources of DNA Damage DNA damage comes from two sources: the intrinsic metabolism of the cell and the environmental insult. Intrinsic metabolism. During aerobic energy generation, about 2% of all the oxygen burned ends up as reactive oxygen species (ROS). DNA is damaged by ROS most frequently by the oxidation of the guanine base to form 8-oxo-guanine (8oGua), which is often misread by the DNA replication machinery, causing a mutation. This is particularly serious for mitochondria, whose DNA is closest to the source of the short-lived ROS. Environmental insult. By far, the most serious damage to skin DNA is from the sun. The cyclobutane pyrimidine dimer (CPD) is formed ten times more frequently than 8oGua, and is caused by direct absorption of UV photons without any ROS intermediate. A second type of base fusion, a 6–4 photoproduct, is similarly formed about one sixth as frequently as CPD. CPDs cause a
characteristic type of DNA mutation produced by no other carcinogen, and these signature mutations are frequently found in key cancer genes in squamous and basal cell carcinomas. This is the smoking gun that connects CPDs to skin cancer. Solar UV also causes the formation of ROS, which results in ROS and then 8oGua. In mitochondria, repeated doses of UVA result in the accumulation of a characteristic deletion mutation in mitochondrial DNA. The frequency of this characteristic mutation in human skin increases with sun exposure, suggesting that it is an internal dosimeter for cumulative sun exposure. Not all DNA damage is from sun exposure. Alkylation is a third type of DNA damage in which an alkyl group is added to DNA. The most prevalent additions are at the 7 position of guanine (N7-meGua) and to the phosphates of the DNA backbone, but a much less common form of damage, alkylation of the 6 position of guanine (O6-meGua) is the most mutagenic and hence the most dangerous. This type of damage is usually caused by side reactions of DNA with normal metabolites such as nitrites, and by toxins like some of the chemicals in cigarette smoke. The premature signs of aging in the skin in smokers may be associated with the alkylation damage from alkylating agents in cigarette smoke transported through the circulation to the skin. Additionally, exposure to other chemicals via exposure to industrial pollution and drugs with DNA alkylating potential may have some role in what has been considered normal skin aging.
Sun Damage to DNA Wavelengths of Sunlight that Damage DNA DNA readily absorbs photons in the UV portion of the solar spectrum. Although the shorter UVC wavelengths (200–280 nm) do not actually reach the earth’s surface due to their absorption by the ozone layer, the longer wavelength UVB (280–315 nm) is still relatively efficient in causing direct damage to the DNA bases and largely penetrates only into the epidermis [4]. The even longer
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DNA Damage and Repair in Skin Aging
wavelength UVA (315–400 nm) penetrates into the dermis; however, since these photons carry less energy, they are relatively less efficient in producing direct damage to DNA than UVB and create proportionately more reactive oxygen species in the skin cells that indirectly damage DNA [5]. Recent evidence suggests that very high doses of visible light can produce indirect DNA damage through the formation of reactive oxygen species [6].
Photoproducts Solar UV directly causes an instantaneous photochemical reaction in DNA that links together adjacent pyrimidine bases (cytosine or thymine) [7]. This cyclobutane pyrimidine dimer (CPD) is the most common form of DNA damage, and is formed by all UV wavelengths, including UVA, UVB, and UVC [8]. After a sunburn dose, on the order of 100,000 CPDs are formed in the DNA of every sun-exposed cell. In a much less common reaction, solar UV can directly link together these bases by a single twisted bond, resulting in a 6–4 photoproduct ( PP) [8]. Solar UV can also cause DNA damage by an indirect method, through the formation of reactive oxygen species that attack DNA, particularly the guanine base. This oxidation reaction most often results in 8-oxo-guanosine (8oGua), but even after UVA exposure CPDs are much more common than 8oGua [9]. Oxidation of DNA can
also result in single-stranded breaks, but under physiological conditions these are very difficult to detect. When single-stranded breaks are found after UV irradiation, they are almost all caused by DNA repair enzymes cutting the DNA in an intermediate step in repair.
Mechanisms of DNA Repair DNA is the rare biomolecule that is not discarded when it is damaged, but rather is repaired. Human cells have developed two fundamental repair strategies to restore DNA to its native sequence and conformation (> Table 31.1).
Nucleotide Excision Repair More than 20 different proteins participate in this multistep process, and many of these proteins also participate in RNA transcription and/or DNA synthesis. In a typical day a cell may have to repair 10,000 damaged bases and after sun exposure each cell of the skin may have to remove 100,000 lesions! This process consumes cellular stores of nicotinamide adenine dinucleotide (NAD), which are used to tag sites of single-stranded breaks and other damages. The depletion of NAD can endanger cell energy reserves, so niacin or niacinamide, members of the vitamin B family and precursors of NAD, are necessary to replenish the NAD reservoir.
. Table 31.1 DNA repair activities. The source of DNA damage and the major form of the damage is shown. The type of DNA repair, the key enzymes in the repair and the relative time for repair is also shown. The means for prevention of the lesions are also shown, including the use of sunscreens, and the natural melanin and antioxidants in skin Source
DNA damage
DNA repair
Key enzymes
Time
Protection
Solar UV
CPD
NER, global and transcription coupled
XPA-XPG
Slow
Sunscreen/Melanin
Solar UV
NER
XPA-XPG
Slow
Sunscreen/Melanin
Solar UV
CPD
Photoreactivators (non-mammation)
Photolyase
Fast
Sunscreen/Melanin
Reactive oxygen species
Single strand breaks
BER
DNA ligase
Fast
Sunscreen/Anti-oxidant
Reactive oxygen species
8-oxo Guanine
BER
OGG1
Fast
Sunscreen/Anti-oxidant
Reactive oxygen species
Double strand breaks
ATM
Slow
Sunscreen/Anti-oxidant
CPD Cyclobutane Pyrimidine Dimers; NER Nucleotide Excision Repair; XPA-XPG Xeroderma Pigmetosum complementation groups A through G; BER Base Excision Repair; OGG1 8-oxo-Guanine Glycosylase; ATM Ataxia Telangiectasia Mutant
DNA Damage and Repair in Skin Aging
Major damage to DNA, such as CPDs or < 6–4 > PPs, interferes with its coding ability and must be repaired in order for the nucleotide sequence to function. Each of these is removed in a patch of about 30 DNA nucleotides by a process termed nucleotide excision repair (NER) [10]. A dozen or more proteins may cooperate to complete NER. One subset of these proteins recognizes CPDs throughout the genome because they distort the regular turns of the DNA helix, and initiate global genomic repair (GGR). However, an additional set of proteins is especially responsive to RNA transcription forks, which are stalled at sites of CPDs in the coding sequence, and are able to more quickly mobilize the NER machinery to these regions of DNA vital to cell function to initiate transcription coupled repair (TCR). Once these recognition proteins bind to the site of DNA damage, they recruit additional enzymes that unwind the DNA, make a single-stranded break on either side of the CPD, and release the 30-nucleotide piece of DNA. The single-strand gap is then filled in by DNA polymerases using the opposite strand of the DNA as a template. Each cell has several varieties of DNA polymerases and most of them copy DNA very accurately. However, a few types are much more error prone and when they are called into service, they introduce mutations by incorporating incorrect bases into the patch [11]. NER of CPDs is not a very efficient process. After UV exposure that produces a sunburn in human skin, it takes about 24h to remove 50% of the damage. NER repair of < 6–4 > PPs is much quicker: about 50% are removed in 30 min. This is due to the fact that < 6–4 > PPs are less frequent, and so they greatly distort DNA that they are easier for the NER proteins to locate and excise.
Base Excision Repair Damage to single bases such as 8oGua is much less distorting to DNA, and is repaired by a second pathway termed base excision repair (BER) [10]. Here a DNA repair enzyme termed oxo-guanine glycosylase-1 (OGG1) specifically recognizes 8oGua and releases it from the DNA backbone, leaving an abasic site. A second enzyme recognizes this baseless site and makes a single-stranded break. A few bases on either side of the break are removed and the short patch is again resynthesized using the opposite strand as a template. This is a speedy process, and half of the 8oGua introduced by solar UV are repaired in about 2h [12]. In human cells, CPDs are not repaired by BER because there is no glycosylase to recognize them. However, the
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bacteriophage enzyme T4 endonuclease V recognizes CPDs and clips one side of the dimer from the DNA, initiating BER. Amazingly, when delivered into human cells this enzyme functions quite well to initiate repair of CPDs by BER [13].
Photoreactivation An additional pathway of DNA repair is used by plants, fish, reptiles, and amphibians, but it is not present in humans or other mammals. This repair is accomplished by the enzyme photolyase that directly reverses CPD by capturing longwavelength UV and visible light, and using the energy to split the bonds that bind together the pyrimidine bases in a CPD [14]. This restores the DNA to normal without producing a single-strand break or removing any DNA. Once again, while human cells have no photolyase enzymes, when these enzymes are introduced into human cells they function quite well in repairing CPDs [15].
Diseases of DNA Repair Much has been learned by studying rare genetic diseases with defects in DNA repair and other diseases in which skin cancer rates are elevated. This has not only clarified the function of many of the DNA repair proteins, but has also revealed that many DNA repair proteins have multiple functions in the cell.
Xeroderma Pigmentosum, Trichothiodystrophy, Cockayne Syndrome Xeroderma pigmentosum (XP) is characterized by mildto-extreme photosensitivity, often with areas of hypopigmentation and hyperpigmentation, an increased risk of skin cancer and a shortened life expectancy [16]. There are seven complementation groups of XP (A–G), corresponding to defects in one of seven genes that code for proteins involved in NER, and a variant group with a defect in repair synthesis. Stringent photoprotection from an early age can greatly reduce actinic damage, but does not prevent neurological defects that are a hallmark of some of the complementation groups. This may be because some of these genes are also involved in non-DNA repair gene transcription. Trichothiodystrophy (TTD) patients have a defect in the same gene as XP-D patients, but at different locations within the gene, so they manifest photosensitivity, stunted growth, and brittle hair, but not an increase in
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skin cancer [16]. This highlights that subtle differences in a DNA repair protein can produce drastic differences in human development and morphology. Patients with Cockayne syndrome (CS) have mutations in one of two genes that code for proteins controlling TCR, and they also have growth and developmental abnormalities, but surprisingly little increased risk of skin cancer [16].
Solid Organ Transplant Patients Organ transplant patients have an elevated rate of skin cancer on sun-exposed skin during the period in which they are on immunosuppressive therapy [17]. There is no doubt that suppression of the immune system plays a significant role in allowing nascent skin cancers to grow out. However, there is increasing evidence that these drugs also impair DNA repair in the skin [18]. The two most widely used drugs, CsA and tacrolimus, target the phosphatase calcineurin. Calcineurin dephosphorylation of the nuclear transcription factor NFAT allows NFAT to localize in the nucleus, where it is a key activator of transcription of several immunoregulatory genes. Immobilization of calcineurin sequesters NFAT in the cytoplasm and shuts down transcription of these genes. Other transcription factors, such as TFHII, are vital to the preferential repair of DNA by targeting the repair machinery to sites of stalled transcription complexes. NFAT may also participate in recovery from transcription blocks.
DNA Repair Gene Polymorphisms The genes implicated in these genetic diseases code for proteins that participate not only in DNA repair, but also in other routine developmental programs and cell functions. The general population carries many forms of these genes with other, less serious, mutations, and these forms are called genetic polymorphisms. While some of these polymorphisms are innocuous, groundbreaking research shows that some gene forms increase the risk of cancer, including skin cancer [19]. One such DNA repair gene polymorphism is in the OGG1 gene coding for the glycosylase that releases 8oGua from DNA. The OGG1 polymorphism S326C has been associated with an increased risk of several types of cancer [20]. However, three separate in vitro biochemical studies of the activity of the protein produced by the variant gene failed to identify any deficit in activity or reduced DNA repair of oxidatively damaged DNA [21–23]. The S326C
variant polymorphism in the OGG1 gene is linked to increased risk of cancers such as prostate cancer, but the protein produced by the variant gene does not have any obvious biochemical defects. The variant polymorphic genotype however, is the most sensitive to cell killing by cytotoxic agents, and the heterozygous genotype was most resistant [24]. Delivery of exogenous OGG1 enzyme to cells increased repair of 8-oxo-guanine in the homozygous variants [25]. Thus, subtle changes in DNA repair genes may alter their activity in cells and increase susceptibility to endogenous and exogenous damage.
Prevention of DNA Damage Melanin The first line of defense against DNA damage is the pigment deposited by melanocytes at the surface of the skin. Melanocytes are pigment-producing cells that are found in the basal layer of the epidermis and disperse melanosomes, containing melanin, among the surrounding keratinocytes. These melanosomes encapsulate two main classes of pigments found in human skin: eumelanin, which is brown or black, and pheomelanin, which is reddishbrown. The relative amounts of these two pigments, and the size and density of the melanosomes largely determine the differences in skin color among humans. The constitutive pigment that is associated with racial groups, is deposited by melanocytes above the nuclei of keratinocytes, thereby shielding them from UV. Skin color has an enormous effect on the risk of skin cancer because this constitutive melanin absorbs and reflects a broad spectrum of UVR. Thus, UV exposure to dark skin produces less DNA damage than in light skin. The induced pigmentation in tanned skin, however, is significantly dispersed as pigment granules, rather than capping nuclei. The result is that tanned skin is much less protective against DNA damage than the equivalent in constitutive color.
Sunscreens Sunscreens are an additional front-line defense against DNA damage by reflecting or absorbing UV at the skin surface. The absorbed energy is released from the sunscreen molecules mostly as fluorescence or heat. Sunscreens are either inorganic, physical sunscreens that largely reflect light, or chemical sunscreens that mostly absorb light. Some sunscreens are less photostable than others
DNA Damage and Repair in Skin Aging
and lose their absorption capacity during UV exposure. Some of the energy absorbed by sunscreen molecules can cause the release of ROS, and this is true for both physical and chemical sunscreens. Recent advances in sunscreen development have been designed to reduce or eliminate these possibilities. To date there is no evidence that ROS released by sunscreens in skin causes significant levels of DNA damage. Of far greater concern is that sunscreens are usually not used properly or in the right amounts, and despite their application, significant DNA damage still results [26]. The most frequently used physical UV filters are the inorganic micropigments, zinc oxide, or titanium dioxide in the range of 10–100 nm in diameter. These micropigments are capable of reflecting a broad spectrum of UV rays in the UVA and UVB regions. Major disadvantages of micropigments are that they also reflect visible light, creating the so called ‘‘ghost’’ effect on skin, and they are difficult to formulate, often resulting in disagreeable preparations in which the micropigments have a strong tendency to agglomerate, which greatly decrease their efficacy. Chemical UV filters have the capacity to absorb shortwavelength UV photons and to transform them into heat by emitting long-wavelength photons (infrared radiation), which are much less likely to damage DNA. Most chemical filters absorb in a relatively small wavelength range. In general, chemical filters may be divided into molecules, which absorb primarily in the UVB region (290–310 nm) and those, which primarily absorb in the UVA region (320–400 nm). Only a few chemical filters absorb both UVB and UVA photons. Although these are relatively easier to formulate into cosmetically elegant textures, combinations of chemical filters are required in order to meet regulatory standards for sunprotection.
Antioxidants A third protection against the formation of DNA damage is antioxidants. They absorb ROS and thereby prevent oxidative DNA damage, primarily 8oGua. Because CPD are not formed by an ROS intermediate, antioxidants cannot prevent them. The natural skin antioxidant system is composed of lipophilic antioxidants such as vitamin E and CoQ10 and hydrophilic antioxidants such as vitamin C, glutathione, and the enzymes catalase and superoxide dismutase. An exciting new finding is that the powerful antioxidant ergothioneine and its receptor (OCNT-1) are found in the suprabasal layer of the epidermis, as
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well as in the dermis. This implicates ergothioneine as a new natural component of the human skin antioxidant system. Antioxidants cooperate to regenerate each other after reacting with ROS. For example, oxidation of vitamin C leads to its fast degradation, but vitamin E can generate oxidized vitamin C. In the same way, vitamin C can regenerate ergothioneine. Complete antioxidant protection requires many types of antioxidants, since ROS can be in the form of singlet oxygen, superoxides, or peroxides, as well as others. They can also be sequestered in the water or lipid compartment of cells. Therefore, the examination of the antioxidant protection system of skin requires consideration of all the antioxidants as a network.
Cellular Effects of DNA Damage A complex system regulates the cell’s progression through division to insure that only undamaged ones replicate, in order to avoid genetic instability and cancer [27, 28]. As cells approach commitment to DNA synthesis (S phase), proteins encoded by checkpoint genes delay entry if DNA damage is present. DNA proteins kinases, such as ATM (Ataxia-Telangiectasia Mutated) and ATR (AtaxiaTelangiectasia Mutated and Rad3 Related), then initiate signaling cascades resulting in DNA damage responses that include activation of the p53 protein. This tumor suppressor plays a central role in whether a cell repairs the damage [29] or is diverted into programmed cell death (apoptosis), cell cycle arrest, or senescence [27]. Mitochondrial DNA is damaged largely as a result of oxidative damage secondary to the production of excess ROS by UV or normal metabolism. Sufficient levels of this damage cause release of mitochondrial factors, such as cytochrome C, which binds to the apoptotic proteaseactivating factor 1 (Apaf-1), resulting in the formation of the apoptosome. This critical event leads to the activation of caspase-9 and the initiation of the mitochondrial apoptotic pathway through caspase-3 activation [30]. Apoptosis is a critical event preventing damaged cells from progressing to malignancy. One new photoprotection strategy is to selectively target DNA-damaged cells for apoptosis while leaving normal cells unaffected. Oral administration of caffeine or green tea (which often contains high levels of caffeine) in amounts equivalent to three to five cups of coffee per day to UVB-exposed mice increased levels of p53, slowed cell cycling, and increase apoptotic sunburn cells in the epidermis [31].
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Signal Transduction The dramatic events that follow DNA damage indicate that DNA is an important sensor of environmental insult and is able to trigger a variety of cell responses. The molecular mechanisms for this sensor-effector mechanism are being unraveled. The UV-induced cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidone photoproducts cause distortions in the DNA helix and halt RNA polymerase II (RNA-PII) transcription of DNA. Protein kinases that activate their downstream targets through phosphorylation play an important role in signal transduction. A group of protein kinases that interact with DNA (ATR, Chk2, DNA-PK,) is implicated in the molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress [32]. ATR (ATM-Rad3related kinase) is a primary DNA sensor and is essential for UV-induced phosphorylation of several G1/S checkpoint proteins. ATR was also shown to bind UVB-damaged DNA, with a resulting increase in its kinase activity, with many proteins as its target [33]. One such target is the RNA-PII itself, where phosphorylation represses further transcription initiation. This stalled RNA polymerase II leads to recruitment of the nucleotide excision repair complex. Another target of ATR is p53. Following phosphorylation, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Next, a conformational change forces p53 to take on an active role as a transcriptional regulator in these stressed cells. p53 is able to transactivate a plethora of genes with an active role in cell cycle arrest, global genomic DNA repair, apoptosis, and cytokine release.
Systemic Effects of DNA Damage Cytokines DNA in the skin acts like a sensor for UV damage on behalf of both exposed and unexposed cells in distal parts. DNA damage triggers the production and release of cytokines that act on the cell itself, as well as other cells with such cytokine receptors, to activate characteristic UV responses, such as wound healing and immune suppression [34]. Keratinocytes are the main source of these cytokines. Other epidermal cells, like Langerhans cells (LHC), and melanocytes, together with infiltrating leukocytes are also active contributors to changed cytokine profile after UV exposure. Keratinocytes are able to secrete a
wide variety of proinflammatory cytokines upon UV exposure, including interleukins IL-1a, IL-1b, IL-3, IL-6, IL-8, granulocytes colony-stimulating factor (G-CSF), macrophage-CSF (GM-CSF), interferon gamma (INF-g), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGF-a), TGF-b, and tumor necrosis factor a (TNF-a) [35–37]. Cytokines such as IL-1 and TNF-a then induce a cascade of other cytokines that can activate collagendegrading enzymes, suppress the immune system, dilate blood vessels, and attract inflammatory T cells [38]. In this way, cells with DNA photodamage, even if they are destined to die, have profound effects on cells in the skin and elsewhere that may not have been exposed to UV. IL-12 plays a curious role in photoprotection. It is an immunostimulatory cytokine that is released by keratinocytes at late times after UV in order to counteract the suppressive effects of IL-10 [39]. Recently, it has also been reported to stimulate the repair of CPDs in the DNA of keratinocytes in a manner yet to be understood [40].
Immune Suppression UV-induced immune suppression is an essential event for skin cancer formation [41]. It is important to note that this is not a generalized immune suppression, but a reduced ability to respond to antigens presented just after exposure. There may be a genetic susceptibility to UV-induced suppression, because skin cancer patients are more easily UV suppressed than cancer-free controls [42]. At lower UV doses, the primary target is the Langerhans cells, which flee the epidermis and those with DNA damage have impaired antigen-presenting ability [43]. Higher doses produce systemic immune suppression, mediated by the generation of suppressor T cells, in which nonexposed skin becomes hampered in responding to antigens [41]. In several experimental models, including humans, reducing DNA damage decreases the degree of immune suppression [44].
Wound Healing and Photoaging UV-induced DNA damage also triggers a wound healing response in the skin, as it tries to eliminate damaged cells and stimulate cell division to replace them. UVR directly to fibroblasts, as well as signals from damaged keratinocytes, cause the release of metalloproteinase (MMP-1), which selectively degrade large collagen cables [45]. Soluble factors released by keratinocytes, including IL-1, IL-6, and TNF-a, are principle actors in this paracrine effect [46].
DNA Damage and Repair in Skin Aging
DNA damage is directly related to the release of soluble mediators since enhanced repair of keratinocyte DNA reduced the release of the mediators and lowered the release of MMP-1 by unirradiated fibroblasts [47]. As part of this response, MMP-2 and MMP-9, which are responsible for digesting small collagen fragments, are downregulated by UVR. This results in the accumulation of collagen fragments, which severs the anchorage of fibroblasts, inhibits their ability to produce new collagen, and degrades the dermal elastic fiber network [48]. This is followed by hyperproliferation among keratinocytes, and together these responses are designed to fill in sites of skin wounds. Repeated rounds of this type of imperfect wound healing produces many of the microscopic hallmarks of photoaged skin, including a corresponding decrease in the biophysical properties of the skin [49], reflected in a loss of both skin strength and elasticity, flattening of the rete ridges, and the appearance of wrinkles and skin folds. Additionally, there are degradative vascular changes in the dermis resulting in telangiectasia and decreases in the capillary network and skin blood flow [50]. These small changes accumulate after repeated rounds of DNA damage to form what is readily recognized as aged skin. These connections, from DNA damage to stalled transcription complexes, resulting in kinase cascades activating metalloproteinases, which degrade skin collagen, explain why photoaging is a product of unrepaired DNA lesions.
Mutations and Skin Cancer Mutations Most of the solar UV-induced DNA damage distorts the double helix. In attempting to replicate past CPD lesions the cell often makes the same mistake of misincorporating two consecutive bases, resulting in mutations characteristic of UV damage [51]. In many cases these mutations have no effect on the cell, but if they occur at critical locations in tumor suppressor genes, they abrogate apoptosis and initiate the process of carcinogenesis. These UV ‘‘signature’’ mutations are often found in mutated p53 genes, a key tumor suppressor gene, in human squamous cell and basal cell carcinomas [51]. This is the key link between UV exposure and skin cancer, and directly implicates CPDs in carcinogenesis. These p53 signature mutations are also frequently found in precancerous actinic keratosis, suggesting that these mutations are an early step in the process of forming squamous cell carcinomas,
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and that later steps, such as additional gene mutations and immune suppression, determine if a cell goes on to malignancy. The situation is less clear in melanoma. There appear to be many different tumor suppressor genes that can be mutated in melanoma, and the frequency of signature mutations is not as common as in squamous cell carcinoma [52]. Mitochondria generate energy for the cell, and they contain DNA that encodes many of the crucial proteins in the energy production machinery. This DNA is also subjected to mutations, and mitochondria develop a peculiar type of mutation called the common deletion, in which a particular 477 base pair section of the DNA is deleted. The frequency of the common deletion in the mitochondria of human skin cells does not correlate with chronological age, but rather with sun exposure and photoaging [53]. This implies that solar UV is responsible for the formation of the common deletion, and its contribution to the signs of photoaging is an active area of research.
Prevention of Skin Cancer with DNA Repair Enzymes The inevitable consequence of the accumulation of DNA damage over a lifetime is an increased incidence of mutations and an elevated risk of skin cancer. The primary strategy for reducing this risk is the attenuation of the UV dose striking the skin, by sun avoidance, pigmentation, and sunscreens. Antioxidants have become a part of the defense by scavenging ROS before they can oxidize DNA. The next step in intervention is the enhanced repair of DNA damage before it can be fixed as a mutation and increase the probability of malignant transformation. Over the past 40 years the field of DNA repair has identified many enzymes that recognize and initiate removal of DNA damage, either by nucleotide excision repair, base excision repair, or direct reversal. The use of some of these enzyme activities for photoprotection became practical with the development of liposomes specifically engineered for delivery into skin [54]. The small protein T4 endonuclease V from bacteriophage recognizes the major form of DNA damage produced by UVB, which is the cyclobutane pyrimidine dimer (CPD). Liposomal delivery of T4 endonuclease V to UV-exposed human skin increased repair from 10% of CPD to 18% over 6 h, but dramatically reduced or eliminated the release of cytokines such as IL-10 and TNF-a [55]. In a randomized clinical study of the effects of daily use of this liposomal
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T4 endonuclease V in XP patients, the rate of premalignant actinic keratosis and basal cell carcinoma was reduced by 68% and 30%, respectively, compared to the placebo control [56].
Conclusion During a lifetime, skin is exposed to chemical challenges generated by its own metabolism as well as the environment, particularly solar UV. There are a variety of defenses in the human body, such as skin color, sunscreens, and antioxidants, to counteract these. Inevitably, however, cells sustain damage. DNA serves the cell not only as the master controller of cell function, and the storehouse of heredity information, but also as a sensor of damage, and consequently a sentinel for danger to the cell and the organism. It is able to convert the distortion caused by altered nucleic acid bases into signals that arrest and redirect its own cell machinery. It also converts that distortion into notification of adjoining cells, whether damaged or not, that significant lesions have occurred. The purpose of these signals is to evoke repair and healing responses. DNA is a unique macromolecule in carrying with it the toolkit for its own repair. DNA repair is focused at the site of actively transcribed DNA by a complex of enzymes, some of which are specifically adapted to recognize modified DNA, and some borrowed from the transcription machinery itself. The repair may have the task of repairing hundreds of thousands of lesions daily, and while it is an efficient process it is not perfect. The resulting mutations in the DNA sequence are the necessary components for the development of skin cancer. Skin aging, therefore, can be viewed as the accumulation of imperfections from repeated rounds of DNA damage and repair, as well as rounds of wounding and healing. Skin cancer is just one manifestation of these cycles. Viewed in this way, it is likely that properly conceived efforts to alleviate skin aging will also have the benefit of reducing rates of skin cancer. Since over long periods of time, people are more motivated by improving their physical appearance than lowering their perceived risk of disease, the most successful anticancer efforts will arrive as treatments for skin aging.
Cross-references > Fibulin-5
Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
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DNA Damage and Repair in Skin Aging 23. Janssen K, et al. DNA repair activity of 8-oxoguanine DNA glycosylase I (OGG1) in human lymphocytes is not dependent on genetic polymorphism Ser326/Cys326. Mutat Res. 2001;486:207–216. 24. Yarosh D, et al. DNA repair gene polymorphisms affect cytotoxicity in the National Cancer Institute Human Tumor Cell Line Screening Panel. Biomarkers. 2005;10:188–202. 25. Yarosh D, et al. After sun reversal of DNA damage: enhanced skin repair. Mutat Res. 2005;571:57–64. 26. Mahroos R, et al. Effect of sunscreen application on UV-induced thymine imers. Arch Dermatol. 2002;138:1480–1485. 27. Funk JO. Cell cycle checkpoint genes and cancer. Encyclopedia Life Sci. 2005;1–5. 28. Harper JW, et al. The DNA damage response: ten years after. Mol Cell. 2007;28:739–745. 29. Sancar A, et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 2004;73: 39–85. 30. Guzman E, et al. Mad dogs, Englishmen and apoptosis: the role of cell death in UV-induced skin cancer. Apoptosis. 2003;8:315–325. 31. Lu Y-P, et al. Effect of caffeine on the ATR/Chk1 pathway in the epidermis of UVB-irradiated mice. Cancer Res. 2008;68:2523–2529. 32. Zhou BB, et al. The DNA damage response: putting checkpoints in perspective. Nature. 2000;408:433–439. 33. Unsal-Kacmaz K, et al. Preferential binding of ATR protein to UVdamaged DNA. Proc Natl Acad Sci USA. 2002;99:6673–6678. 34. Kondo S. The roles of keratinocyte-derived cytokines in the epidermis and their possible responses to UVA-irradiation. J Invest Dermatol Symp Proc. 1999;4:177–183. 35. Ansel J, et al. Cytokine modulation of keratinocyte cytokines. J Invest Dermatol. 1990;94:101S–107S. 36. Luger TA, et al. Evidence for an epidermal cytokine network. J Invest Dermatol. 1990;95:100S–104S. 37. Enk AH, et al. Early molecular events in the induction phase of contact sensitivity. Proc Natl Acad Sci USA. 1992;89:1398–1402. 38. Heck DE, et al. Solar ultraviolet radiation as a trigger of cell signal transduction. Toxycol Appl Pharmacol. 2004;195:288–297. 39. Barr R, et al. Suppressed alloantigen presentation, increased TNF-a, IL-1, IL-1RA, IL-10, and modulation of TNF-R in UV-irradiated human skin. J Invest Dermatol. 1999;112:692–698. 40. Schwarz A, et al. Interleukin-12 suppresses ultraviolet radiationinduced apoptosis by inducing DNA repair. Nature Cell Biol. 2002;4:26–31.
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41. Kripke M. Immunologic unresponsiveness induced by UV radiation. Immunol Rev. 1984;80:87–102. 42. Streilein J. Immunogenetic factors in skin cancer. N Engl J Med. 1991;325:884–887. 43. Vink A, et al. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers. Proc Natl Acad Sci USA. 1997;94:5255–5260. 44. Kuchel J, et al. Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans. Photochem Photobiol Sci. 2005;4:577–582. 45. Brennan M, et al. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol. 2003;78:43–48. 46. Wlaschek M, et al. UVA-induced autocrine stimulation of fibroblastderived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol. 1994;59:550–556. 47. Dong K, et al. UV-Induced DNA damage initiates release of MMP-1 in human skin. Exp Dermatol. 2008;17:1037–1044. 48. Fisher GJ, et al. Looking older. Fibroblast collapse and therapeutic implications. Arch Dermatol. 2008;666–672. 49. Leveque J-C, et al. Aging Skin: Properties and Functional Changes. Aulnoy-sous Bois: Informa Health Care, 1993. 50. Ryan T. The ageing of the blood supply and the lymphatic drainage of the skin. Micron. 2004;35:161–171. 51. Brash D. Sunlight and the onset of skin cancer. Trends Genet. 1997;13:410–414. 52. High W, et al. Genetic mutations involved in melanoma: a summary of our current understanding. Adv Dermatol. 2007;23:61–79. 53. Berneburg M, et al. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. 54. Yarosh D, et al. Localization of liposomes containing a DNA repair enzyme in murine skin. J Invest Dermatol. 1994;103:461–468. 55. Wolf P, et al. Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-a, J. Invest Dermatol. 2000;114:149–156. 56. Yarosh D, et al. Effect of topically applied T4 endonuclease V in liposomes on skin cancer in xeroderma pigmentosum: a randomized study. Lancet. 2001;357:926–929
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Endogenous and Exogenous Factors in Skin Aging
41 Effect of Ozone on Cutaneous Tissues Giuseppe Valacchi
Introduction Living organisms are continuously exposed to environmental pollutants. Depending on their state, pollutants can be taken up by ingestion, inhalation, or contact with the skin. Because the skin is an interface between the body and the environment, it is chronically exposed to several forms of stress such as ultraviolet (UV) radiation and other environmental oxidants such as cigarette smoke and ozone (O3). UVB and, to a lesser degree, UVA induce various skin pathological conditions, including erythema, edema, hyperplasia, ‘‘sunburn cell’’ formation, photoaging, and photocarcinogenesis. There is abundant information that reactive oxygen species (ROS) such as hydroxyl radicals are involved in UV-induced skin damage, both by direct effects of UV and by subsequent phagocyte infiltration and activation. Oxidative environmental pollutants, such as cigarette smoke, O3, and oxides of nitrogen that have been studied in the respiratory tract [1], also represent a potential oxidant stress to the skin. In order of importance, the skin is the second most frequent route by which chemicals can enter into the body. The skin is the major target of liquid and gaseous pollutants and the pollutant that reacts most specifically with the cutaneous tissues beside UV radiation, hydrocarbon, and organic compounds in O3. Ozone represents one of the major oxidants in photochemical smog, levels being highest in heavily polluted areas where exposure to UV is also high. In the last decade, many studies have shown the toxic effect of O3 on the skin [2, 3].
a stable structure but exists in several mesomeric states in dynamic equilibrium.
In the liquid and solid states, O3 is highly explosive, and among oxidant agents, it is the third strongest (O3, E = +2.076 V) after fluorine (+3.0353 V) and persulfate (+2.866 V).
Ozone: The Good and the Bad O3 is naturally present in the atmosphere surrounding the Earth. In the upper part of the atmosphere, the stratosphere, 20–30 km from the earth’ surface, the O3 layer can reach the concentration of 10 ppm. The O3 occurring in the stratosphere, where the majority of atmospheric ozone is found, forms a ‘‘filtering layer’’ that acts as a barrier to the dangerous radiation from the sun.
Physicochemical Properties of Ozone The word ozone derives from the Greek word deo, which means ‘‘to give off a smell.’’ It is an unstable gas of a soft sky-blue color, with a pungent, acrid smell already perceptible at a concentration of 0.01 ppb. The molecule is composed of three oxygen atoms (O3) and has a molecular weight of 48 kDa. It has a cyclical structure assessed by the spectrum absorption in the infrared region, with a distance of 1.26 A˚ among oxygen atoms. O3 does not have
In contrast, O3 present within the lower troposphere (10 miles from the ground level) is hazardous and dangerous to the terrestrial health. It is an ubiquitous
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_41, # Springer-Verlag Berlin Heidelberg 2010
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pollutant of the urban environment, and it is not emitted directly by any man-made source in significant quantities. Ozone arises from chemical reactions in the atmosphere through the action of sunlight on oxygen molecules. The photochemistry involved in the generation of O3 usually involves several reactions such as photo activation, photodecomposition, and free radical chain reaction [4]. The most common molecules that lead to O3 formation at the ground level are nitric oxides (NOx). NO2 can be photolyzed by solar UV resulting in NO and the atomic oxygen that can react with molecular oxygen leading to the formation of O3. Ozone can also be destroyed by nitric oxide. NO can react with O3 to form NO2 and O2. Under these steady state conditions, the concentration of O3 cannot increase until most of NO has been converted to NO2 by additional reactions occurring within the complex. This accumulation occurs as the rate of NO2 photolysis is much faster than that of O3. Other species in photochemical smog also undergo photodecomposition to yield free radicals that may participate either directly or indirectly in the conversion of NO to NO2. Hydroxyl and hydroperoxyl radicals are the examples of compounds that can react with nitrogen radicals with the destruction of O3 by NO.
The average tropospheric amount of ozone ought to be far less than 40 ppb, which is much lower than that present in the stratosphere. Yet in large metropolises such as Mexico City, and also in European cities such as Rome, Milan, and Paris, O3 can reach toxic concentrations (0.8 ppm) especially during the summer. Anthropogenic emissions, mainly of NOx and also methane (CH4), carbon monoxide (CO), and sulfuric compounds, have caused a progressive increase of O3 concentration up to 1,000 ppb or more [5]. At the street level, O3 has become the main toxicant not only for the respiratory tract, but also for the cutaneous tissues.
Ozone in Life Reports of hazardous effects induced by smog reach as far back as the thirteenth century when, during the reign of Richard III (1377–1399), human diseases were attributed
to severe air pollution. Trends in tropospheric O3 are poorly documented. The O3 level in the northern hemisphere increased significantly during the periods of industrialization. In the late nineteenth century, O3 was measured near Paris (Montsouris) to follow a seasonal cycle and to be in the range of 10–20 ppb. Such values are considerably lower than present-day with background concentration values of 40–50 ppb observed over the continents in the Northern hemisphere. The presence of O3 in air was well recognized in Los Angeles during the early 1940s, based on its damaging effects on rubber products (http://www.who.int/en/). In the 1950s, London and other major cities in UK suffered a series of smog episodes that caused the death of 4,000 people in a week [6]. During pollution episodes, O3 mixing ratios higher than 80 ppb can be observed locally in the industrialized regions of North America, Europe, and Asia. It is likely that, on an average, the O3 abundance has increased by a factor of 2 or more since the preindustrial era (http:// www.who.int/en/), and it is, therefore, conceivable that oxidizing power of the atmosphere has changed during the same period. It has been estimated by Zeng and Pyle that the level of tropospheric O3 will be increased fivefold at the end of this century because of the increase of cars and industrial fumes, leading to dangerous consequences to the terrestrial life [7]. The average environmental O3 levels, that vary considerably for many reasons need to be known, in order to understand the effects of a daily 8-h O3 exposure (April–October). The US Clean Air Act has set an O3 level of 0.06 ppm (120 mg/m3) as an 8-h mean concentration to protect the health of workers (US Environmental Protection Agency, 2005). Evaluation of recent studies [8, 9] allows establishing an average environmental O3 concentration of 90 10 ppb. However, O3 concentration in urban air can exceed 800 ppb in high-pollution conditions [10], reducing not only pulmonary functions and enhancing the risk of cardiovascular death, but also affecting skin physiology.
Ozone as an Oxidant It is generally understood that, although O3 is not a radical species per se, the toxic effects of O3 are mediated through free radical reactions and they are achieved either directly by the oxidation of biomolecules to give classical radical species (hydroxyl radical) or by driving the radicaldependent production of cytotoxic, nonradical species (aldehydes) [11].
Effect of Ozone on Cutaneous Tissues
Furthermore, the formation of the oxidation products characteristic of damage from free radicals has been shown to be prevented by the addition of the antioxidant vitamins E and C, though the mechanism is not fully understood. The target specificity of O3 toward specific compounds together with its physicochemical properties of fairly low aqueous solubility and diffusibility must be taken into account when a target tissue like the skin is exposed to O3. As it was hypothesized [12], O3 does not penetrate the cells, but oxidizes available antioxidants and reacts instantaneously with surfactant’s polyunsaturated fatty acids (PUFA) present at the air–cellular interface to form reactive oxygen species (ROS), such as hydrogen peroxide and a mixture of heterogenous LOPs including lipoperoxyl radicals, hydroperoxides, malonyldialdeyde, isoprostanes, the ozonide radical, O3 [13], and alkenals, particularly 4-hydroxy-2,3-trans-nonenal (HNE) [14]. As cholesterol is a component of the upper layer of the skin and because its double bond is readily attacked by O3, it can give rise to biologically active oxysterols [15]. A major ozonation product of cholesterol, 3b-hydroxy-5-oxo-5,6-secocholestan6-al, induces apoptosis in H9C2 cardiomyoblasts of which 3b-hydroxy-5-oxo-5,6-secocholestan-6-al has been implicated in pulmonary toxicity, Alzheimer’s disease, and atherosclerosis. Cell membranes and their lipids are relevant potential targets of environmental stressors such as O3. Using a spin trapping technique, the formation of radicals in the SC upon exposure to O3 was detected. The spin adduct could arise from an alkoxyl radical formed during lipid peroxidation. Furthermore, lipid radicals (L●) are generated in epidermal homogenates that have been exposed to environmental stressors. The organic free radical L● reacts with O2, forming peroxyl radical LOO● and hydrolipoperoxides (LOOH). Transition metals, and in particular iron, play a key role in the reactions of LOOH and in the subsequent generation of alkoxyl radicals (RO● can amplify the lipid peroxidation process). Moreover, the toxicity is certainly augmented by the presence of NO2, CO, SO2, and particles (PM10). On this basis, it appears clear how the O3-generated ROS and LOPs at the tissue level, after being only partly quenched by the antioxidants, will act as cell signals able to activate transcription factors (nuclear factor-kappa B (NF-kB), NO synthase and some protein kinases, thus enhancing the synthesis and release of proinflammatory cytokines (TNFa, IL-1, IL-8, IFNg, and TGFb) and the possible formation of nitrating species. With a possible increasing inflow into the cutaneous tissues of neutrophils and activated macrophages, a vicious circle will start,
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perpetuating the production of an excess of ROS including also hypoclorous acid [16], LOPs, isoprostanes, tachykinins, cytokines, and proteases, which will self-maintain the inflammation after O3 exposure.
The Stratum Corneum as the First Target of Environmental Stressors Within the skin, the SC has been identified as the main target of oxidative damage. As the outer skin barrier, the SC has important functions, limiting transepidermal water loss and posing a mechanical barrier to penetration by exogenous chemicals and pathogens. It comprises a unique two-compartment system of structural, nonnucleated cells (corneocytes) embedded in a lipid-enriched intercellular matrix, forming stacks of bilayers that are rich in ceramides, cholesterol, and free fatty acids. The effects of O3 on cutaneous tissues have been evaluated using a murine model and in a few studies using even human subjects [17, 18]. While no effect of O3 on endogenous antioxidants was observed in fullthickness skin (dermis, epidermis, and SC), it could be demonstrated that a single high dose of O3 (10 ppm for 2 h) significantly depleted topically applied vitamin E. When the skin was separated into upper epidermis, lower epidermis, papillary dermis, and dermis, O3 induced a significant depletion of tocopherols and ascorbate followed by an increase in the lipid peroxidation measured as malondialdehyde (MDA) content. O3 is known to react readily with biomolecules and does not penetrate through the cells; therefore, it was hypothesized that O3 mainly reacts within the SC [19]. This hypothesis was supported by further experiments, where hairless mice were exposed to varying levels of O3 for 2 h. Depletion of SC lipophilic (tocopherols) as well as hydrophilic (ascorbate, urate, GSH) antioxidants was detected upon O3 exposure and it was accompanied by a rise in lipid peroxidation as an indicator of increased oxidative stress. Furthermore, a recent study has shown the increase of 4-hydroxylnonenal (4-HNE) content in murine SC using both Western blot and immunohistochemical analysis. Finally, the increase of protein oxidation was also shown in in vivo studies [19, 20]. It is well known that oxygen radicals and other activated oxygen species generated as by-products of cellular metabolism or from environmental sources like O3 cause modification of the amino acids of proteins and therefore modifying their functions. Besides the modification of amino acid side chains, oxidation reactions can also alter the protein cross-linking with peptides. Protein
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carbonyls may be formed by oxidative cleavage of proteins or by direct oxidation of lysine, arginine, proline, and threonine residues. In addition, carbonyls groups may be introduced into proteins by reaction with aldehydes (4-HNE) produced during lipid peroxidation generated as a consequence of O3 reactivity with the cutaneous tissues. This explains the use of carbonyls formation as a marker of oxidative stress. Protein oxidation has been also associated with skin aging and other skin pathologies. Thiele et al. were able to detect protein carbonyls formation in the upper layer of the stratum corneum (SC) and in whole skin homogenates exposed to environmental insults. The main protein oxidized was keratin 10 and it showed an increasing level of oxidation from the lower SC to the upper level. This protein oxidation gradient was inversely correlated with the gradient of the antioxidant vitamin E. There are studies that have shown that protein oxidation can be quenched by antioxidants such as tocopherol and thiols. Of note is the fact that the keratin in SC contained dramatically more carbonyl groups than the keratin present in keratinocytes, indicating that the baseline levels of keratin oxidation are considerably higher in the SC as compared to the epidermal layers. Furthermore, the oxygen partial pressure, a ratelimiting factor for the formation of reactive oxygen intermediates in the skin, decreases gradually from the outer to the inner SC layers. All these may have important implications in the desquamation process of the SC because of the role that the proteins in corneodesmosomes play in cell cohesion. It should also be taken into account that while protein degradation increases proteolytic susceptibility up to a protein-specific degree, further damage actually causes a decrease in proteolytic susceptibility and leads to cross-linking and aggregation [19, 20].
Skin Antioxidant Defenses To protect itself against oxidative stress, the skin is equipped with an elaborate system of antioxidant substances and enzymes that includes a network of redox active antioxidants. Antioxidant enzymes such as glutathione reductases and peroxidases, superoxide dismutases, and catalase interact with the low-molecular-weight antioxidant substances such as vitamin E isoforms, vitamin C, glutathione (GSH), and ubiquinol [21]. The presence of tocopherol, ascorbate, urate, and glutathione has been demonstrated in the SC [21]. Interestingly, the distribution of antioxidants in the SC is not uniform, but follows a gradient with higher
concentrations in deeper layers and decreasing concentrations toward the skin surface. This may be explained by the fact that SC layers move up in time as a part of the physiological turnover, and are replaced by newly differentiated keratinocytes. Therefore, the superficial layer has been exposed longer to chronic oxidative stress than a deeper layer. Compared with the SC, the surface lipids contain high levels of tocopherol due to the secretion of vitamin E by sebaceous glands [22]. Eventually, the uppermost layer of the SC desquamates and the remaining antioxidants and reaction products will be eliminated from the body. In general, the outermost part of the skin, the epidermis, contains higher concentrations of antioxidants than the dermis. In the lipophilic phase, tocopherol is the most prominent antioxidant, while vitamin C and GSH have the highest abundance in the cytosol.
Ozone-Induced Cellular Responses in Cutaneous Tissues Inflammatory Responses Ozone, like many other environmental challenges, is able to activate redox-sensitive transcriptional factors such as nuclear factor kappa B (NF-kB). This transcriptional factor acts as an activator for a multitude of proinflammatory genes (IL-8, TNFa, and TGFb) and adhesion molecules (ICAM and VCAM). It has been assessed that O3 is able to activate NF-kB using both in vitro and in vivo systems. Thiele et al., using a immortalized human keratinocytes (HaCaT cells) was able to show that O3 induced the activation of NF-kB by electrophoretic mobility shift assay (EMSA). O3 induced a dosedependent activation of the transcription factor. This effect was likely to be mediated by ROS because it was inhibited by the incubation of the cells with lipid soluble antioxidants (tocopherol). Finally, using a murine model, an increase of proinflammatory marker cyclooxygenase-2 (COX-2) was detected confirming the role that O3 can play in skin inflammation [17].
Induction of Heat Shock Protein As mentioned above, O3 exposure was shown to induce antioxidant depletion as well as lipid and protein
Effect of Ozone on Cutaneous Tissues
oxidation in the SC. Recent studies have investigated the effects of O3 in the deeper functional layers of the skin. To evaluate the effect on cutaneous tissues of O3 exposure, hairless mice were exposed for 6 days to 0.8 ppm of O3 for 6 h/day and the skin responses were analyzed using the whole skin homogenates. Under these experimental conditions an increase in the protein level of heat shock protein (HSP)32, also known as hemoxygenase-1 (HO-1), confirming that HSPs are sensitive markers of O3-induced stress in cutaneous tissues. The author’s group was the first to document the upregulation of HSPs 27, 32, and 70 in homogenized murine skin upon O3 exposure. HSP27 showed the earliest (2 h) and highest (20-fold) response to O3 compared with the delayed induction (12 h) of HSP70 and HO-1. Increased expression of HSP27 has been demonstrated following heating of both keratinocyte cell lines and organ-cultured human skin. HSP27 is expressed predominantly in the suprabasal epidermis in human skin, whereas HSP70 predominates in the dermis compared with the epidermis. These differences in location between HSP27 and HSP70 might explain the different time course of induction of these stress proteins upon O3 exposure. Interestingly, O3 induction of HO-1 showed a delayed time course compared with that for HSP27 and 70, in line with a previous study, which showed a peak of HO-1 induction at 18–24 h in rat lungs after O3 treatment [17]. It is therefore possible that bioactive compounds generated by the products of O3 exposure may be responsible for the induction of HO-1 as was also shown after UV radiation. As HSPs are involved in cell proliferation, apoptosis, and inflammatory response, O3-mediated HSPs induction can affect normal skin physiology. Thus, HSPs might provide an adaptive cellular response to O3; enhancing the expression of HSPs might turn out to be a new way to deal with the immediate and long-term consequences of O3 exposure. A prerequisite for the utilization of this concept is the development of nontoxic HSP inducers and their evaluation for clinical efficacy and safety.
Ozone and MMPs Among the multiple systems altered in the skin by environmental pollutants, MMPs are among the major targets. Indeed, O3 exposure is able to affect their synthesis and/or activity with logical consequences on tissue remodeling and wound healing. Within the MMP family, MMP-2 and MMP-9 are the only members able to degrade type-IV
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collagen of the basal membranes [23]. MMP-2 is involved in pathological processes such as photoaging and precancerous/cancerous skin lesions after UV exposure; moreover, MMP-2 is capable of cleaving other substrates, in addition to type-IV collagen, including other MMPs and therefore can (indirectly) control extracellular matrix degradation and remodeling. MMP-9, like MMP-2, plays a role in human skin aging and tumor development as well as in other cutaneous lesions such as psoriasis and dermatitis [24]. In a recent study, it was demonstrated that the environmental pollutant O3 was able to affect specific types of MMP activity in whole skin homogenates from hairless mice. Specifically, the exposure to 0.25 ppm of O3 for 4 days (6 h/day) clearly induced MMP-2 activity in cutaneous tissues. In this case, the generation of ROS can be the cause of such activation, as it has been shown that MMPs can be activated by reactive oxygen species. It has been also demonstrated that O3 is able to induce NO production via the activation of iNOS in cutaneous tissues. NO, while playing regulatory roles in the skin at physiological levels, when produced in excess, may combine with superoxide to form peroxinitrite (derived from other sources) that can activated MMPs especially MMP-9. Thus, the increase of oxidative stress after O3 exposure, plus the interaction between oxygen and nitrogen active molecules might be the main mechanism that leads to the enhanced MMPs activities in skin tissues. It has been shown in a number of cases that photoaging and precancerous/cancerous lesions can result from an imbalance between MMPs and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [24]. In fact the activities of MMPs are regulated by TIMPs, which can be produced by a multitude of cell types present in the cutaneous tissue. While MMP activity resulted to be altered by the O3, neither TIMP-1 nor TIMP-2 level expression was affected. The lack of changes in TIMP-1 and 2 levels, combined with the increased activity of MMPs suggest that O3 can cause a net increase in matrix degradation. Furthermore, there are other MMPs involved in skin diseases; for example, MMP-12, the human macrophage metalloelastase, accumulates in skin granuloma and in other inflammatory skin diseases such as dermatitis herpetiformis and pityriasis lichenoides. Moreover, MMP-7 or Matrilysin is very efficient in elastin degradation and increased elastolytic activity by both MMP-12 and MMP-7 has been reported upon oxidative stress exposure in hairless mice skin. Enhanced MMP-7 expression has also been detected in benign sweat gland tumors and aggressive basal and squamous cell carcinomas.
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Skin Age-Related Responses to O3 Exposure: MMPs It is well known that oxidative stress occurring after oxidant stressor such as O3 or UV radiation is implicated in the pathogenesis of skin-related diseases, and that the levels of antioxidant defenses decrease with aging. Consistently, it has been reported that aged mice are more susceptible to oxidative stress than young mice [25] and previous reports have suggested that oxidant pollutant exposure and age interact and potentiate each other [20]. Therefore, it can be concluded that among the multiple consequences of oxidative stress, an increase in the MMP/TIMP ratio also occurs. The question of whether a cause–effect relationship exists between oxidative stress and MMPs release, or they are two independent responses is not still clear and needs further studies to be better demonstrated. However, by integrating the results from recent works, the redox-associated signal transduction pathways that lead to MMPs induction can be easily reconstructed. Indeed, oxidative stress, through both receptor and non-receptor protein tyrosine kinases (PTK) activates several signaling proteins, such as ERK and PKB, which, in turn, mediate the transcriptional regulation of MMPs, via Ap-1, Ets, and NF-kB [26]. In closing, it is now documented that the interaction between aging and oxidative pollutant exposure can impair resistance of cutaneous tissues to environmental oxidative stress in elderly subjects.
Skin Age-Related Responses to O3 Exposure: Wound Healing Wound healing is a critical process in the skin and has been known to be affected by oxidative stress and also to decline with increasing age. Although the exact sequence of wound healing is not completely understood, cutaneous wound healing begins with wounding induced signaling factor-based transformation of stationary keratinocytes into cells capable of both replication and migration. Upon transformation, these cells express a host of molecules that promote the invasion of the injured epithelial matrix and re-epithelialization of the wound surface. Delayed wound healing in the elderly has been well described [27]. Among the elderly, the SC transit time was delayed 10 days compared to young adults. More recently, Hellemans and coworkers published that older skin, subjected to UVA-induced deactivation of catalase, requires a longer time to replenish
the antioxidant capacity than in younger skin, furthermore, it was shown that aged skin strongly differs from young skin in the ability to cope to oxidative environmental insults [20–28]. In the recent literature, it has been shown that hydrogen peroxide (H2O2) (molecules involved in the induction of oxidative stress) induced vascular endothelial growth factor (VEGF) expression in human keratinocytes and therefore can be able to stimulate wound healing [29]. As mentioned above, O3 exposure is also associated with activation of transcription factor NF-kB, which is important to regulate inflammatory responses and eventually entire wound healing. O3 exposure increased levels of tumor growth factor (TGFb) that is a critical factor in tissue remodeling [30]. The roles of the multiple, coordinated processes involved in the injured skin repair, as well as the signals that initiate and terminate skin responses remain ill defined. Furthermore, the age-related differences in the response of the skin wound healing to particular environmental insults are poorly documented. Given the documented role of oxidants in wound healing [29], the potential effects of O3 on cutaneous wound healing in combination with aging represent a poorly understood area. It has been suggested that O3 as an oxidant might also stimulate wound healing but aging with O3 would be detrimental due to increased oxidative stress and have biological as well as practical implications. In a recent study the detrimental effects of O3 on cutaneous wound healing in the aged animals was demonstrated. In fact when young hairless mice (8-week old) and aged mice (18-month old) with full-thickness excisional wounds were exposed to 0.5 ppm O3 for 6 h/day the rate of wound closure was significantly delayed in the aged group. It was also shown that O3 exposure induces protein and lipid oxidation assessed as changes in protein oxidation (carbonyls) and lipid peroxidation (4-hydroxynonenal, HNE adducts) in the old mice compared to the young mice during the later stage of cutaneous wound healing. The extent of wound closure in young and old animals with full-thickness excisional wounds exposed to a relevant concentration of O3, was monitored until day 9 (complete wound closure) [20]. These data suggest that O3 exposure has different effects depending on the age of the mice. In fact, it significantly delayed wound closure in old mice, while in young mice, it had no significant effect, although an accelerated trend during the first few days of the exposure was detected. This might be attributed to the antibacterial properties of O3, as it has been shown that application of hydropressive
Effect of Ozone on Cutaneous Tissues
ozonization provides fast cleansing of wound surface from pyonecrotic masses, promotes elimination of infection, and thus substantially reduces the period of treatment of the patients. Recently, clinical treatments using hyperbaric oxygen therapy demonstrated that increased oxygen tension at the wound site increases the formation of granulation tissue and enhanced accelerated wound closure and ameliorated impairs dermal wound healing; therefore, accelerated trend of wound closure shown in young population may be due to decreased bacterial infection and/or increased oxygen tension by O3 exposure in wound area [31]. One of the possible driving processes of the effect of O3 on wound healing can be, also in this case, the modulation of the transcription factor NF-kB. Although NF-kB is an immune regulator in inflammatory stage, it maybe critical to modulate later stage of healing process in injury. Consistent with this one, a recent study reported that human airway epithelium inflammatory response to inhaled O3 has been shown to be in part controlled by free radical-mediated NF-kB activation. Further, very recently, it has been reported that overexpression of superoxide dismutase not only prevents O3-related changes in bronchoalveolar lavage fluid protein, macrophage content, and 4-hydroxyalkenals, but also O3-dependent activation of NF-kB [32]. These researchers have also reported that O3-induced lung injury is mediated by NF-kB. These results clearly link O3 exposure to NF-kB activation and suggest that intracellular oxidants such as superoxide and related free radicals are important components of these responses. Interestingly, the dose– effect relationship between level of oxidative stress and NF-kB exhibits a biphasic profile: while moderate levels of oxidative stress activate NF-kB through an IkB kinaseindependent mechanism, extremely high levels of oxidative stress have been shown to inhibit NF-kB activation by blocking IkBa phosphorylation. Furthermore, the levels of oxidative stress were increased in aged rats and the content of activated forms, p50, and p65 subunits of NF-kB increased with age. One potential explanation for the differential effect of O3 in the older animals is that the level of oxidative stress generated by O3 exposure combined with aging causes levels of oxidative stress that inhibits IkBa phosphorylation, thereby resulting in a decline in NF-kB activation. The existence of a higher basal level of oxidative stress in old mice is proved by the higher levels of protein carbonyls and 4-HNE. These data fit with studies that have shown that old rats had higher lipid peroxides and superoxide dismutase activity tended to decrease. This finding is consistent with what mentioned previously that O3 exposure induced skin antioxidants
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depletion. In addition, Gilhar and coworkers have reported that human epidermis showed reduced proliferation and increased keratinocyte apoptosis with aging [33]. This could be interpreted as an additional evidence of increased load of oxidative stress burdens in the keratinocytes of old mice, as apoptosis has been linked to elevated levels of cellular oxidants. The evidences and considerations reported above are controversial because the levels of cutaneous oxidative stress in response to O3 treatment should be higher in aged skin and these levels may be further increased by O3 exposure so as to raise levels of skin oxidative stress in old mice to levels above those that maximally evoke NF-kB activation. This study suggests that although O3 exposure increased NF-kB activation in the young and old mice it may differently modulate wound healing process by aging. Furthermore, NF-kB also have important roles in later tissue remodeling stage as well as in initial inflammatory stage during cutaneous wound healing. This interpretation is also bolstered by the data on TGFb, a crucial modulator of tissue remodeling and is linked to both NF-kB status and levels of oxidative stress during entire wound healing process. The reduced TGFb levels in both air- and O3-exposed old mice, as well as the lower induction of TGFb by O3 exposure in the old animals suggest that the noted delays in wound closure might be related to defects in oxidative stress-dependent NF-kB status as well as levels of oxidative stress and TGFb signaling in aged mice during later stage of wound healing. In summary, given the role of oxidative stress in wound healing, an interaction between O3 and aging is of great interest to be explored in cutaneous wound healing process. The ability of O3 to alter wound healing indicates that environmental effects of pollutants need to be taken into account when damaged skin repair is explored in human subjects.
Ozone Potentiates UV-Induced Oxidative Stress in the Skin Although exposure of cutaneous tissues to either UV or O3 alone is known to deplete vitamin E and induce lipid peroxidation, it is of interest to evaluate the possible additive effects of sequential or simultaneous exposure of skin to these important environmental oxidants stresses. It should be taken into consideration that the skin is continuously exposed to several environmental pollutans each day and UVand O3 are among the most toxic and noxious of them. While UV radiation penetrates into the epidermis (UVB) or into the dermis (UVA), and is known to induce the release of tissue-degrading enzymes even at
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suberythemal levels, O3 oxidizes biological systems only at the surface. Therefore, since O3 and UV cooperatively damage SC components, it can be speculated that they exert an additive effects in the deeper layers of the epidermis. Products of O3-induced lipid oxidation penetrate the outer skin barrier and cause effects to constituents of the deeper epidermis. This can lead to the activation of NF-kB. On the other hand, NF-kB activation has also been implicated in the expression of collagenases by solar-simulated UV radiation and in cutaneous responses to wounding. UV radiation has been shown to compromise the skin barrier. O3 may enhance this phenomenon by perturbing SC lipid constituents in the SC, which are known to be critical determinants of the barrier function. Thus, O3 may cause a disturbance of the barrier function, increase the transepidermal water loss, and provokes epidermal repair responses, as can be also seen after barrier perturbation. Since O3 enhances UV-induced oxidation in the SC, it cannot be excluded that potentially O3 also enhances other UV effects such as photoaging [34]. In conclusion, the ‘‘additive’’ data demonstrate that O3 and UV radiation, two common sources of environmental oxidant stressors, exhibit additive effects in terms of oxidative damage to the skin barrier.
Health Implication Being lipids, the first target of O3, the consequent induction of lipid peroxidation in the upper layers of the skin can affect the physiology of cutaneous tissues. In fact, oxidation of the lipids present in the SC will change the skin barrier integrity and this has been shown to be a leading factor for several skin pathologies such as psoriasis, atopic dermatitis, and irritant dermatitis. The increase of peroxidation markers such as 4-HNE, MDA, and TBARS in the upper layers of the skin after O3 exposure is a consequence of the PUFA peroxidation like arachidonic acid and linolenate and this could consequently affect also the lower layers of the skin trigging all a cascade of noxious biological processes. The toxicity of O3 is the results from the effects of a cascade of products that are produced in the reactions of O3 with target molecules that lie close to the air–tissue interface. Ozone is too reactive to penetrate far into the tissue, only a small fraction of environmentally relevant doses of O3 is believed to pass through a bilayer membrane, and none pass through the cell. Therefore, the products that derive from the oxidation of the SC, which have longer lifetime and lower reactivity will transmit the effect of O3 beyond the air–tissue interface. Peroxidation products such as
4-HNE and alkenals are relatively stable and can damage or alter cells and tissues at more distant sites not directly exposed to O3.
Therapeutical Approaches Because the SC is the main target of O3 reactivity, therapeutic strategies should involve the more accessible skin layers via a topical antioxidant application. In a murine model, topical application of vitamin E reduced the peroxidation induced by O3 exposure, demonstrating that topical application could be a way to counteract ozone-induced skin toxicity. This indicates a key role for vitamin E both as an indicator and in the prevention of skin oxidative damage. In addition to physical or chemical measures for protection against environmental stressors consistent with the ‘‘free radical theory of aging,’’ the use of low-molecularweight antioxidants for preventing premature skin aging and skin disease seems appropriate. Vitamin E and other antioxidants can only be supplied to the skin to some extent via a diet rich in fruits and vegetables. Moreover, vitamin E supplementation and/or its topical administration will substantially enhance skin vitamin E concentrations. Since oxidant skin alteration occurs mainly in the SC and outer epidermal layers, this is relevant for a preventive and/or therapeutic approach.
Conclusion The results summarized in this chapter support the concept first advanced by Pryor et al. [4] that O3 exposures to noncellular constituents of surface epithelial cells are capable of generating potentially toxic peroxidation products. Extrapolation of this concept to cutaneous tissues would suggest that O3 reacts directly with SC lipids that contribute to cutaneous tissue protective barrier, generating products that are able to penetrate the SC and target keratinocytes. It is concluded that O3 not only affects antioxidant levels and oxidation markers in the SC, but also induces cellular responses in the deeper layers of the skin (> Fig. 41.1a, b). It is recognized that exposure of the skin to environmental stressors causes injury to the skin due to oxidants and free radicals, which leads to ‘‘oxidative stress,’’ also defined as imbalance between oxidants and antioxidants. Low-molecular-weight antioxidants are present in high concentrations especially in the epidermis. Oxidative stress can overwhelm the skin antioxidants and increase
Effect of Ozone on Cutaneous Tissues
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. Figure 41.1 (a) Summarizing scheme showing the effects of ozone exposure to the cell membrane of the Stratum Corneum and consequently generation of bioactive compunds such as aldehydes and H2O2, etc that can transmit the oxidative effects to the deeper layer of the skin. (b) Scheme showing the possible events as a consequence of SC oxidation by Ozone exposure
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the formation of oxidized cell components. Topical exposure to tropospheric O3 induces skin oxidative stress. Oxidative damage to the SC may result in a barrier perturbation, and in the production of lipid oxidation products that can act as ‘‘second messengers’’ in the deeper layers of the skin, which, in turn, elicits repair responses and/or the induction of defense enzymes such as HSPs. Oxidative injury to the outermost layers of the skin may initiate localized inflammatory responses, resulting in the recruitment of phagocytes and their cell-specific, tightly regulated NAD(P)H-oxidase systems for generating oxidants, thus amplifying oxidative stress and inducing activation of MMPs.
Cross-references > Global
Warming and its Dermatologic Impact on Aging Skin > Skin Photodamage Prevention: State of the Art and New Prospects
References 1. Kelly FJ, et al. Air pollution and the elderly: oxidant/antioxidant issues worth consideration. Eur Respir J Suppl 2003;40:70s–75s. 2. Valacchi G, et al. Induction of stress proteins and MMP-9 by 0.8 ppm of ozone in murine skin. Biochem Biophys Res Commun. 2003;305 (3):741–746. 3. Valacchi G, et al. The dual action of ozone on the skin. Br J Dermatol. 2005;153(6):1096–100. 4. Mudway IS, et al. Ozone and the lung: a sensitive issue. Mol Aspects Med. 2000;21(1–2):1–48. 5. Zimmermann PH. Tracing the sources of tropospheric ozone, Proceedings of the International Ozone Symposium, 21 and 22 October 1999. Basel (IOA – EA3G Ed), 1999, pp. 157–160. 6. Weber SU, et al. Ozone: an emerging oxidative stressor to skin. Curr Probl Dermatol. 2001;29:52–61. 7. Dentener F, et al. The global atmospheric environment for the next generation. Environ Sci Technol. 2006;11:3586–3594. 8. Mortimer KM, et al. The effect of ozone on inner-city children with asthma: identification of susceptible subgroups. Am J Respir Crit Care Med. 2000;162(5):1838–1845. 9. Tager IB, et al. Chronic exposure to ambient ozone and lung function in young adults. Epidemiology. 2005;16(6):751–759. 10. Mustafa MG. Biochemical basis of ozone toxicity. Free Radic Biol Med. 1990;9(3):245–265. 11. Pryor WA. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic Biol Med. 1994;17(5):451–465. 12. Pryor WA, et al. The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products. Free Radic Biol Med. 1995;19(6):935–941.
13. Ballinger CA, et al. Antioxidant-mediated augmentation of ozoneinduced membrane oxidation. Free Radic Biol Med. 2005;38 (4):515–526. 14. Esterbauer H, et al. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11 (1):81–128. 15. Sathishkumar K, et al. A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts. FEBS Lett. 2005;597(28):6444–6450. 16. Spickett CM, et al. The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids. Acta Biochim Pol. 2000;47(4):889–899. 17. Valacchi G, et al. In vivo ozone exposure induces antioxidant/stressrelated responses in murine lung and skin. Free Radic Biol Med. 2004;36(5):673–681. 18. He QC, et al. Effects of environmentally realistic levels of ozone on stratum corneum function. Int J Cosmet Sci. 2006;28(5):349–357. 19. Thiele JJ, Schroeter C, Hsieh SN, et al. The antioxidant network of the stratum corneum. Curr Probl Dermatol. 2001;29:26–42. 20. Lim Y, et al. Modulation of cutaneous wound healing by ozone: differences between young and aged mice. Toxicol Lett. 2006;160(2):127–134. 21. Packer L, et al. Antioxidants and the response of skin to oxidative stress: vitamin E as a key indicator. Skin Pharmacol Appl Skin Physiol. 2002;15(5):282–290. 22. Thiele JJ, et al. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin. J Invest Dermatol. 1999;113 (6):1006–1010. 23. Brenneisen P, et al. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann N Y Acad Sci. 2002;973:31–43. 24. Hofmann UB, et al. Matrix metalloproteinases in human melanoma. J Invest Dermatol. 2000;115(3):337–344. 25. Stadtman ER. Role of oxidant species in aging. Curr Med Chem. 2004;11(9):1105–1112. 26. Galis ZS. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002;90 (3):251–262. 27. Crooks A. How does ageing affect the wound healing process? J Wound Care. 2005;14(5):222–223. 28. Fortino V, et al. Cutaneous MMPs are differently modulated by environmental stressors in old and young mice. Toxicol Lett. 2007;173(2):73–79. 29. Sen CK, et al. Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing. J Biol Chem. 2002;277(36):33284–33290. 30. Valacchi G. Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells. Mediators Inflamm. 2000;9(6):271–276. 31. Gajendrareddy PK, et al. Hyperbaric oxygen therapy ameliorates stress-impaired dermal wound healing. Brain Behav Immun. 2005;19(3):217–222. 32. Fakhrzadeh L. Ozone-induced production of nitric oxide and TNFalpha and tissue injury are dependent on NF-kappaB p50. Am J Physiol Lung Cell Mol Physiol. 2004;287(2):L279–285. 33. Gilhar A, et al. Ageing of human epidermis: the role of apoptosis, Fas and telomerase. Br J Dermatol. 2004;150(1):56–63. 34. Valacchi G, et al. Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum. FEBS Lett. 2000;466(1):165–168.
45 Environmental and Genetic Factors in Facial Aging in Twins David J. Rowe . Bahman Guyuron
Introduction The etiologic factors contributing to facial senescence have been investigated for decades if not centuries. In essence, the causes of facial aging can be divided into two broad categories: intrinsic and extrinsic [1]. Intrinsic aging is that which occurs as a response to the deterioration of tissues over time [2]. This process is ubiquitous throughout all organs and tissues, although the methods of ‘‘deterioration’’ may vary from system to system. Intrinsic changes of the face include those to the skin, subcutaneous tissue, dermal appendages, facial musculature, as well as the facial skeleton. The process of extrinsic facial aging is, theoretically, a distinct entity. Typically, extrinsic aging is induced by external factors, such as UV radiation, causing progressive damage at both the molecular and cellular levels. Unlike intrinsic aging, most extrinsic factors exert their effect at the skin level only. Discriminating between the levels of involvement of both aging processes, extrinsic and intrinsic, is problematic. As both processes continually occur, the ability for the body to resist change depends on the amount of cumulative damage of each individual process. Furthermore, there may be a dynamic interplay between extrinsic and intrinsic modalities that lead to variable amounts of aging depending on the levels of each and the ability of the body to repair or resist these. For example, a minor intrinsic defect, such as that in a DNA repair mechanism, may cause slightly accelerated aging from an intrinsic standpoint; however, it could cause an uncommonly aberrant response for extrinsic factors. A preponderance of information exists on the presence and types of extrinsic factors that may influence aging; however, the intrinsic causes are likely as important or more important on skin and facial aging. Multiple genetic disorders exist that corroborate the fact that premature aging can be genetic. Diseases with defects in DNA repair mechanisms, such as xeroderma pigmentosa, Werner Syndrome, and Cockayne Syndrome, all show accelerated aging [3]. Hutchinson-Gilford Progeria Syndrome is caused by a defect in Lamin A, a protein used in
the cell nuclear envelope, leading to abnormal morphology in the cell nucleus. This too leads to an accelerated aging phenotype. Theoretically, variable penetrance, or a less severe genetic aberration, may either increase or decrease the signs of aging, independent of external factors. Most data regarding the influence of external factors on aging have been conducted as epidemiological collections on specific patient populations. Epidemiological studies have intimated associations between environmental factors and aging; however, the influence of genetic differences cannot be controlled when dealing with a diverse genetic population. One research tool that may be implemented in the study of skin aging is the investigation of twin sets. Examination of monozygotic, as well as dizygotic twins, allows a unique opportunity to control for genetic differences. In the analysis of facial senescence, this gives the investigator the ability to control for most of the causes of intrinsic aging. Despite the relative benefit of genetic control, the statistical study of twins requires a relatively large patient population, as many twin sets have very similar lifestyles. The rest of the chapter attempts to analyze the findings of the several twin investigations in the current literature and identify possible risk factors for extrinsic aging.
Extrinsic Causes of Aging Smoking The association of skin aging with smoking was first scientifically proposed in 1856 when Solly postulated that facial appearance in smokers is markedly different than nonsmokers [4]. Since this time, epidemiological data have shown a possible correlation between smoking and facial aging, although the amount of associated aging due to smoking has been debated. Results have been equivocal, with several investigations showing little to no aging difference due to smoking status [5, 6] while others have seen a significant association [7–12]. Several analyses of twins have corroborated the influence of smoking on facial aging (> Fig. 45.1). Doshi et al.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_45, # Springer-Verlag Berlin Heidelberg 2010
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reported a significant difference in facial aging of a singular set of twins [13]. Each of the twins in this report had similar lifestyles, body habitus, and sun exposure; however, one of the twins had a greater than 50 pack-year history of smoking. The differences with regards to superficial and deep rhytides, lentigines, tissue laxity, and pigmentary changes were disparate. Antell and Taczanowski also provided anecdotal evidence on 34 sets of identical twins, showing a possible correlation to smoking differences [14]. Rexbye et al., in an investigation of 1826 Danish twin sets over the age of 70, demonstrated that smoking was a significant determinant of facial aging in men, yet less so in women [15]. Smoking 20 cigarettes per day per year for 20 years increased perceived age by one year. For women, this number was increased to 20 cigarettes per day for
. Figure 45.1 Twins (natural age 52) with difference in smoking history. Twin a, c had a 20 year greater smoking history than Twin b, d. Perceived age difference of the twins was 6.25 years
40 years (> Fig. 45.2). The authors attribute this discrepancy to the difference of smoking habits between the sexes in the Netherlands. Fewer women were smokers, and those who did smoke, actually smoked less than their male counterparts. Guyuron et al. conducted an investigation of 186 sets of identical twins at an annual international festival for twins [16]. Females from the ages of 18 to 76 were analyzed in this study. A comprehensive questionnaire was filled out and standardized high resolution photographs were taken of each twin set. Smoking was found to be significantly correlated with perceived facial age. In analysis of the data, approximately 10 years of smoking difference led to a 2 ½ years older appearance.
. Figure 45.2 Twins (natural age 57) with difference in smoking history. Twin b, d had a 40 year greater smoking history than Twin a, c. Twin a, c had 2 years of hormone replacement therapy. The perceived age difference was 8.25 years
Environmental and Genetic Factors in Facial Aging in Twins
The mechanism for smoking induced damage in the skin is unclear, although many theories exist. One postulate is that the increase of matrix metalloproteinases (MMPs) in the skin of smokers contributes to skin damage [17]. Matrix metalloproteinases are proteases responsible for degrading dermal collagen and other extracellular matrix material. This effect may be synergistic with the effects of UV radiation, as irradiation also induces MMPs. Other likely changes include the decrease of the skin microvasculature, leading to the increase of reactive oxygen species, and thus free radicals [13, 18]. These processes are also known to occur in the face of photodamage, therefore, there may be additive effects of sun and smoking on skin aging.
Sun Exposure Sun exposure is perhaps the most investigated cause of extrinsic aging. There is incontrovertible evidence that ultraviolet irradiation damages the skin and induces premature aging. The pathophysiology of photodamage is multifactorial, involving the upregulation of matrix metalloproteinases, reversible and irreversible damage to DNA, and creation of reactive oxygen species [18]. Details on these and other mechanisms are beyond the scope of this chapter and are discussed in subsequent chapters. Controversy does exist, however, on the importance of sun damage on perceived age. Chronological or intrinsic skin aging shares many of the features and possible mechanisms of photoaged skin. In both photoaging and chronological aging of the skin, elevated concentrations of degraded collagen are present. Furthermore, both mechanisms of aging have been theorized to occur as a result of oxidative damage [19]. Although the actual pathways involved in chronological aging may be distinct from those in photoaging, the two may share common central mediators. Many studies have been performed evaluating the importance of photoaging with respect to skin changes and alteration of perceived age [20]. While there is no question that UV irradiation induces age-related changes in the skin, the amount of change is a topic of considerable debate. When evaluating an elderly (> 60 years) population, Leung and Harvey found that sun exposure alone did not have a considerable effect on perceived age [2]. In their multivariate regression analysis, 30 years of sun exposure for 5 hours a day only produced 1.5 years of perceived age difference. Guinot et al., in designing a skin age score in a prospective analysis of Caucasian women from 18 to 80 years of age, found that visual signs of chronic
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photodamage did not contribute significantly to the age score [21]. These investigations do not refute the contribution of ultraviolet irradiation in skin aging but they do underscore the difficulty in attributing visible skin changes to a singular extrinsic or intrinsic cause. One method to investigate the importance of photoaging, and control for chronological (intrinsic) aging, is the analysis of identical twins. The analysis of twin sets has shown significant perceived differences in aging with respect to sun exposure (> Fig. 45.3). Guyuron et al. investigated hours of sun exposure as well as the participation in outdoor hobbies and the use of sunscreen [16]. The increase in sun exposure as well as the participation in outdoor activities both significantly increased perceived
. Figure 45.3 Twins (natural age 61) with significant difference in sun exposure. Twin b, d had approximately 10 h/week greater sun exposure than twin a, c. Twin a, c had a BMI 2.7 points higher than twin b, d. The perceived age difference was 11.25 years
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age. The use of sunscreen did significantly decrease perceived age; however, the level of SPF used was not assessed. Sun exposure was also correlated with increased perceived age in a study of elderly (70+) Danish twins [15]. Here, sun exposure was evaluated on the type of employment the individual performed during the longest period of working. Interestingly, sun exposure was statistically significant in men, however not in women. The authors attributed this effect to the likely high rate of sun exposure in working men. Only 8.3% of women in this study were exposed to sun during the working hours of the day.
. Figure 45.4 Twins (natural age 58) with differences in BMI. Twin a,c had a 14.7 point higher BMI than twin b, d. No other differences were discerned from the questionnaire. Perceived age difference was 5.25 years
Diet (BMI) BMI may have a not-so-indirect effect on perceived age, although this may not necessarily be due to changes at the skin level. For years, plastic surgeons have recognized that facial atrophy and soft tissue descent are harbingers for an increase in perceived facial age. Various surgical and injectable modalities have been introduced that restore facial volume and resuspend tissues to their previous anatomical position. Several studies have identified an inverse relationship between BMI and facial wrinkling. Guinot et al. prospectively analyzed a cohort of 361 white females in the process of developing a skin age score [21]. They found that body mass index did significantly affect their skin age score in an inverse relationship. Purba et al. evaluated skin wrinkling in elderly (> 70) patients from multiple countries [22]. As with the previous study, this investigation found that wrinkling was significantly negatively correlated with BMI. Analysis of twins’ data has further substantiated these findings. In Guyuron et al.’s study, the influence of body mass index was highly dependent on the age of the twin set [16]. In twins that were younger than 40 years of age, a four-point increase in BMI was associated with a perceived older appearance. A four-point increase in BMI in twins older than 40 years of age, however, was associated with a perceived younger appearance (> Fig. 45.4). The latter finding was supported by the Rexbye et al. investigation [15]. Here, an increase of BMI in both elderly men and women was associated with a younger appearance. A decrease of BMI of 2 in males and 7 in females was associated with a one year older perceived age.
Hormone Replacement Therapy (HRT) Estrogens have a profound effect on the skin, as evidenced by the cutaneous alterations in skin characteristics
following menopause. Although the mechanisms are poorly understood, a decrease in circulating estrogen is associated with decreased skin elasticity, decreased dermal thickness, as well as increased dryness [23]. Conversely, analyses of estrogen replacement therapy have suggested possible increases in the dermal thickness, concentration of dermal collagen, elasticity, and fewer fine wrinkles [24]. Epidemiological investigations have nonetheless been equivocal in their findings of hormone replacement therapy on perceived aging. With regard to twins’ analysis, hormone replacement therapy has been associated with a younger perceived age [16]. In addition, the effect of HRT on age increased as the age of the twin sets increased and as the difference of years of treatment increased (> Fig. 45.5).
Adverse Social Factors External social factors have been found to significantly alter biological aging. Several aspects that have been associated with aging include depression, divorce, socioeconomic status, and alcohol consumption [25, 26]. Additionally, there is usually an interaction or intermingling of these deleterious social factors. Osler et al. investigated the role of marital status, BMI, depression, and smoking in 1,175 sets of identical Danish twins [27]. The twins that were divorced, widowed, or never married had
Environmental and Genetic Factors in Facial Aging in Twins
. Figure 45.5 Twins (natural age 71) with difference in HRT. Twin b, d had 22 more years of HRT than twin a, c. Twin b, d had a 1.2 lower BMI. Perceived age difference was 7.25
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past, led to an increase in perceived age [16]. Rexbye et al. used a depression symptomatology score to analyze depression in their study [15]. They found that an increase in the depression score was significantly associated with an increase in perceived age. Specifically, an increase in depression symptomatology score from 17 to 49 was associated with a perceived age increase of 2.4 years for men and 3.9 years for women. The length and type of depression, however, has not been investigated with relation to facial aging. In both of these studies, only the presence of depression, either in the past or current was analyzed.
Alcohol Consumption A significant amount of data on the effects of alcohol on twins does not exist. Guyuron et al. investigated alcohol avoidance, but did not quantify alcohol consumption [16]. Despite this caveat, the twins who ‘‘avoided alcohol’’ had a younger perceived age. Rexbye et al. did not notice a significant effect of substantial alcohol consumption, however their number of positive responders was small enough to limit the power of this study [15].
Marital Status
higher depression scores and smoked more than their married counterparts. It is no surprise that perceived facial aging may also be adversely affected by these same factors. Limited epidemiological data exists on perceived facial aging and skin aging relating to factors such as depression, divorce, and alcohol consumption. However, several of the twins investigations previously discussed in this chapter have evaluated these social facets and will be discussed below.
Depression In the Guyuron et al. study, depression was indirectly investigated by measuring the utilization of antidepressants. Here, the use of antidepressnats, either current or
In the analysis of twins, women who were divorced were perceived to be approximately 1.7 years older than those who were either single or married [16]. Interestingly, widows appeared approximately two years younger than their non-widowed counterparts. Although Rexbye et al. also reported a 1.9-year difference in age between married and unmarried women, this was not statistically significant.
Conclusion The etiological delineation of skin and facial aging is complicated, given the large number of possible contributing factors. Epidemiological data has been helpful, however it does not allow for control of the intrinsic factors of aging that are inherently different in all individuals. The analysis of twins gives the investigator the ability to control for genetic causes. In the study of skin and perceived facial aging, one is able to investigate purely the environmental causes of skin and facial aging, extrinsic aging. From the data presented in the several twins investigations in the current literature, there appear to be several
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factors important to extrinsic skin aging and perceived facial age. Smoking and sun exposure, the two most epidemiologically studied factors, do appear to have a significant role in perceived age. Other factors such as hormone replacement therapy, BMI, depression, and the use of alcohol also have an influence on facial aging. Larger series of twins’ analyses are needed to further delineate the importance of each of these facets.
Cross-references > Effect
of Ozone on Cutaneous Tissues Warming and its Dermatologic Impact on Aging Skin > In Vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model > Skin Photodamage Prevention: State of the Art and New Prospects > Tobacco Smoke and Skin Aging > Global
References 1. Uitto J. Understanding premature skin aging. New Eng J Med. 1997;337:1419–1428. 2. Leung W, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol. 2002;147(6):1187–1191. 3. Pesce K, Rothe M. The premature aging syndromes. Clin Dermatol. 1996;14:161–170. 4. Solly S. Clinical lectures on paralysis. Lancet. 1856;130(2):167–173. 5. O’Hare P, et al. Tobacco smoking contributes little to facial wrinkling. J Eur Acad Dermatol Venereol. 1999;12(2):133–139. 6. Allen H, Johnson B. Diamond S Smokers wrinkles? JAMA. 1973;225: 1067–1069. 7. Daniell H. A study in the epidemiology of ‘crows feet’. Ann Intern Med. 1971;75(6):873–880. 8. Model D. Smoker’s face: an underrated clinical sign? BMJ. 1985;291 (6511):1760–1763. 9. Ernster V, et al. Facial wrinkling in men and women, by smoking status. Am J Public Health. 1995;85:78–82.
10. Keadunce D, et al. Cigarette smoking: risk factor for premature facial wrinkling. Ann Intern Med. 1991;114(10):840–844. 11. Chung J, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol. 2001;137 (8):1043–1051. 12. Helfrich Y, et al. Effect of smoking on aging of photoprotected skin: evidence gatheres using a new photonumeric scale. Arch Dermatol. 2007;143(5):397–402. 13. Doshi D, Hanneman K, Cooper K. Smoking and skin aging in identical twins. Arch Dermatol. 2007;143(12):1543–1546. 14. Antell D, Taczanowski E. How environment and lifestyle choices influence the aging process. Ann Plast Surg. 1999;43:585–588. 15. Rexbye H, et al. Influence of environmental factors on facial ageing. Age Ageing. 2006;35(2):110–115. 16. Guyuron B, et al. Factors contributing to the facial aging of identical twins. Plast Reconstr Surg. 2009;123:1–11. 17. Lahmann C, et al. Matrix Metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357:935–936. 18. Fisher G, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138(11):1462–1470. 19. Sohal R, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59–63. 20. Fisher G, et al. Pathophysiology of premature skin aging by ultraviolet light. New Engl J Med. 1997;337:1463–1465. 21. Guinot C, et al. Relative contribution of intrinsic vs extrinsic factors to skin aging as determined by a validated skin age score. Arch Dermatol. 2002;138(11):1454–1460. 22. Purba M, et al. Can skin wrinkling in a site that has received limited sun exposure be used as a marker of health status and biological age? Age Ageing. 2001;30:227–234. 23. Pierard G, et al. Effect of hormone replacement therapy for menopause on the mechanical properties of the skin. J Am Geriatr Soc. 1995;43(6):662–665. 24. Callens A, et al. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology. 1996;193(4):289–294. 25. Nilsson P, et al. Adverse social factors predict early ageing in middleaged men and women: the Ebeltoft Health Study, Denmark. Scand J Public Health. 2003;31(4):255–260. 26. Demakakos P, et al. Socioeconomic status and health: the role of subjective social status. Soc Sci Med. 2008;67(2):330–340. 27. Osler M, et al. Marital status and twins’ health and behavior: an analysis of middle-aged Danish twins. Psychosom Med. 2008;70 (4):482–487.
Rheology
27 Facial Skin Rheology Ge´rald E. Pie´rard . Fre´de´rique Henry . Pascale Quatresooz
Introduction The precise determination of the physical properties and functions of facial skin and its constituent parts remains an open question. In contrast, much more attention has been paid to the molecular biological characterization of skin components. In part, this may reflect the comparatively late development of bioengineering and biophysics and the intrinsic difficulty of obtaining relevant reproducible physical data from the skin. In addition, some ambient environmental conditions profoundly and specifically influence the physical attributes of the skin. As a result, the variations in physical parameters owing to body region, age, gender, and ethnicity greatly outweigh the variability of the corresponding molecular composition of the cutaneous structures. The term properties of the skin implies assessments similar to any other physical material, and this may give relatively little information about clinical or biological relevance. It is most applicable to in vitro testing. By contrast, clinicians and cosmetologists are primarily concerned with a more restricted range of functions rather than properties of the skin. It is of course true that functions are largely dependent on properties, but the conceptual and practical differences are important. Testing of a function must be performed in vivo under a fairly narrow range of ambient conditions. However, it does not follow that in vitro testing has no importance for clinicians. By contrast, some data are only obtained by using the in vitro approach, although this requires caution in interpretation.
Structure and Mechanical Functions of Skin Skin is a complex five-layered composite structure (stratum corneum, stratum Malpighi, papillary dermis, reticular dermis, and hypodermis), whose functions depend on the mutual interdependence of the constituent tissues. Overall, the mechanical properties and functions of skin are mainly governed by the dermal and hypodermal connective tissues, with a possible minimal contribution from
the stratum corneum [1, 2]. In addition, the skin structures differ largely according to the body site [1, 3]. Thus, regional variations have profound influences on the biomechanical characteristics. Moreover, the chronic and cumulative environmental threats including ultraviolet light and near infrared radiations [4] are dissimilar in distinct body regions, and according to age, phototype, and behavior with regard to sun exposure. The balance between these factors determines and distinguishes the intrinsic and extrinsic aging processes [5]. In these respects, facial skin is particularly susceptible to the diverse aspects of weathering and photoaging. Thus, aging of facial skin is not similar to that occurring on most other body sites. Such regional anatomical variation was not always fully acknowledged in the past. The mechanical properties and functions of skin are time dependent. They are also anisotropic as they differ according to the direction in which the load is applied. Both these characteristics of skin add complications in obtaining descriptors of its mechanical properties and functions. A further major problem in this field is the lack of standardization among investigators. Different research groups have used a variety of devices and measuring units, as well as different conditions of measurement. In addition, they have employed differing test modes for obtaining what was expected to be a similar information. It is clear that progress in the field of mechanical bioengineering will not be made until some attempt at uniformity is made, and acceptable, controlled, and standardized practices are developed.
Facial Skin Aging and Its Physical Attributes Facial skin is bound to deeper structures, but is allowed some mobility. Thus, it allows both movements and temporary compression and distension of a part. Flexibility and elasticity are important attributes of skin while firmness is also an essential component. These skin qualities are relevant to the visual and tactile features of facial skin [6]. Aging of facial skin takes place gradually over 2–4 decades. In its early stages, little clinical evidence is
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_27, # Springer-Verlag Berlin Heidelberg 2010
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present with the exception of the mosaic faint (subclinical) melanoderma [7]. The incipient clinical signs of facial aging usually begin to be recognized by individuals with the emergence of discrete furrows and wrinkles, together with a loss of firmness [8–11]. Additionally, various cutaneous signs and lesions develop with aging, and some of them are the result of more severe photoaging. In addition, changes in the deep cutaneous tissues distinct from sun-induced damages are responsible for deepening of facial creases and sagging [10–13]. As a rule, the results given by in vivo mechanical testing of facial skin may prove to be difficult to interpret because of the multiple and complex relationships between the various components of the skin, and the illdefined intrinsic properties of each tissue. In addition, there is commonly an unknown influence of previous mechanical solicitations at the test sites [1]. Both the mechanical functions and the surface contours of the skin reflect the structural organization of the tissues [10]. As a result, the combination of rheological and profilometric assessments provides a relevant and noninvasive characterization of the overall aging process of facial skin [1, 14, 15]. However, the diversity of bioengineering methods of evaluation combined with a wide variation in experimental designs has brought a number of uncertainties and discrepancies in the information.
Fibrous Structures Mainly Responsible for the Mechanical Properties On a mechanical viewpoint, the dermis has often been compared to rubber, and a series of the tests used were adaptations from those used in the rubber industry. In fact, the dermis does not exhibit similar properties and functions. The bulk of the cutaneous connective tissue consists of a network of collagen fibers, the organization of which determines the mechanical characteristics of the tissue and its resistance to deformation [1]. The elastic fibers present in smaller amounts serve to recoil the stretched collagen bundles to their relaxed position. This complex network of fibers is permeated by highly hydrated proteoglycans, glycoproteoglycans, and glycoproteins embedding the connective tissue cells. Fibroblasts and dermal dendrocytes are responsible for maintaining or remodeling the quantitative and structural steady state of the connective tissue fibers and the amorphous matrix as well. There are considerable differences in the relative proportions and organization of each of these components in different skin regions, as well as variations during aging and diseases [1].
With respect to the major mechanical functions of the connective tissues, and to structural features similar to all body regions, the dermis is conveniently divided into two major superposed layers. The adventitial dermis corresponds to a superficial zone of loose connective tissue adjacent to the epidermis and encasing its follicular adnexae as well. It corresponds to the papillary and the periadnexal dermis. The rest of the dermis is identified as the reticular dermis because of the netlike appearance of its fiber bundles. Still a third deeper layer corresponds to the connective septae partitioning lobules of adipocytes in the hypodermis. The differences and limits between the three layers are not always sharply identified. It is the relative concentration and arrangement of fibers rather than any absolute differences in composition that enable these regions to be distinguished. In mechanical terms, it is expected that the physical functions of the adventitial dermis somewhat resemble those of the hypodermal septae because they are conditioned by thin collagen fibers arranged in a rather similar loose open meshwork running perpendicular to the surface of the skin. The reticular dermis is more rigid because the collagen fiber bundles are coarser, tightly connected each another, and most often closely packed in planes parallel to the skin surface. It must be emphasized that this descriptive view of the connective tissue varies tremendously according to the body site. In its structural organization, the dermis of the face, scalp, back, forearm, palms, and soles differs greatly. It should be stressed that the rheological functions of facial skin are markedly influenced by the presence of a high density of terminal hairs in men and by abundant and large sebaceous glands. These structures likely put the surrounding fiber networks of the dermis under tension. In addition, facial muscles impose some anisotropic tensions to the skin, which are in turn responsible for some wrinkles such as facial frown lines and glabellar rhytids [9, 10, 16]. Moreover, age is important to consider as the skin presents marked differences during fetal life, childhood, climacteric period, and senescence [3, 14, 17]. Nonionizing radiations from the environment [4] superimpose their effects on those of the natural chronological aging [5, 18].
Physiological Interferences with Skin Mechanical Properties Skin withstands forces originating from the body and reacts to those imposed by the environment. These features govern the global skin mechanobiology. The perception of normal, loose, or tight skin depends on the
Facial Skin Rheology
ability of the connective tissue to resist and transmit the various forces. When assessing the in vivo skin mechanical functions, intrinsic tension forces are hardly measurable, and they should ideally be reduced to a minimum in order to prevent interference with testing [1]. This is tentatively achieved by muscle relaxation and a comfortable, controlled posture of the concerned body region. These controlled conditions do not abrogate the relaxed skin tension lines and the Langer’s lines [19–21]. Facial skin is quite unique as far as skin mechanical functions are concerned [14, 22–24]. Skin is anisotropic with regard to the variability of mechanical functions according to the direction of the forces applied [19, 25]. There is some complexity associated with sorting out some straightforward relevant information from most in vivo testings. In fact, measuring the overall mechanical characteristics of skin provides a rough estimate of the resultant of multiple features acting on various parts of the skin. The distinction between the specific dermal and epidermal properties is not accessible to accurate measurement. One of the major challenges resides in the interpretation of the combination of histological, instrumental, and biological variations that are found [1, 19]. In all circumstances, when a force is applied to the dermis, fibers are first reoriented in parallel to the force. At completion, some elongation is obtained for elastic fibers, while collagen fibers remain almost inextensible. In the physiological range of tension, the structural organization of collagen bundles, their orientations, the anchorage of bundles together, as well as their relation to elastic fibers and proteoglycans should be considered as predominant in determining the natural tension lines in the skin [20]. When skin is elongated, the fibers become aligned and slip over one another. With increasing forces the extensibility of the collagen fibers themselves is being tested. With sustained forces, there is a gradual change in the bonding of the collagen fibers or some other form of molecular realignment. During persistent compression, the interfiber matrix is squeezed out of the site. There is both an alignment of the fiber bundles along the lines of stress and a decrease in their convolution [20]. This first phase is largely due to straightening of the usually convoluted fibers but not to their lateral contraction.
Basic Viscoelastic Properties of Skin Basically, skin exhibits viscoelastic functions and properties. However, the literature may appear quite confusing with regard to the expression and interpretation of
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mechanical testing of the skin. This situation is due to the absence of a well-recognized nomenclature, of a uniform system of units, and of standardized techniques. However, the basic terms and units used in physics should be applied to biology, and these prove to be useful in clinical practice [1]. A series of experimental devices have been developed in research laboratories in order to measure the mechanical functions of the skin perpendicular or parallel to the skin surface. Mechanical functions of facial skin are conveniently assessed noninvasively by a series of methods. Skin may be pulled upward, pressed, twisted, extended parallel to its surface in one or several directions, and submitted to vibrations and to many other types of mechanical stimuli. Forces applied to the skin vary in direction, intensity, and time of application. These different approaches provide different information on the functions of the various connective tissue frameworks. In practice, the experimental approaches to determining the mechanical properties of skin are divided into six types: the tensile and torsional types, as well as those based on elevation, indentation, suction, and vibration. In general, the most relevant information is gained with testing facial skin at low stress. When a force is applied to the skin using a conventional testing device, the tension created is calculated in newtons (N) or in millibars (mbar). Stress corresponds to the ratio between the force (load) and the cross-sectional area of the skin in a plane at right angles to the direction of the force. It is expressed in newtons per square meters (N/m2). Strain is the ratio between elongation and the original length of the tissue submitted to the force. It is dimensionless, since measured as mm per mm. Usually, the crude information received from an experiment is the relationship of force (or stress) to deformation (or strain) over time. However, the maximum deformation for a given force is not gained immediately as some elongation still takes place under stable traction after certain periods of time. In addition, the deformation is not completely reversed within a short period of time in the absence of compressive force. These features explain the complexity of stress–relaxation curves. Facial skin is a viscoelastic material characterized by a nonlinear stress–strain properties with hysteresis (HY) [1, 26]. This means that the stress–strain curve obtained during loading is not identical to the curve obtained during unloading. Furthermore, the deformation of skin as a function of time shows an immediate incomplete elastic deformation and a creeping viscoelastic deformation followed by an immediate elastic recovery and a creeping recovery with a residual deformation. In any
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instance, the accuracy, repeatability, and reproducibility of the data rely on strictly controlled and validated experimental conditions. The selection of the most relevant biological viscoelastic parameters benefits from standardized modalities of assessments. Attempts were made to derive constants from the experimental data. The Hook constant is obtained in the portion of the curve where a fixed ratio is obtained between load and extension. The Young’s modulus refers to the value of the stress–strain ratio. When skin is stressed by a load, rapid extension takes place at first but then gives way to a region of much less extension. The overall response is nonlinear, although there is a linear portion of the slope. Where extension is directly proportional to the load applied and the material tested will return to its original length, when the load is removed the relationship is said to be ‘‘elastic’’ or ‘‘Hookean.’’ The second phase is typical of an ‘‘elastic’’ material, but the first indicates that the skin becomes ‘‘stiff.’’ The shape of the curve is such that it is very difficult to describe it mathematically. As a residual deformation is commonly present, it interferes with subsequent testing at the same site during the next hours. These changes in mechanical characteristics are sometimes referred to as ‘‘creep,’’ ‘‘viscous extension,’’ or ‘‘viscous slip.’’ Hence, the concept of ‘‘preconditioning procedure’’ is achieved by applying a series of preliminary stresses to the tissue before measuring its mechanical functions. When a series of stress cycles are consecutively applied and removed, slightly different curves are obtained on each occasion. Because of the above considerations, the results of mechanical testing are clearly time dependent. Indeed, the results obtained depend to some extent on the rate at which the stress is applied, the duration for which it is applied, and the previous stress history of the site. While performing in vivo mechanical testing of skin, information is expected in line with other biological parameters. The measurements should provide data consonant with the usual functions of the skin. In fact, in normal and pathological conditions, the relevant mechanical functions of skin represent only a small part of its maximum mechanical capacity. Results unrelated to biological functions of the skin should be disregarded. Such a concept covers several questions to be answered successively when mechanical testing of the skin is to be performed in vivo. ● What is the relevant range of mechanical function in the condition being studied? ● What is the nature of information expected? ● What is the most relevant parameter to be measured? ● What region of the skin is being tested?
● What section of the skin is being tested? ● How do the tissues respond to the forces exerted? ● What is the interpretation to be given in a fourdimensional concept of skin volume and time? In the past, many methods were used to assess the mechanical functions of the skin. They measured different parameters, and the results were hardly comparable because they differed considerably by many qualitative and quantitative aspects. Indeed, the crude data must be interpreted with respect to the method used and the type of test performed. Detailed information about the testing conditions is mandatory. It includes any eventual preconditioning, the orientation, time of application and magnitude of the force exerted, the deformations gained for several load intensities, the body site, and the geometry of the device.
Viscoleastic Function of Facial Skin Using the Suction Method One of the most popular method for measuring skin biomechanics in vivo relies to the so-called suction method [1, 14, 17, 27, 28]. On facial skin, the upper part of the cheeks and the forehead are the sites commonly chosen for the assessments. The Cutometer1 SEM 575 (C + K, Cologne, Germany) is a convenient device equipped with a hand-held probe applied to the skin at constant pressure. The probe has a central suction aperture of 2–6 mm diameter. The diameter of the probe and the intensity and duration of the suction are the critical parameters influencing the results. The accuracy of measurements reaches 0.01 mm in vertical skin extension under stress. Two main operating modalities are possible. One is the fluage test using the time/strain mode (> Fig. 27.1). In this mode, for a given aperture of the probe, the choice of vacuum (from 50 to 500 mbar), the total duration of suction (stress on) and relaxation time (stress off), and the number of measurement cycles are selected. The quantitative parameters describe the elastic deformation and recovery of the skin, the viscoelastic creep after the initial deformation and the initial recovery and the residual deformation. Ue is defined as the immediate elastic distention (ED) corresponding to the steep linear part of the curve computed at a very short interval after application of the suction, usually around 0.1 s. Uv refers to the delayed viscoelastic part of the skin deformation (creep). Uf corresponds to the maximum deformation (MD) combining the elastic distention Ue and the following viscoelastic deformation Uv. Uf is computed after various time intervals ranging from 1 to 10 s. This value
Facial Skin Rheology
. Figure 27.1 Fluage test showing the relationship between the skin extension (E) and time (T). The curve shows the effect of a suction applied for 5 s followed by a release of the suction for 5 s. The immediate elastic distension (ED = Ue) is followed by a delayed viscoelastic phase Uv to reach the maximum distension (MD = Uf). The immediate elastic recovery (ER: Uf – Ur) is followed by a delayed viscoelastic phase ending with a residual distension (RD) following the Ua recovery
of maximum deformation depends on the probe aperture and the applied suction. Ur is defined as the immediate elastic recovery (ER) of the skin after removal of the suction. It is measured in the steep linear part of the recovery, mostly 0.1 s after stopping the suction. Ua is equal to the total recovery deformation of the skin at the endpoint of the recovery phase. RD is the residual deformation of the skin persisting after completion of the stress-off measuring time. All these determinations of parameters are computed by the device according to a predetermined timing of measurement. It is the investigator who makes the choice and there is no standardized procedure as yet. As a result, caution should be taken before comparing data from different studies. From the computed biomechanical parameters, different elastic and viscoelastic ratios have been proposed in order to characterize the mechanical functions of the skin: ● Ua/Uf is defined as the overall (biologic) elasticity. It is expressed in percentage, and it corresponds to the ratio of the total deformation recovery to the total deformation. ● Ur/Ue is defined as the basic elasticity ratio; it is equal to the ratio of the immediate recovery to the immediate deformation without the contribution of the viscoelastic part.
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● Ur/Uf is defined as the relative elastic recovery; it is equal to the ratio of immediate recovery to the total deformation. ● Uv/Ue is defined as the viscoelastic ratio; it is equal to the ratio of the viscoelastic deformation to elastic deformation. Under repetitive measuring cycles, the deformation versus time curves obtained for the second, third, and subsequent deformation cycles are similar to the first, but they progressively shift upwards as a consequence of the residual deformation. The differential distension (DD, m) is calculated as the difference between MDs reached at the last and the first cycles. For example, three to five load (traction) cycles of 2–5-s tractions under negative pressure of 400 mbar are separated by identical relaxation periods (> Fig. 27.2). The second modality test corresponds to the hysteresis procedure using the stress/strain mode. For example, one cycle of progressively increasing suction at a linear rate of 25 mbar/s for 10–20 s is followed by a release of the depression at the same rate (> Fig. 27.3). In this procedure, nonlinear curves are obtained. The suction curve on loading is not superposed by the relaxation curve. During the relaxation period the values of strain do not return to zero and the curve intercepts the strain axis. Hysteresis (HY) represents the area delimited by the traction and relaxation curves given by the stress/strain method. It is measured using image analysis of the graphs yielded by the time/strain method.
Viscoelastic Function of Facial Skin Using the Torque Method The torque method at low stress provides information that may be presented as being similar to that described for the suction method. However, the skin layers subjected to the stress are difficult to identify. It is claimed that the contribution of the stratum corneum to the overall mechanical functions of skin is increased using the torque method. Some equivalence in the mechanical functions of the face and the volar forearm was reported [28]. Such finding await confirmation.
Ultrasound Speed Propagation and Airblown Technique Subtle variations in tensile functions of facial skin is conveniently studied by measuring the speed of propagation
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. Figure 27.2 Repetitive fluage tests showing the effect of preconditioning the skin with a progressive increase in the skin deformation
. Figure 27.3 Hysteresis (HY) procedure showing the evolution of the stress (S) – skin extension (E) under progressive but regular suction until the maximum distension (MD) is gained followed by a progressive relaxation phase. Hysteresis represents the area between the suction and the relaxation curves
corneum suppleness/stiffness influence the data [20, 29]. It is assumed that the velocity of ultrasound propagation is affected by the orientation of the resting tension lines or Langer’s lines [20]. The airblown technique is another ingenious procedure for measuring the skin mechanical functions under low stress [30].
Physiological Changes in Mechanical Functions There is general agreement that age, gender, skin thickness, and location on the body are the four main parameters that influence the rheological functions of the skin. They have to be taken into account before interpreting any given physio-pathological process.
Age Influence
of ultrasound shear waves. The Reviscometer RVM 600 (C + K electronic, Cologne, Germany) is available for that purpose. The resonance running time measurement (RRTM) is inversely correlated with the skin stiffness. Both the dermal mechanical functions and the stratum
Cutaneous aging encompasses distinct features. Chronologic or intrinsic aging depends on genetic factors, lapse of time, and the sum of various effects of diseases and desmotropic drugs (i.e., corticosteroids, phenitoin), as well as physiologic variations and environmental influences with the exception of sun exposure. Photoaging deals with all these features to which chronic exposure
Facial Skin Rheology
to ultraviolet light and near infrared radiations are superimposed [4, 5]. Some authors regard aging of facial skin as a single and direct result of actinic insult, but this opinion may be an oversimplification. In fact, dermatoheliosis is only one aspect of variable importance among subjects of the same age and same phototype. On the face, it is superimposed to both the overall intrinsic aging process mainly responsible for tissue atrophy, and to unrelated opposite hypertrophic changes consisting of compact solar elastosis. In addition, focal hyperplasia develop in the subcutaneous connective tissue where striated muscles are anchored. Such tissue remodeling resulting from distinct origins is responsible for the progressive deepening of the natural expression lines [11]. It is clinically obvious that the mechanical functions of skin are quite different in children and the elderly [1, 7, 13, 18, 22]. They appear correlated with the skin surface patterns and wrinkling [31]. However, measuring them by different test modalities provides controversial findings regarding the nature of the changes and the moment they take place. From the available information, it is probable that the resistance of the dermis to forces exerted parallel to the skin surface increases with age at least until 60 years. Conversely, the vertical resistances at the dermo– epidermal junction, as well as within the dermis and the hypodermis progressively weaken. All age-related changes in skin obviously influence the mechanical functions. Moreover, mechanical stimuli applied to the skin throughout life affect the structure of the cutaneous tissues, which in turn modifies the mechanical functions. Such multiple interrelationships between the various structures of facial skin, the innumerable factors influencing aging, and the complexity of the mechanical properties likely preclude any clear-cut understanding of the problem. Most of the studies on rheological functions of aging skin were focused on the forearms. Little is known about corresponding changes on facial skin. It has been shown that the elevation due to suction depends on the force exerted, the body site, and the area of contact between the probe and the skin. It is particularly difficult to assess the influence of subcutaneous tethering. However, it is likely that the tests using a small hollow probe and producing small elevations of the skin are little influenced by subcutaneous attachments. In these instances, skin extensibility increases while elasticity decreases with aging of facial skin [1]. However, the bulk of elevation experiments reveals wide variations in the deformability of skin in aged individuals. Changes in the elastic rebound of the skin (BE and HY) are more constant, indicating a progressive decrease in these parameters over time.
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Gender Influence The influence of gender on the mechanical functions of skin is a subject of contradictory reports in the literature. Some consider that the skin extensibility is higher, or that the modulus of elasticity is lower, in women than in men, but the reverse opinion has been expressed as well. The difference is in part due to different test modalities.
Skin Thickness Influence The thickness of the dermis is a function of age and gender. It influences the mechanical functions of the skin [1, 32]. This contention holds true when considering the extracellular matrix of the connective tissue. However, facial skin thickness is considerably influenced by the size of the sebaceous glands. Such a double-component structure exhibits peculiar biomechanical functions. The importance of the facial skin thickness is further complicated by the variable extent in solar elastosis.
Credentiating Anti-aging Treatments During the past decades, the dermocosmetic science applied to facial skin has considerably influenced the clinical presentation of aging. Specific cosmetics, cosmeceuticals, and drugs are designed for corrective purposes. A few studies have been conducted to assess the rheological changes following the regular use of specific topical treatments [24, 33, 34]. The effects were correlated with the histological nature of wrinkles and improvement of the skin relief. Other minimally invasive methods are available for improving the appearance of aging face [35]. Among them, the filling procedures, the peelings, the botulinum toxin, the photorejuvenation, and still other nonablative resurfacing procedures are very popular. No information is available regarding the induced changes in mechanical functions of skin. The same lack of information applies to the effects of skin lifting, the lipoaugmentation, and other invasive surgical procedures. Active cosmetic products, called cosmeceuticals in some countries and quasi-drugs in others are rapidly expanding and becoming increasingly sophisticated. The potential value of these formulations for skin health is undisputable. Yet, both the consumer and the dermocosmetologist are challenged while evaluating their benefits. Many prescription dermatologics fall short of patient
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expectations, opening the way for the use of cosmeceuticals to enhance the outcome. However, the physician has some guarantee that the pharmaceuticals are at least moderately effective. When dealing with facial skin, testing the effects of products on the rheological functions of skin is difficult to predict when using the suction method on the model of the forearm skin. Only indirect and partial information is obtained. By contrast, the torque method has been reported to give equivalent information on the face and the forearms [28].
Conclusion The evaluation of the mechanical functions of the skin is useful in the dermocosmetic field of investigation. A series of different methods using dedicated devices provide different information, although those useful in practice are limited in number. The lack of standardization often precludes the comparison of results obtained by different groups of workers. In terms of bioengineering, the skin withstands and transmits mechanical forces through specific deformations. Creep and stress relaxation effects are well recognized. This fact implies that the time rate of application of forces onto the skin also influences the data while assessing the load transmitting capabilities of the tissue. The comparison between different rheological methods applicable to the face indicates that the progressive increase and release of suction in the stress/strain mode yields the greatest relative variations with age. Hence this methodological approach appears well suited to study facial skin aging and the efficacy of products aiming at its correction. Tests performed in vivo are likely to have the most value for clinical purposes, but have special problems of their own. ● The dermis in vivo is under a variable degree of resting tension. Langer’s lines are an expression of the state of resting tension in the skin and indicate the orientation of maximum tension. ● There are intimate connections between the dermis, the epidermis, and its pilosebaceous adnexae, as well as between the dermis and the hypodermis. It is virtually impossible to isolate the dermis from its intimately associated neighboring structures when tests are performed in vivo. Inevitably, the test performed are, in part, also testing the epidermal, adnexal, and hypodermal functions. It is, however, quite evident that in
most cases the results mainly reflect the properties and functions of the dermal collagen fiber bundles. The scope of these tests must be borne in mind while interpreting their results. ● Clearly, the results of mechanical testing depend on the dimensions of the site being investigated. It is relatively easy to define the length and breadth of the area tested, but the skin thickness decreases with age and is greater in men than women. It is susceptible to endocrine and environmental influences. In addition, the mechanical functions of the dermis are time dependent.
References 1. Pie´rard GE. EEMCO guidance to the in vivo assessment of tensile functional properties of the skin. Part 1: relevance to the structures and ageing of the skin and subcutaneous tissues. Skin Pharmacol Appl Skin Physiol. 1999;12:352–362. 2. Hendriks FM, Brokken D, Oomens CW, Bader DL, Baaijens FP. The relative contributions of different skin layers to the mechanical behaviour of human skin in vivo using suction experiments. Med Eng Phys. 2006;28:259–266. 3. Ryu HS, Joo HY, Kim SO, Park KC, Youn SW. Influence of age and regional differences on skin elasticity as measured by the Cutometer1. Skin Res Technol. 2008;14:354–358. 4. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008;43:629–632. 5. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 6. Ambroisine L, Ezzedine K, Elfakir A, Gardinier S, Latreille J, Mauger E, et al. Relationships between visual and tactile features and biophysical parameters in human facial skin. Skin Res Technol. 2007;13:176–183. 7. Pie´rard-Franchimont C, Paquet P, Quatresooz P, Pie´rard GE. Smoothing the mosaic subclinical melanoderma by calcipotriol. J Eur Acad Dermatol Venereol. 2007;21:657–661. 8. Akazaki S, Nakagawa H, Kazama H, Osanai O, Kawai M, Takema Y, et al. Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis. Br J Dermatol. 2002;147:689–695. 9. Batisse D, Bazin R, Baldeweck T, Querleux B, Le´veˆque JL. Influence of age on the wrinkling capacities of skin. Skin Res Technol. 2002;8:148–154. 10. Pie´rard GE, Uhoda I, Pie´rard-Franchimont, C. From skin microrelief to wrinkles. An area ripe for investigation. J Cosmet Dermatol. 2003;2:21–28. 11. Quatresooz P, Thirion L, Pie´rard-Franchimont C, Pie´rard GE. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet Sci. 2006;28:389–395. 12. Fukuda Y, Fujimura T, Moriwaski S, Kitahara T. A new method to evaluate lower eyelid sag using three-dimensional image analysis. Int J Cosmet Sci. 2005;27:283–290. 13. Saito N, Nishijima T, Fujimura T, Moriwaki S, Takema Y. Development of a new evaluation method for cheek sagging using a Moire 3D analysis system. Skin Res Technol. 2008;14:287–292.
Facial Skin Rheology 14. Pie´rard GE, Henry F, Castelli D, Ries G. Ageing and rheological properties of facial skin in women. Gerontology. 1998;44:159–161. 15. Weiss RA, McDaniel DH, Geronemus RG, Weiss MA, Beasley KL, Munavalli GM, et al. Clinical trial of a novel non-thermal LED array for reversal of photoaging: clinical, histologic, and surface profilometric results. Lasers Surg Med. 2005;36:85–91. 16. Staloff IA, Guan E, Katz S, Rafailovitch M, Sokolov A, Sokolov S. An in vivo study of the mechanical properties of facial skin and influence of aging using digital image speckle correlation. Skin Res Technol. 2008;14:127–134. 17. Pie´rard-Franchimont C, Cornil F, Dehavay J, Deleixhe-Mauhin F, Letot B, Pie´rard GE. Climacteric skin ageing of the face. A prospective longitudinal intent-to-treat trial on the effect of oral hormone replacement therapy. Maturitas. 1999;32:87–93. 18. Pie´rard GE, Kort R, Letawe C, Olemans C, Pie´rard-Franchimont C. Biomechanical assessment of photodamage. Derivation of a cutaneous extrinsic ageing score. Skin Res Technol. 1995;1:17–20. 19. Pie´rard GE, Lapie`re ChM. Microanatomy of the dermis in relation to relaxed skin tension lines and Langer’s lines. Am J Dermatopathol. 1987;9:219–224. 20. Hermanns-Leˆ T, Jonlet F, Scheen A, Pie´rard GE. Age – and body mass index-related changes in cutaneous shear wave velocity. Exp Gerontol. 2001;36:363–372. 21. Jacquet E, Josse G, Khatyr F, Garcin C. A new experimental method for measuring skin’s natural tension. Skin Res Technol. 2008;14:1–7. 22. Takema Y, Yorimoto Y, Kawai M, Imokawa G. Age-related changes in the elastic properties and thickness of human facial skin. Br J Dermatol. 1994;131:641–648. 23. Hermanns-Leˆ T, Uhoda I, Smitz S, Pie´rard GE. Skin tensile properties revisited during ageing. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 24. Pie´rard-Franchimont C, Castelli D, Van Cromphaut I, Bertin C, Ries G, Cauwenbergh G, et al. Tensile properties and contours of aging facial skin. A controlled double-blind comparative study of the effects of retinol, melibiose-lactose and their association. Skin Res Technol. 1998;4:237–243.
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25. Khatyr F, Imberdis C, Vescovo P, Varchon D, Lagarde JM. Model of the viscoelastic behaviour of skin in vivo and study of anisotropy. Skin Res Technol. 2004;10:96–103. 26. Delalleau A, Josse G, Lagarde JM, Zahouani H, Bergheau JM. A nonlinear elastic behavior to identify the mechanical parameters of human skin in vivo. Skin Res Technol. 2008;14:152–164. 27. Khatyr F, Imberdis C, Varchon D, Lagarde JM, Josse G. Measurement of the mechanical properties of the skin using the suction test. Skin Res Technol. 2006;2:24–31. 28. Bazin R, Fanchon C. Equivalence of face and volar forearm for the testing of moisturizing and firming effect of cosmetics in hydration and biomechanical studies. Int Cosmet Sci. 2006;28:453–460. 29. Xhauflaire-Uhoda E, Fontaine K, Pie´rard GE. Kinetics of moisturizing and firming effects of cosmetic formulations. Int J Cosmet Sci. 2008;30:131–138. 30. Fujimura T, Osanai O, Moriwaki S, Akazaki S, Takema Y. Development of a novel method to measure the elastic properties of skin including subcutaneous tissue: new age-related parameters and scope of application. Skin Res Technol. 2008;14:504–511. 31. Ahn S, Kim S, Lee H, Moon S, Chang I, et al. Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity. Skin Res Technol. 2007;13:280–284. 32. Smalls LK, Wickett RR, Visscher MO. Effect of dermal thickness, tissue composition, and body site on skin biomechanical properties. Skin Res Technol. 2006;12:43–49. 33. Pie´rard GE, Henry F, Pie´rard-Franchimont C. Comparative effect of short-time topical tretinoin and glycolic acid on mechanical properties of photodamaged facial skin in HRT-treated menopausal women. Maturitas. 1996;23:273–277. 34. Uhoda I, Faska N, Robert C, Cauwenbergh G, Pie´rard GE. Split face study of the cutaneous tensile effect of a 2-dimethylaminoethanol (deanol) gel. Skin Res Technol. 2002;8:164–167. 35. Bogle MA. Minimally invasive techniques for improving the appearance of the aging face. Expert Rev Dermatol. 2007;2:427–435.
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32 Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis Satoshi Amano
Introduction Skin aging is classified into two types, intrinsic aging and photoaging. Intrinsic aging is a basic biological process common to all living things, and can be characterized as age-dependent deterioration of skin functions and structures, such as epidermal atrophy and epidermal–dermal junction flattening [1]. Histologically, intrinsically aged skin has an atrophied extracellular matrix with a reduced amount of elastin [2]. On the other hand, photoaging is well known to be a consequence of chronic exposure to sunlight. Sun-exposed skin, such as the skin on the face or neck, is apparently prematurely aged compared with the relatively sun-protected skin of the trunk, and is characterized by various clinical features, including wrinkles, sagging, roughness, sallowness, pigmentary changes, telangiectasia, and neoplasia [3, 4], and histological features of sun-exposed skin including cellular atypia, loss of polarity, epidermal–dermal junction flattening, a decrease in collagen, and dermal elastosis, with abnormal deposition of elastotic material in the dermis [1, 5]. Damage to skin collagen and elastin (extracellular matrix) is the hallmark of long-term exposure to solar ultraviolet irradiation, and is believed to be responsible for the wrinkled appearance of sun-exposed skin [5]. The fibulin gene family comprises five distinct genes that encode more than eight protein products via alternative splicing [6]. Fibulins are widely expressed secreted proteins found in the blood and in the basement membranes and stroma of most tissues, where they self-associate [7, 8], and/or interact with a variety of extracellular matrix components, including fibronectin, laminin, nidogen, aggrecan, versican, endostatin, fibrillin, and elastin [6, 9–11]. Fibulins are thought to be involved in the assembly and stabilization of extracellular matrix structures, and have also been implicated in regulating organogenesis, vasculogenesis, fibrogenesis, and tumorigenesis [12–14].
The newest member of the fibulin family is fibulin-5 (known as EVEC [15] or DANCE [16]), a 448-amino acid glycoprotein with interesting structural features; it contains an integrin-binding RGD motif, six calcium-binding epidermal growth factor-like repeats, a Pro-rich insert in the first calcium-binding epidermal growth factor-like repeat, and a globular C-terminal domain [15, 16]. Functionally, fibulin-5 binds avb3, avb3, and a9b1 integrins [10], and mediates endothelial cell adhesion via its RGD motif [16]. Inactivation of the fibulin-5 gene in mice, produces profound elastinopathy in the skin, lung, and vasculature [10, 11]. In humans, mutations in fibulin-5 have been found to cause cutis laxa [17]. Elastic fibers are composed of an amorphous elastin core surrounded by a peripheral mantle of microfibrils. Soluble tropoelastin monomers are polymerized and crosslinked to form insoluble elastin, which is essential for the assembly of elastic fibers [18]. However, self-association of tropoelastin monomers alone is not sufficient to form elastic fibers indicating the need for other processes or substances. Microfibrils are 10–12 nm filaments in the extracellular matrices, and composed of many proteins such as fibrillin-1 and -2 [19–21], microfibril-associated glycoproteins (MAGPs) [22, 23], and latent transforming growth factor b-binding proteins (LTBPs) [24, 25]. Microfibrils are considered to provide a scaffold for the polymerization of elastin, and play an essential role in elastogenesis. The aim of this study was to explore the changes of elastic fibers during the skin aging process. Fibulin-5 expression in both normal and actinically damaged skin when compared with expression of elastin, fibulin-2, and fibrillin-1 showed that fibulin-5 decreased in an age-dependent manner in the reticular dermis, and the reduction was enhanced by UVB irradiation. Moreover, acute UVB irradiation markedly reduced fibulin-5. However, fibulin-5 was also found to accumulate in solar
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_32, # Springer-Verlag Berlin Heidelberg 2010
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elastosis, together with other elastic fiber components, such as elastin, fibulin-2, and fibrillin-1. The results indicated that fibulin-5 is an early marker of skin aging, and that the early loss of fibulin-5 in the dermis may prefigure the age-dependent reduction of other elastic fiber components.
Localization of Fibulin-5 in the Dermis of Young Human Sun-Protected Skin In the reticular dermis of young sun-protected skin from the upper arm of a 17-year-old female, fibulin-5 was colocalized with other elastic fiber components, such as elastin, fibrillin-1, and fibulin-2 (> Fig. 32.1a–d). In contrast with fibrillin-1, fibrillin-2 was not detected in skin from children and adults (data not shown). In the papillary dermis, fibulin-5 showed candelabra-like structures perpendicular to the epidermis, resembling those of the other elastic fiber components (> Fig. 32.1a–d). In areas just beneath the epidermis, fibulin-5 does not go up to the dermal–epidermal junction like fibrillin-1 which was observed there as fiber structures that may be inserted into the epidermal basement membrane [26, 27] (> Fig. 32.1b, c). The fiber structure of fibulin-2 was not as sharp in the papillary dermis (> Fig. 32.1a) as those of elastin, fibrillin-1, or fibulin-5 (> Fig. 32.1a–c), although it was clearer in the reticular dermis (> Fig. 32.1d).
Age-Dependent Changes of Fibulin-5 Distribution in the Dermis of SunProtected or Sun-Exposed Skin In the dermis of sun-protected thigh skin from 5-, 13-, and 16-year-old subjects, fibulin-5 showed candelabralike structures in the papillary dermis, and was associated with elastic fibers composed of other elastic fiber components, such as elastin, fibrillin-1, and fibulin-2 (> Fig. 32.2a–l) in the reticular dermis. However, the staining intensity of fibulin-5 (> Fig. 32.2n) was reduced as compared with that of the other elastic fiber components (> Fig. 32.2m, o, and p) in the reticular dermis of skin from a 34-year-old subject. Fibulin-5 (> Fig. 32.2r) was almost absent in the reticular dermis of skin from a 75-year-old women. Similarly, fibulin-5 associated with elastic fibers was reduced in the reticular dermis of sun-protected upper arm skin from a 36-year-old subject as compared with those from 11-, 17-, and 24-year-old subjects (data not shown). Moreover, fibulin-5 associated
with elastic fibers was markedly reduced as compared with fibrillin-1 and fibulin-2 in the reticular dermis of abdomen skin from 34- and 75-year-old subjects (data not shown). On the other hand, in the papillary dermis, fibulin-5 maintained its staining intensity, although the number of stained fibers seemed to be reduced with age (> Fig. 32.2n, r), whereas elastin was age-dependently reduced much more markedly in papillary dermis than in reticular dermis (> Fig. 32.2m, q). In sun-exposed skin, fibulin-5 was mostly lost in the dermis of cheek skin even from 20- and 45-year-old subjects (> Fig. 32.3b, f ), and this change occurred much earlier than that in sun-protected skin (> Fig. 32.2). Elastic fiber structures in the dermis of 45-year-old skin (> Fig. 32.3e, g, and h) appeared to be thicker than those of 20-year-old skin (> Fig. 32.3a, c, and d), and was intermediate in pattern between the 20-year-old skin, and the elastic fibers observed in the dermis of a 76-year-old subject (> Fig. 32.3i, k, and j), suggesting that the 45-year-old skin may be progressing to solar elastosis. However, while increased deposition of fibulin-5 was observed in solar elastosis (> Fig. 32.3j), as was observed for other elastic fiber components, fibulin-5 decreased with age in sun-exposed skin before solar elastosis appeared (> Fig. 32.3b, f ). Fibulin-5-deficient mice were reported to develop marked elastinopathy owing to the disorganization of elastic fibers, resulting in loose skin, vascular abnormalities, and emphysematous lung [10, 11]. Since fibulin-5 has an integrin-binding N-terminal domain, fibulin-5 is thought to stabilize the attachment of cells to elastic fibers, and to contribute to the organization of elastic fibers [10]. Recently, fibulin-5 is reported to be a key protein for the induction of elastic fiber formation and full intact form of fibulin-5 diminishes with age [28]. Therefore the loss of fibulin-5 may decrease the stability of elastic fibers by disturbing the interactions between dermal cells and elastic fibers or among elastic fiber components, and may contribute to the atrophy of elastic fibers during aging. The hallmark of actinic damage of the skin changes is associated with deposition of elastotic materials in the dermis [1]. Previous immunohistochemical studies reported an increased deposition of elastin, versican, hyaluronic acid, fibrillin, and fibulin-2 in areas of solar elastosis [29–31]. Fibulin-5 deposition also increases in solar elastosis. The mechanism of the increase in the expression of these elastic fiber components, leading to abnormal deposition in the dermis of actinically damaged skin, remains unknown. However, since all elastic fiber components, including fibulin-5, are increased in solar
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
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. Figure 32.1 Localization of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of young human sun-protected skin. The localization of elastin (a and e), fibulin-5 (b and f), fibrillin-1 (c and g) or fibulin-2 (d and h) in sun-protected skin from upper arm of a 17-year-old female was examined by means of immunohistochemistry using specific antibodies
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. Figure 32.2 Age-dependent changes of expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of sun-protected skins. The localization of elastin (a, e, i, m, and q), fibulin-5 (b, f, j, n, and r), fibrillin-1 (c, g, k, o, and s) or fibulin-2 (d, h, l, p, and t) in the dermis of sun-protected thigh skin was examined by means of immunohistochemistry using specific antibodies. Age-dependent changes of the elastic fiber components were examined in the dermis of skins from subjects in the age range from 5 to 75 year old. It should be noted that fibulin-5 decreased especially markedly in the reticular dermis with aging (Image reproduced, with permission, from Kadoya et al. [35]
elastosis, the mechanism may activate the elastic fiber developmental program. Since fibulin-5 was observed to be extremely reduced in the aging dermis, it is possible that the control of gene expression, protein synthesis, or
deposition of fibulin-5 may be different from those of other elastic fiber components. Further studies are needed to clarify the role of fibulin-5 in normal aging and in the pathogenesis of solar elastosis.
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
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. Figure 32.3 Age-dependent changes of expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of sun-exposed skin. The localization of elastin (a, e, and i), fibulin-5 (b, f, and j), fibrillin-1 (c, g, and k) or fibulin-2 (d, h, and l) in the dermis of sun-exposed cheek skins was examined by means of immunohistochemistry using specific antibodies, as described in Materials and Methods. Age-dependent changes of the elastic fiber components were examined in the dermis of skin from subjects in the age range from 20 to 76 year old. It should be noted that the reduction of fibulin-5 in the reticular dermis occurred earlier in sun-exposed skin than in sun-protected skin. Increased deposition of fibulin-5, as well as the other elastic fiber components, was observed in solar elastosis (Image reproduced, with permission, from Kadoya et al. [35]
Reduction of Fibulin-5 in the Dermis After UVB Irradiation Since fibulin-5 in the dermis was reduced in sun-exposed skin earlier than that in sun-protected skin, the effect of UVB irradiation on the fibulin-5 distribution in buttock skin from two male volunteers was explored. A single UVB irradiation at 2 MED decreased fibulin-5 (> Fig. 32.4i, j), fibulin-2 (> Fig. 32.4k, l ), and elastin (> Fig. 32.4g, h) levels in the dermis markedly, moderately, and weakly, respectively, as compared with those in non treated skin (> Fig. 32.4a–f ). Fibulin-5 deposition decreased much earlier in sunexposed skins than in sun-protected skins. Furthermore, UVB-irradiation induced the degradation of fibulin-5 deposited in the dermis. Matrix-degrading metalloproteinase messenger RNAs, proteins and activities are known to be induced in human skin in vivo within hours of
exposure to UVB irradiation, and may degrade collagen and elastin in skin [32]. Fibulin-2 was reported to be degraded by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leucocyte elastase, and mast cell chymase [33]. Smaller degradation products of fibulin-2 were also detected in actinic elastosis, presumably reflecting increased proteinase activity in photodamaged skin [31]. The sensitivity of fibulin-5 to proteinases has not yet been reported, but proteinases induced by the exposure of skin to UV may be involved in the early loss of fibulin-5 observed in the dermis.
Conclusion Fibulin-5 content in the reticular dermis decreases with age, and decreases earlier than other elastic fiber
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. Figure 32.4 Reduction of fibulin-5 in the dermis after UVB irradiation. The localization of elastin (a, b, g, and h), fibulin-5 (c, d, I, and j), or fibulin-2 (e, f, k, and i) in the dermis of buttock skin was compared between non treated sites (a–f) and UVB-irradiated sites (g–l) by means of immunohistochemistry using specific antibodies. It should be noted that fibulin-5 was markedly reduced in the dermis by UVB-irradiation (Image reproduced, with permission, from Br J Dermatol. 2005;153(3):607–612)
components, such as elastin, fibrillin-1, and fibulin-2. The reduction of fibulin-5 was enhanced by UVB exposure, and occurred in sun-exposed skin much earlier than in sun-protected skin. Therefore, UVB is likely to be one of the major factors causing impairment of elastic fibers during aging, and the early loss of fibulin-5 may signal
the later changes of elastic fibers during aging, especially photoaging. Therefore, fibulin-5 is proposed to be a good marker of skin aging, especially photoaging. Interestingly, fibulin-5 deposition is enhanced in solar elastosis, suggesting that solar elastosis involves the global activation of genes for elastic fiber components.
Fibulin-5 Deposition in Human Skin: Decrease with Aging and UVB Exposure and Increase in Solar Elastosis
Fibulin-5-overexpressing cells enhanced the assembly of elastic fibers in cultured normal human dermal fibroblasts, suggesting that fibulin-5 was an important microfibril constituent for the assembly of elastic fibers [34]. Thus, fibulin-5 may be a potential target to prevent or delay the deterioration of elastic fibers during the skinaging process in human.
Cross-references > DNA
Damage and Repair in Skin Aging
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43 Global Warming and its Dermatologic Impact on Aging Skin Young Hui . Haw-Yueh Thong . Howard I. Maibach
Introduction The large-scale impacts of global warming are widely debated in both mainstream and academic literatures [1], but the specific consequences of global warming for individual scientific disciplines are poorly discussed. Many scientific disciplines are yet to plan for global warming; not only for changing temperature gradations, but also for the effects on plant, animal, and man. It is suspected that global climate change will alter skin – in ways not yet determined. Little research has been done to assess the challenges that dermatology might face in the future in the light of global warming; much less research has been done in the area of skin care for old people, but such studies can nevertheless be extrapolated from some upcoming trends.
Ultraviolet Effects Decades of ozone depletion have focused scrutiny on the effects of increased ultraviolet (UV) exposure. While not a direct consequence of global warming, increased UV exposure may result from factors such as cloud cover and aerosols [2]. Warmer temperatures also appear to increase the incidence of skin cancer from UV. Studies suggest that a rise of just 2 C may increase the rates of such cancer by up to 10% [3]. As people retire and temperatures rise, more may seek recreation outdoors. Not only the intensity, but also the duration of UV that people experience will potentially increase. Beyond the increase in UV penetration, such changes in social habits may be the greatest factor in future skin cancer rates [4, 5].
The Hot Zone: Skin Infections A shift in temperature would change the dynamics of vector-borne diseases, such as malaria and mosquitoes or leishmaniasis and sand flies [6–9]. Cases of leishmaniasis,
in particular, increased during the warming phase of El Nino and decreased during the cooling phase of La Nina in Columbia [10]. Tick-borne diseases, susceptible to climate change, have also increased [11]. But current data cannot prove a causal relationship for either of these cases. To give a convincing proof one requires more detailed and direct evidence of climate change impacts. The impact of global warming on pathogen outbreaks appears more definite. The Monteverde harlequin frog was driven to extinction by outbreaks of the pathogenic chytrid fungus Batrachochytrium, which occurred when highland locales warmed [12]. Similar outbreaks among humans have not yet been witnessed, but the increased proclivity of Staphylococcus, Streptococcus, and enteric bacteria to colonize humans in warmer climes has been observed [13, 14]. Other research has also suggested that increased temperature and humidity generally favor bacterial growth [15]. Such challenges await dermatologists as humans venture into the fires of global warming. Pathogen outbreaks, with inherent potential for antimicrobial resistance, will stare at dermatologists if global warming proceeds. Those who are most at risk, such as the elderly or infirm, should be advised most of all.
Worsening Weather for Dermatologists Global warming may change the patterns of severe weather events. These changes would generate wide effects upon public health and disease transmission. This broader transmission can only be detrimental to a vulnerable, aging society. If global warming will bring more extreme weather in the future, it may be informative to examine recent weather for indications as to the future trends in the weather. Hurricane Katrina brought a deluge of skin infection reports. The Centers for Disease Control and Prevention (CDC) highlighted methicillin-resistant Staphylococcus aureus (MRSA), Vibrio vulnificus, and V. parahaemolyticus infections among the rescued, and tinea corporis, folliculitis, miliaria, and arthropod bites among rescuers [16].
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Septic shock was reported among victims with V. vulnificus [17]. The Indian Ocean tsunami of December 2004 is another case to consider. Although the cause of this calamity may not be global warming, it still carries consequences similar to any massive precipitation event. A sampling of survivors showed 515 (66.3%) of them suffering from skin or soft-tissue infection [18]. Flooding contaminated freshwater with a variety of exotic pathogens, as well as multidrug-resistant bacteria and polymicrobial infections [19–21]. Such challenges are faced by all rescue efforts and doctors should familiarize themselves with those challenges if global warming leads to more occasions for rescue work.
Conclusion The peril of global warming, if it comes to pass, is dangerously clear. It is evident why the reduction of greenhouse gases has become a top social and political priority. Its impact on the biodiversity of the Earth may be tremendous; some predict over a million extinctions of animal and plant species by the middle of this century [22]. While in all likelihood humanity will not be among them, the potential toll on humanity remains immense. Health professionals should be prepared for worst-case scenarios; thus, it is imperative for all, including dermatologists, to investigate and ready themselves for likely future challenges. At this time, what is needed to act is knowledge of what will be faced. Taken together, one or more global-oriented research institutes dedicated to understanding global warming’s skin-related issues should be initiated to define the extent of the problem and create prophylactic and therapeutic interventions. Time does not wait; so action has to begin now.
Cross-references > Effect
of Ozone on Cutaneous Tissues Vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model > Skin Photodamage Prevention: State of the Art and New Prospects > In
References 1. Flannery T. The Weather Makers: How Man is Changing the Climate and What It Means for Life on Earth. New York: Atlantic Monthly Press, 2006.
2. Kerr JB, Seckmeyer G. Surface ultraviolet radiation: past and future. In: Scientific Assessment of Ozone Depletion, Geneva: Global Ozone Research and Monitoring Project, 2002. 3. van der Leun JC, de Gruijl FR. Climate change and skin cancer. Photochem Photobiol Biol Sci. 2002;1:324–326. 4. Diffey BL. Human exposure to ultraviolet radiation. In: Hawk JLM (ed) Photodermatology. London: Arnold, 1999, pp. 5–24. 5. Diffey BL. Climate change, ozone depletion and the impact of ultraviolet exposure on human skin. Phys Med Biol. 2004;49: R1–R11. 6. Kolodynski J, Malinowska A. Impacts of climate change on infectious diseases. Wiad Parazytol. 2002;48:29–37. 7. Kovats RS, Campbell-Lendrum DH. McMichael AJ, et al. Early effects of climate change: do they include changes in vector-borne disease. Philos Trans R Soc Lond B Biol Sci. 2001;356:1057–1068. 8. Bormane A, Lucenko I, Duks A, et al. Vectors of tickborne diseases and epidemiological situation in Latvia in 1993–2002. Int J Med Microbiol. 2004;293(Suppl 37): 36–47. 9. Sutherst RW. Global change and human vulnerability to vectorborne diseases. Clin Microbiol Rev. 2004;17:136–173. 10. Cardenas R, Sandoval CM. Rodriguez-Morales AJ, et al. Impact of climate variability on the occurrence of leishmaniasis in Northeastern Colombia. Am J Trop Med Hyg. 2006;75:273–277. 11. Randolph SE. Evidence that climate change has caused ‘‘emergence’’ of tick-borne diseases in Europe? Int J Med Microbiol. 2004;293: (Suppl 37): 5–15. 12. Pounds JA, Bustamante MR, Coloma LA, et al. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature. 2006;439:161–167. 13. Taplin D, Lansdell L, Allen AM, et al. Prevalence of streptococcal pyoderma in relation to climate and hygiene. Lancet. 1973; 1:501–503. 14. Yildirim A, Erdem H, Kilic S, et al. Effect of climate on the bacteriology of chronic suppurative otitis media. Ann Otol Rhinol Laryngol. 2005;114:652–655. 15. McBride ME, Duncan WC, Knox JM. Physiological and environmental control of Gram negative bacteria on skin. Br J Dermatol. 1975;93:191–199. 16. Centers for Disease Control and Prevention (CDC). Infectious disease and dermatologic conditions in evacuees and rescue workers after Hurricane Katrina – multiple states, August–September, 2005. MMWR Morb Mortal Wkly Rep. 2005;54:961–964. 17. Rhoads J. Post-Hurricane Katrina challenge: Vibrio vulnificus. J Am Acad Nurse Pract. 2006;18:318–324. 18. Hiransuthikul N, Tantisiriwat W, Lertutsahakul K. Skin and softtissue infections among tsunami survivors in southern Thailand. Clin Infect Dis. 2005;41:e93–96. 19. Garbino J, Garzoni C. Unusual pathogens and multidrugresistant bacteria in tsunami survivors. Clin Infect Dis. 2006;42:889–890. 20. Nieminen T, Vaara M. Burkholderia pseudomallei infections in Finnish tourists injured by the December 2004 tsunami in Thailand. Euro Surveill. 2005;10:E050303.4. 21. Petrini B, Farnebo F, Hedblad MA, et al. Concomitant late soft tissue infections by Cladophialophora bantiana and Mycobacterium abscessus following tsunami injuries. Med Mycol. 2006;44:189–192. 22. Thomas CD, Cameron A, Green RE, et al. Extinction risk from climate change. Nature. 2004;427:145–148.
2 Histology of Microvascular Aging of Human Skin Peter Helmbold
Introduction In this chapter, various histological studies regarding the role of pericytes in the dermis will be summarized, focusing on dermal microvascular aging [1–4]. Aging of the dermis proceeds under special conditions. In addition to chronological aging, a powerful extrinsic factor – chronic UV light – leads to photo-aging (actinic or solar aging). Some known facultative intrinsic or extrinsic factors that influence dermal aging include diabetes mellitus, alcohol, cigarette smoking, and genodermatoses like progeria [5–8]. Previous studies have shown that human dermal microvessel densities depend on age with reduction of functioning reserve capillaries, and there are typical ultrastructural changes in the microvasculature of elderly individuals [5, 6, 9]. Most efforts in microvascular research focus on endothelial cells (EC). By contrast, progress in knowledge on PC which cover microvascular capillaries and venules on their abluminal surfaces has been slow. In the microvasculature, EC and PC are anatomical and functional neighbors. They are separated from each other by the EC basal lamina, which allows punctate direct contact and interdigitation [10]. Endothelin-1 [11] and vascular endothelial growth factors are thought to be the most important cytokines responsible for the interaction of the two cell types [11–13]. Pericytes have contractile function and they are thought to regulate local blood flow [14]. Moreover, they are essential for microvessel stability and control of angioneogenesis [15, 16]. Pericytes are involved in the pathogenesis of diabetic microangiopathy [17, 18], hypertension [19], tumor growth [20], and retinopathy of prematurity [21]. In the skin, PC hyperplasia has been reported in chronic venous insufficiency and in scleroderma [22, 23]. Because of methodological difficulties, most of the dermatological research performed on PC was restricted to ultrastructural or unspecific identification of this cell type by their smooth muscle actin expression [10, 18, 24–26]. One of the most striking methodological problems in this field was identification and counting of a sufficient
number of PC and EC in dermal microvessels. Ultrastructurally, 90–130 ultra-thin sections are required for the reconstruction of one vessel segment with one to four PC [27]. Thus, two methods for identification of cutaneous PC and EC were recently developed: a direct but relatively expensive technique that allows identification of PC and EC nuclei in cryosections by 3G5 antigen and von Willebrand factor expression, and an indirect method that uses identification of PC and EC nuclei according to their anatomical relationship with the collagen IV-positive microvascular basal lamina [2, 22]. The indirect technique in particular allows rapid identification of all key microvascular parameters that were used in this study. From these studies, it can be concluded that the PC/EC ratio is a crucial ‘‘functional-morphological’’ parameter in the dermal microvasculature [22]. In the first study, 120 biopsies from normal skin of 87 patients were obtained from surplus areas (i.e., Burow’s triangle) of routinely excised and histologically controlled benign nevus cell nevi of normal skin (it was previously verified that PC numbers or total microvascular counts were not influenced by non-inflammatory nevus cell nevi, unpublished results). Biopsies with inflammatory cells (infiltrated nevus cell nevi) or histological conditions other than normal skin, and biopsies from patients with known vasculopathies were strictly excluded from the study. To eliminate the influence of latent venous insufficiency, skin samples from the lower legs of patients older than 14 years were generally excluded. Known vascular diseases and diabetes mellitus were additional exclusion criteria. Each specimen was characterized by a set of clinical data: age, sex of the patient, and body location of the biopsy. Further methods are stated in [1]. The relative number of capillaries and venules as well as PC/EC ratios were counted in the upper horizontal dermal plexus including papillar and the upper reticular dermis in collagen IV stained paraffin sections (hematoxylin counterstained) as reported previously [22] (> Fig. 2.1a, b). In short, intra-luminal nuclei (within the lumen that is surrounded by the inner layer of the microvascular basal lamina) were ascribed to EC. By contrast, nuclei found between the two
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layers of microvascular basal lamina were ascribed to PC. Segmented nuclei or nuclei without contact with the basal lamina were excluded. Only clearly recognizable cross or longitudinal sections with unequivocal assignment of the nuclei to the cell types to be determined were selected for examination (> Fig. 2.1a, b). The mean coefficient of variation of this method was 5.7 3.9% [22]. In another experimental study, the ratio PC/EC was studied in clearly cross- or longitudinal-sectioned capillaries by co-localization analysis of 3G5 or vWF binding sites with nuclei in crysoctions by triple-staining with anti-3G5 (a pericyte marker), anti-von Willebrand factor (endothelial marker), and DNA fluorochrome (Hoechst 33258, Sigma) as reported before (> Fig. 2.1c, d) [2, 22]. In a third study part, different TGFs, VEGFs, and PDGFreceptors in the upper dermal plexus were studied in paraffin sections (> Fig. 2.1e, f).
Results and Discussion Two periods of vascular aging – childhood and adulthood. Densities of capillaries and venules in the upper dermal plexus showed dramatic decrease during childhood and slow decrease during adulthood. Results showed a mean of 4.9 2.8 capillaries per HPF and 2.4 1.4 venules per HPF. The density of capillaries was highly negatively dependent on chronological age (r = 0.572, p < 0.001) (> Figs. 2.1a, b and > 2.2). In young children (0–4.99 years) capillary density was 9.7 2.9 per HPF decreasing with adolescence to 4.4 1.5 (15–19.99 years). Thereafter, there was further slow decrease to 2.3 1.4 (range 0.7–5.8) in the age group 70 + years. By contrast, the density of venules showed no significant change during life. At higher ages, the densities of capillaries and venules were comparable. Pericyte loss during childhood and adult chronological life. Mean PC/EC ratios were 0.125 0.054 and 0.132 0.067 in the capillaries and venules, respectively. There was a negative correlation between chronological age and PC/EC ratio of the capillaries (r = 0.560, p < 0.001) or venules (r = 0.594, p < 0.001). Similar to capillary density, the most dramatic changes occurred during adolescence (> Fig. 2.3). In the youngest group (0–4.99 years), PC/EC was twice that in the age group 15–19.99 years, Thereafter, no significant correlation of PC/EC ratio and chronological age was detectable. Studies of the area-based densities of PC and EC showed that the values for PC were highly correlated to the PC/EC ratio, while there was no correlation between total EC counts and PC/EC ratio. It can be concluded that only life-time changes of absolute PC densities (but not changes of EC densities) are responsible for age-dependent
decrease in PC/EC ratio. Fluorescence microscopy analysis of PC and EC distribution by anti-pericyte, anti-endothelial antibodies, and DNA fluorochrome (details see above) brought similar results to those shown in the paraffin imbedded material [1]. Photo-aging. Body regions that reflect typical actinic exposure (photo-aging) showed a negative correlation to each of the key microvascular parameters (capillary density: Spearman r = 0.203, p = 0.039; capillary PC/EC ratio r = 0.242, p = 0.042; venular PC/EC ratio r = 0.255, p = 0.010). The effect of photo-aging was more clearly demonstrable by a newly introduced technique, the histological scoring of dermal basophilic degeneration (DBD, see Chapter 99). The influence of DBD on key microvascular parameters was studied in 84 biopsies of normal skin of subjects 15 years or older. In connection with chronological aging, additional actinic aging could be demonstrated: the capillary density and the PC/EC ratios of the capillaries or venules showed clear diminution with the degree of DBD – a significant photoeffect that is ‘‘added’’ individually to the chronological aging (> Fig. 2.4). Logistic regression demonstrated that PC/EC ratio of the capillaries and venules was predicted by DBD, and, in contrast to younger ages, the chronological age had only weak independent influence in any subjects 15 years or older [1]. Study of TGF-b, PDGFR, and VEGF expressions showed that there was a correlation between microvascular TGF-b1 expression and the PC/EC ratios of capillaries or venules (Spearman r = 0.583, p = 0.006, or r = 0.857, p < 0.001) (> Figs. 2.1e, f and > 2.5), but there was no correlation between the microvascular expression of TGF-b2 and the anatomical parameters of the microvessels. Constitutive microvascular PDGFR-a and -b as well as VEGF expressions were very low and not correlated with any of the microvascular anatomical parameters. In summary, two key parameters of microvasular aging were identified: capillary density and the quantitative ratio of pericytes and endothelial cells (PC/EC ratio). During the first 15 years of life, the number of capillaries of the upper dermis and the PC/EC ratio of the capillaries and venules decrease dramatically by nearly one half. This is called juvenile aging, which might be a process of maturation. Obviously, this maturation is finished with the end of longitudinal body growth. Nevertheless, decrease in capacity of wound healing at the same time advocates classification of this process as an early aging process. This would explain the known deceleration of wound healing and angioneogenesis as well as the reduction of local microvascular reactivity due to aging of the skin by reduction of PC-dependent microvascular
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. Figure 2.1 Age dependency of microvascular density, pericyte-to-endothelial cell ratio (PC/EC), and microvascular TGF-b1 expression Left column (a, c, e), younger; right column (b, d, f), older skin biopsies. (a) Nuclei of microvascular pericytes (arrows) are identifiable between the two layers of the collagen IV-positive microvascular basal lamina (red) in a collagen IV/hemalaun stained paraffin section of a biopsy of a 21-month-old girl (7.0 capillaries per HPF, PC/EC ratio 0.345). (b) Biopsy from the de´collete´ of a 30-year-old woman with low capillary and pericyte densities (3.1 capillaries per HPF, PC/EC ratio 0.081). (c) Fluorescence microphotograph of pericytic surface 3G5 mAB-binding sites (red), endothelial cell von Willebrand factor (green), and DNA fluorochrome (blue). The figure shows a venular capillary of the upper dermis of a 5-year-old boy with almost complete covering of endothelial cells by pericytes (10.75 capillaries per HPF, PC/EC ratio 0.250). (d) By contrast, a capillary of the upper dermal plexus of a thoracic biopsy of a 31-year-old woman demonstrates sparse PC density (4.25 capillaries per HPF, PC/EC ratio 0.075). (e) High number of microvascular cells express cytoplasmatic TGF-ß1 (arrows) in a PC-rich biopsy of a 7-year-old boy (7.1 capillaries per HPF, PC/EC ratio 0.191). (f) By contrast, a biopsy from the back of a 20-year-old woman with low microvascular and PC densities is lacking microvascular TGF-ß1 (3.5 capillaries per HPF, PC/EC ratio 0.121). The epidermis serves for intrinsic positive control. Scale bar: A, B = 50 mm, C, D = 15 mm, E = 100 mm, F = 200 mm. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
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. Figure 2.2 Age dependency of the densities of capillaries and venules in the upper dermal plexus age groups: ‘‘0–4’’ means 0–4.99 years etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
. Figure 2.3 Age dependency of the pericyte-to-endothelial cell ratios (PC/EC) of the capillaries and venules of the upper dermal plexus Age groups: ‘‘0–4’’ means 0–4.99 years etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
angioneogenic plasticity and functional loss of physiological microvascular contractility [28–30]. Thereafter, chronological aging alone has a comparatively low influence on both parameters. However, after puberty, the microvascular parameters are modified severely by photo-aging, resulting in further decrease of capillary densities and PC/EC ratios. Regarding the capillary densities, this is consistent with previous
investigations showing higher influence of photo-aging than chronological aging on the upper dermal microvasular plexus during adult life [5, 6, 9]. In summary, two phases of microvascular aging can be postulated in human dermis: a juvenile phase finished by the onset of puberty, when an ‘‘adult plateau’’ is reached, and an adult phase that highly reflects photo-aging with interindividual sunexposure-specific alterations.
Histology of Microvascular Aging of Human Skin
. Figure 2.4 PC/EC ratios of capillaries and venules of the upper dermal plexus in the context of basophilic degeneration, an indicator of photo-aging Age groups: ‘‘0–4’’ means 0–4.99 years, etc. (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
Most important, PC loss, but not changes in EC density, is the cause of the changes in the PC/EC ratio in both the juvenile and the photo-aging of microvessels. PC express several cytokines, particularly TGF-b [31, 32]. The classical members of the TGF-b family belong to a much larger group. In humans, this family consists of almost 30 members, including bone morphogenic proteins, activins, and Mullerian inhibiting substance [33]. These TGF-b family members have effects during development, affect proliferation, differentiation, and cell death, and are important for the development of many tissues. Dermal TGF-b was constitutively active in and around microvessels. A correlation was found between microvascular TGF-b1 expression and the PC/EC ratio. This agrees with previous papers hypothesizing that PCs are the main source of constitutional TGF-b expression within the microvasculature [22,34].
Conclusion Thus, it is concluded that TGF-b1 expression reflects the functional state of the microvessels. TGF-b has different
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. Figure 2.5 Microvascular TGF-b1 expression and EC/PC ratios of the capillaries and venules of the upper dermal plexus White, capillaries; gray, venules. Level 1 and 2 expressions were summarized to ‘‘positive’’ (pos.), and level 0 is represented in the figure as ‘‘negative’’ (neg.). (Published in Helmbold et al. [1]. Reprinted with permission of J Invest Dermatol)
effects on microvessels: it inhibits proliferation and migration of endothelial cells, stimulates in-vivo angiogenesis in the presence of an inflammatory response and increases the stability of blood vessels. Furthermore, it has great impact on fibroblasts and connective tissue through chemotaxis of monocytes and fibroblasts, supporting anchorage-independent growth of fibroblasts, production of antiproteolytic activity via modulation of uPA/PAI-1 expression levels, inhibition of the production of proteases, and stimulation of the production of protease inhibitors [data reviewed [34]]. Thus, results on microvascular TGF-b expression link the relative and absolute absence of microvascular PC in adult and photo-aged skin to proteolytic degradation of dermal connective tissue and reduction of fibroblast function. PC loss might be crucial for dermal connective fiber aging. In contrast to TGFs, significant expression of PDGFR and VEGF seems to be limited to (neo)angiogenesis or cell proliferation, respectively. There was no evidence for the necessity of these cytokines for physiological microvessel maintenance in normal skin.
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Cross-references > Basophilic
(Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photoaging
References 1. Helmbold P, Lautenschlager C, Marsch W, et al. Detection of a physiological juvenile phase and the central role of pericytes in human dermal microvascular aging. J Invest Dermatol. 2006;126:1419–1421. 2. Helmbold P, Wohlrab J, Marsch WC, et al. Human dermal pericytes express 3G5 ganglioside – a new approach for microvessel histology in the skin. J Cutan Pathol. 2001;28:206–210. 3. Helmbold P, Fiedler E, Fischer M, et al. Hyperplasia of dermal microvascular pericytes in scleroderma. J Cutan Pathol. 2004;31: 431–440. 4. Helmbold P. Methodische Grundlagen zur Erforschung von Perizyten der Haut. In: Medizinische Fakulta¨t. Halle (Saale): MartinLuther-Universita¨t Halle – Wittenberg. 2002. 5. Braverman IM. Elastic fiber and microvascular abnormalities in aging skin. Clin Geriatr Med. 1989;5:69–90. 6. Korkushko OV, Sarkisov KG. Age-specific characteristics of microcirculation in middle-and old age. Kardiologiia. 1976;16:19–25. 7. Herrick AL, Moore T, Hollis S, et al. The influence of age on nailfold capillary dimensions in childhood. J Rheumatol. 2000;27:797–800. 8. Leung WC, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol. 2002;147:1187–1191. 9. Chung JH, Yano K, Lee MK, et al. Differential effects of photoaging vs intrinsic aging on the vascularization of human skin. Arch Dermatol. 2002;138:1437–1442. 10. Braverman IM. Ultrastructure and organization of the cutaneous microvasculature in normal and pathologic states. J Invest Dermatol. 1989;93:2S–9S. 11. Dehouck MP, Vigne P, Torpier G, et al. Endothelin-1 as a mediator of endothelial cell-pericyte interactions in bovine brain capillaries. J Cereb Blood Flow Metab. 1997;17:464–469. 12. Takagi H, King GL, Robinson GS, et al. Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells. Invest Ophthalmol Vis Sci. 1996;37:2165–2176. 13. Kim Y, Imdad RY, Stephenson AH, et al. Vascular endothelial growth factor mRNA in pericytes is upregulated by phorbol myristate acetate. Hypertension. 1998;31:511–515. 14. Hirschi KK, D’Amore PA. Pericytes in the microvasculature. Cardiovasc Res. 1996;32:687–698. 15. Lindahl P, Johansson BR, Leveen P, et al. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277: 242–245. 16. Hirschi KK, D’Amore PA. Control of angiogenesis by the pericyte: molecular mechanisms and significance. Exs. 1997;79:419–428. 17. de Oliveira F. Pericytes in diabetic retinopathy. Br J Ophthalmol. 1966;50:134–143.
18. Braverman IM, Sibley J, Keh A. Ultrastructural analysis of the endothelial-pericyte relationship in diabetic cutaneous vessels. J Invest Dermatol. 1990;95:147–153. 19. Wallow IH, Bindley CD, Reboussin DM, et al. Systemic hypertension produces pericyte changes in retinal capillaries. Invest Ophthalmol Vis Sci. 1993;34:420–430. 20. Schlingemann RO, Rietveld FJ, Kwaspen F, et al. Differential expression of markers for endothelial cells, pericytes, and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol. 1991;138:1335–1347. 21. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development. 1998;125:1591–1598. 22. Helmbold P, Fiedler E, Fischer M, et al. Hyperplasia of dermal microvascular pericytes in scleroderma. J Cutan Pathol.2004;31: 431–440. 23. Laaff H, Vandscheidt W, Weiss JM, et al. Immunohistochemical investigation of pericytes in chronic venous insufficiency. Vasa. 1991;20:323–328. 24. Lugassy C, Eyden BP, Christensen L, et al. Angio-tumoral complex in human malignant melanoma characterised by free laminin: ultrastructural and immunohistochemical observations. J Submicrosc Cytol Pathol. 1997;29:19–28. 25. Tsukamoto H, Mishima Y, Hayashibe K, et al. Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors. J Invest Dermatol. 1992;98:116–120. 26. Sundberg C, Ivarsson M, Gerdin B, et al. Pericytes as collagenproducing cells in excessive dermal scarring. Lab Invest. 1996;74: 452–466. 27. Braverman IM, Sibley J. Ultrastructural and three-dimensional analysis of the contractile cells of the cutaneous microvasculature. J Invest Dermatol. 1990;95:90–96. 28. Gendron RL. A plasticity for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Surv Ophthalmol. 1999;44:184–185. 29. Scho¨nfelder U, Hofer A, Paul M, et al. In situ observation of living pericytes in rat retinal capillaries. Microvasc Res. 1998;56:22–29. 30. Erber R, Thurnher A, Katsen AD, et al. Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J. 2004;18:338–340, Epub 2003 Dec;2004. 31. Sato Y, Rifkin DB. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol. 1989;109:309–315. 32. Antonelli-Orlidge A, Saunders KB, Smith SR, et al. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci USA. 1989;86:4544–4548. 33. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell. 2000;103:295–309. 34. Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol. 2002;282:947–970.
22 Hyaluronan and the Process of Aging in Skin Robert Stern
Introduction The process of aging of human skin is not well understood. However, loss of apparent moisture is one of the hallmarks of aging skin, with its attendant wrinkling and deterioration in appearance. Hyaluronan (HA, hyaluronic acid) is the predominant mechanism for skin moisture, and must be involved in the aging process. Hyaluronan has an enormous volume of associated water-of-hydration. The water that surrounds the HA molecule is not in equilibrium with the remaining water of the body, but comprises its own compartment. A 70 kg individual has 15 g of HA, half of which is contained in skin. There is also rapid turnover of HA, with a half-life in skin of 1–2 days [1, 2]. However, the biology of skin HA and its bound water has never been thoroughly studied as a function of age. Understanding the metabolism of HA, its reactions within skin, and the interactions of HA with other skin components will facilitate understanding of skin aging as well as decrease in skin hydration. In the past several decades, the constituents of skin have become well characterized. The earliest studies were devoted to the cells that make up skin: epidermis, dermis, and the underlying subcutis. Now it is appreciated that the materials that lie between cells, the matrix components, have major instructive roles for cell and tissue activities. Though the extracellular matrix (ECM) appears amorphous by light microscopy, it forms a highly organized structure of glycosaminoglycans (GAGs), proteoglycans, glycoproteins, peptide growth factors, and structural proteins such as collagen and to a lesser extent, elastin. It is in this ECM that most of skin HA is located. In fact, the predominant component of skin ECM is HA. Recent progress in the details of HA metabolism can also clarify the long appreciated observation that the oxidative damage from free radicals and reactive oxygen species, and the sun damage caused by ultraviolet light cause premature aging of skin. These processes utilize mechanisms similar to that of normal aging, with HA being a common denominator. Attempts to enhance the moisture content of skin, in the most elemental terms, require increasing the level and
the length of time HA is present in skin, preserving optimal chain length of the polymer, and inducing expression of the best profile of HA-binding proteins to decorate the molecule. All of these components are examined in this chapter.
Structure of Hyaluronan Hyaluronan was identified by Karl Meyer [3] in 1938 as a hexuronic acid-containing material that provided the turgor for the vitreous of the eye. It required 20 years, however, before the chemical structure of HA was established. It was later found to be present in virtually every vertebrate tissue. Hyaluronan is a high molecular weight, very anionic polysaccharide. It is a straight chain GAG composed of repeating alternating units of glucuronic acid and N-acetylglucosamine, all connected by b-linkages, GlcAb (1 ! 3) GlcNAc b (1 ! 4), that can reach 107 Da in molecular size. Hyaluronan is the simplest of the GAGs, the only one not covalently linked to a core protein, not synthesized by way of the Golgi pathway, and the only nonsulfated GAG [4, 5]. The b-linkage is of more than passing interest and not merely a curiosity relevant only to carbohydrate chemists. Glycogen is a polymer of a-linked glucose. Changing to a b-linkage converts the polymer to cellulose. A high molecular weight chain of b-linked N-acetylglucosamine is the structure of chitin. Chitin and cellulose are the most abundant sugar polymers on the surface of the earth. Yet such b-linked sugar polymers are not abundant in vertebrate tissues, and the enzymes for their catabolism exist in some suppressed state, for their substrates can survive eons of time. Hyaluronan occurs covalently bound to proteins such as inter-alpha trypsin inhibitor, a plasma protein that also functions as a stabilizer of HA-rich structures, such as the cumulus mass surrounding the mammalian ovum. The molecular domain of HA encompasses a large volume of water that expands extracellular space, hydrates
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_22, # Springer-Verlag Berlin Heidelberg 2010
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tissues, and in the dermis is responsible for skin moisture. It is also a major component in the edema of the inflammatory response. Hyaluronan is capable of expanding its solvent domain over 1,000 times its actual polymer volume. Even at low concentrations, solutions of HA have very high viscosity. By electron microscopy, HA is a linear polymer. It is polydisperse, but usually has a molecular mass of several millions. In solution at physiological pH and salt concentrations, HA is an expanded random coil with an average diameter of 500 nm. Existing models suggest that for high molecular mass HA, the super molecular organization consists of networks in which molecules run parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and then join again into similar aggregates. There is strong evidence that an H2O bridge between the acetamido and carboxyl groups is involved in the secondary structure. The hydrogen-bonded secondary structure also shows large arrays of contiguous –CH groups, giving a hydrophobic character to parts of the polymer that may be significant in the lateral aggregation or self-association, and for interaction with membranes. This hydrophobic character is perhaps involved in the extrusion of newly synthesized HA chains from the cytoplasmic surface of the plasma membrane where the HAS are located, through the membrane to the exterior of the cell. The unusually stiff tertiary polymeric structure is also stabilized by such hydrophobic interactions.
Functions of Hyaluronan Hyaluronan, despite the simplicity of its structure, has a surprisingly wide range of functions. In high concentrations, as found in the ECM of both the dermis and the epidermis, it regulates water balance and osmotic pressure, functions as an ion exchange resin, and regulates ion flow. It functions as a sieve, to exclude certain molecules, to enhance the extracellular domain of cell surfaces, particularly the luminal surface of endothelial cells. It can function as a lubricant and as a shock absorber. Hyaluronan can also act as a structural molecule, as in the vitreous of the eye, in joint fluid, and in Wharton’s jelly. Hyaluronan promotes cell motility, suppresses cell– cell interactions, and regulates cell–matrix adhesion, promotes proliferation, and suppresses differentiation. It participates in such fundamental processes as embryological development and morphogenesis, wound healing, repair and regeneration, and inflammation. Hyaluronan levels increase in response to severe stress, and in tumor
progression and invasion. Recent studies indicate that HA can also exist intra-cellularly [6]. The intracellular functions of HA are unknown. The persistent presence of HA also inhibits cell differentiation, creating an environment that instead promotes cell proliferation. The elevated levels of anti-adhesive surface HA that promotes cell detachment, also permits the embryonic cell to migrate or the tumor cell to move and metastasize. The water-of-hydration also opens up spaces creating a permissive environment for such cell movements. The ECM that surrounds cells also contains variable levels of HA. It is composed predominantly of structural proteins such as collagen and elastin, as well as proteoglycans, and a number of glycoproteins. The HA content is greatest in embryonic ECM, and in tissues undergoing rapid turnover and repair. The basal lamina or basement membrane that separates dermis and epidermis is also considered an ECM structure. The basal lamina contains HA, though the precise structural position is not known. Loss of basement membrane HA in the skin of diabetic patients correlates with skin stiffness. A number of growth factors are embedded in ECM, concentrated by ECM components where they are protected from degradation. Such factors are presented to cells as mechanisms for growth control and modulators of cell function. Heparan sulfate-containing proteoglycans bind members of the FGF and EGF family, while HA can bind growth factors such as TGF-beta, and also protect them from proteolytic digestion [7]. A complex picture is emerging suggesting that the two classes of GAGs, HA, and heparan sulfate, have opposing functions. An HA-rich environment is required for the maintenance of the undifferentiated, pluripotential state, facilitating motility and proliferation, and abundant in the stem cell niche, while the heparan sulfate proteoglycans promote differentiation. However, the concentration of HA in the ECM can vary widely. Even when the levels are decreased, as in areas of marked fibrosis, HA functions as an organizer of the ECM, as a scaffold about which other macromolecules of the ECM orient themselves. Diameters of collagen fibers can be modulated by levels of HA, the thinner more delicate fibers being favored in regions of high HA concentrations. In fibroblast cultures, the addition of exogenous HA to the medium decreases the diameter of the collagen fibers that accumulate. The ability of HA to promote cell proliferation is dependent in part on the size of the HA molecule, opposite effects being achieved at high and intermediate sizes. High molecular weight HA is anti-angiogenic, while
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intermediate molecular weight HA moieties are highly angiogenic, stimulating growth of endothelial cells, attracting inflammatory cells, and also inducing expression of inflammatory cytokines. Partially degraded HA may have the opposite effect, possibly because it is no longer able to retain and release growth factors such as TGF-b [7]. Such observations are relevant for understanding aspects of skin pathology. For example, the intense staining for HA in psoriatic lesions may in part be due to partially degraded highly angiogenic HA, and may be the mechanism for the marked capillary proliferation and inflammation that characterizes these lesions [8]. Attempts to stimulate HA deposition for purposes of promoting skin hydration and to reverse the effects of aging must use caution that the HA deposited should be of a high molecular weight. This can be done by preventing free radical catalyzed chain breaks and by restricting the catabolic reactions of the hyaluronidases carefully. The most recent development is the realization that HA and associated hyaldherins are intracellular, and have major effects on cellular metabolism. Much of the recent advance comes from the ability to remove the ECM of cultured cells using the highly specific Streptomyces hyaluronidase. Permeabilizing such cells and using confocal microscopy makes it possible to use localization techniques for the identification of intracellular HA and its associated proteins. Some of these intracellular HA complexes appear to be a component of the nuclear matrix in a wide variety of cells. They may have importance in regulating the cell cycle and gene transcription. But no definitive functions have been demonstrated to date. The abilities of HA to associate with itself, with cell surface receptors, with proteins, or with other GAGs speak to the versatility of this remarkable molecule. The tight regulation required for HA deposition in association with these multiple and diverse processes depends on net levels of synthesis and degradation. Hyaluronan is generally produced in the interstitium, in the mesenchymal connective tissue of the body, and is largely a product of fibroblasts. It reaches the blood through the lymphatics. Most of the turnover of HA, approximately 85%, occurs in the lymphatic system. The remaining 15% that reaches the blood stream has a rapid turnover with a t1/2 of 3 to 5 min, being rapidly eliminated by receptors in the liver, and also, by unknown mechanisms in the kidney. When the hepatic or renal arteries are ligated, there is an immediate rise in the level of circulating HA [8]. Thus, humans synthesize and rapidly degrade several grams of HA daily. During acute stress, such as in shock, septicemia, major trauma, and in burn patients there is a rapid rise
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in circulating HA [9]. Such HA may function as a volume expander, as a survival mechanism to prevent circulatory collapse. Some of this rapid rise in HA represents HA recruited from interstitial stores and from lymphatics, and not entirely a reflection of increased synthesis or decreased degradation. However, higher plasma levels of HA do correlate with decreased turnover rates, the t1/2 reaching 20 to 45 min in situations of acute stress. The mean serum and plasma level of HA in healthy young people is 20 to 40 mg/L. This value increases with age and probably reflects slower clearance, and decreased HA degradative capacity, though this has not been carefully investigated. Hyaluronan also increases in the circulation in liver disease, particularly cirrhosis, and in renal failure reflecting aberrant degradation, in rheumatoid arthritis and consistently in some malignancies as a result of increased tumor tissue synthesis.
Hyaluronan Oligomers Size-Specific Activities The extracellular high molecular weight HA polymers are space-filling molecules that hydrate tissues, and are antiangiogenic. These HA polymers are also anti-inflammatory and immunosuppressive. This derives in part from the space-filling polymers’ ability to prevent ligand access to cell surface receptors. The 20 kDa fragments, are highly angiogenic, and stimulate synthesis of inflammatory cytokines. These HA fragments induce transcription of MMPs (matrix metalloproteases), and stimulate endothelial recognition of injury. Oligomers, in the 6 to 20 kDa size range induce inflammatory gene expression in mononuclear and in dendritic cells. Hyaluronan fragments thus are highly angiogenic, inflammatory, and immunostimulatory. Very small HA oligosaccharides also have specific activities. Tetrasaccharides induce expression of heat shock proteins, are antiapoptotic, suppressing cell death. These smallest fragments of HA catabolism thus ameliorate the effects of the intermediate angiogenic and inflammatory fragments. Nature has apparently devised mechanisms to control the extent of stress reactions, to keep them in check. From all of these observations, it can be concluded that fragmentation of HA in the course of its catabolic pathway generates products that are involved in essential processes, with size-specific and widely differing and sometimes opposing biological activities. The working assumption is that HA catabolism is a highly ordered, carefully controlled process, the mechanism for which relies on regulation of the individual enzyme activities. It can be
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concluded that HA fragments are an information-rich system [10].
The Hyaluronan Binding Proteins, the Hyaladherins Hyaluronan exists in a number of states in a vertebrate body. Within the ECM, it can be firmly intercalated within proteoglycans and binding proteins in a bottlebrushlike configuration. It can be bound to cells by means of cell surface receptors. Some of the HA exists in a free form circulating in the lymphatic or cardiovascular system. However, even in this relatively free form, there are a number of binding proteins that decorate HA. These are referred to collectively as hyaladherins [11]. The hyaladherins associate with HA through electrostatic or covalent bonds. It is likely that some of the properties attributed to HA are also a function of the hyaladherins bound to HA. Growth factors, collagens, and many other proteins have been identified. Changes in hyaladherin profiles as a function of aging, and in particular, aging skin, have not been investigated, but are obviously important if the mechanism of skin aging has to be understood.
Hyaluronan Receptors The HA receptors, exist in a myriad of forms, owing their diversity to both variant exon expression as well as to multiple post-translational modifications. The multiple sites for the control of HA synthesis, deposition, celland protein-association, and degradation is a reflection of the complexity of HA metabolism. Their relationships are becoming clarified through the ability to sequence rapidly using the newer techniques of molecular genetics. There promises to be an enormous increase in information and in the understanding of HA biology, as the genes for these enzymes and proteins become known, and rapid sequence analysis carried out.
CD44 There are varieties of HA-binding proteins that are broadly distributed, and with wide variations in locations, in the ECM, cell surface-associated, intracellular, both cytoplasmic and nuclear. The same molecule may occur in multiple locations. However, it is those that attach HA to the cell surface that constitute receptors. The most prominent among these is CD44 [12], a transmembrane glycoprotein
that occurs in a wide variety of isoforms, products of a single gene with variant exon expression. CD44 is coded for by ten constant exons, plus from 0 to 10 or 12 variant exons, depending on species, all inserted into a single extracellular position near the membrane insertion site. Additional variations in CD44 can occur as a result of posttranslational glycosylation, addition of various GAGs, including chondroitin sulfate and heparan sulfate. CD44 is able to bind a variety of other ligands, some of which have not yet been identified. CD44 has been shown, however, to interact with fibronectin, collagen, and heparan-binding growth factors. CD44 is distributed widely, being found on virtually all cells except red blood cells. It plays a role in cell adhesion, migration, lymphocyte activation and homing, and in cancer metastasis. The appearance of HA in dermis and epidermis parallels the histolocalization of CD44. The nature of the CD44 variant exons in skin at each location has not been described. The ability of CD44 to bind HA can vary as a function of differential exon expression. It would be of intrinsic interest to establish what modulation occurs in CD44 variant exon expression with changes in the state of skin hydration, and as a function of age. Only one of the many possible examples of the importance of CD44-HA interactions in normal skin physiology is given here. The HA in the matrix surrounding keratinocytes serves as an adhesion substrate for the Langerhans cells with their CD44-rich surfaces, as they migrate through the epidermis. In skin pathophysiology, the effect of local and systemic immune disorders on such interactions between Langerhans cells and keratinocytes also awaits explication.
RHAMM The other major known receptor for HA is the receptor for HA-mediated motility (RHAMM). This receptor is implicated in cell locomotion, focal adhesion turnover, and contact inhibition. It also is expressed in a number of variant isoforms [13]. The interactions between HA and RHAMM regulate locomotion of cells by a complex network of signal transduction events and interaction with the cytoskeleton of cells. It is also an important regulator of cell growth. The TGF-b stimulation of fibroblast locomotion utilizes RHAMM. TGF-b is a potent stimulator of motility in a wide variety of cells. In fibroblasts, TGF-b triggers the transcription, synthesis, and membrane expression of not only RHAMM, but also the synthesis and expression of
Hyaluronan and the Process of Aging in Skin
the HA, all of which occurs coincident with the initiation of locomotion. In summary, both RHAMM and CD44 may be among the most complex of biological molecules, with locations in an unusually wide variety of cell compartments, and associated with a spectrum of activities involving signal transduction, motility, and cell transformation. The apparent inconsistency of observations between different laboratories regarding the receptors CD44, and RHAMM reflects the subtle ways HA exerts its broad spectrum of biological effects and the myriad of mechanisms for controlling levels of HA expression and deposition. Particularly in the experimental laboratory situation, minor changes in culture conditions, differences in cell passage number, length of time following plating, variations in growth factors contained in lots of serum, or differences in stages of cell confluence have major repercussions in expression of HA, its receptors, or the profile of that decorate the HA molecule. This makes age-related changes in cultured skin cells all the more daunting. One of the major challenges is to identify the profile of hyaladherins specific for the HA of epidermis and dermis, to characterize these proteins and to understand their function in relation to age-related changes. In an examination of skin as a function of age, the levels of HA did not decrease, as would be expected, but rather the binding of HA to tissue proteins became more tenacious, and the HA became increasingly more difficult to extract [14]. Another challenge is to understand how HA as a substrate for degradation by hyaluronidases is affected by associated hyaladherins. It is also reasonable to assume that the secondary structure of the HA polymer is modulated, in part, by the hyaladherins bound to it. A CD44-deficient mouse has been obtained that has a reasonably normal phenotype, suggesting that other HA receptors may substitute for CD44 [15]. In fact, it has been documented that RHAMM is up-regulated when CD44 is deficient. Other receptors including layillin, endothelium receptor (LYVE-1), and others have now been identified using database mining approaches. A convenient tabulation of hyaladherins and HA receptors including database information has recently become available [16].
Hyaluronan and Skin General Observations Hyaluronan occurs in virtually all vertebrate tissues and fluids, but skin is the largest reservoir of body HA, containing more than 50% of the total. Earlier studies on the
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distribution of HA in skin, using histolocalization techniques, seriously underestimated HA levels. Formalin is an aqueous fixative, and much of the soluble tissue HA is eluted by this procedure. The length of time tissue in the formalin is a variable that may explain the conflicting results that are often encountered. Acidification and addition of alcohol to the fixative causes the HA to become more avidly fixed, so that subsequent aqueous steps are unable to elute HA out of the tissue [17]. Comparisons have been made of HA localization in skin sections fixed with acid–formalin/ethanol and conventional formalin fixation. Much of the HA, particularly in the epidermis, is eluted during the process of formalin fixation. This suggests that epidermal HA is more loosely associated with cell and tissue structures than is dermal HA. A further incubation of 24 h in aqueous buffer further increases the disparity between the acid–formalin/alcohol and the conventional fixation technique. Once the tissue has been exposed to the acid–formalin/alcohol, the HA association with tissue becomes tenaciously fixed, with little loss of apparent HA observed following additional aqueous incubation, while the formalin-fixed tissues demonstrate progressive loss of HA.
Epidermal Hyaluronan Until recently, it was assumed that only cells of mesenchymal origin were capable of synthesizing HA, and HA was therefore restricted to the dermal compartment of skin. However, with the advent of the specific techniques for the histolocalization of HA, the biotinylated HA-binding peptide, evidence for HA in the epidermis became apparent. In addition, techniques for separating dermis and epidermis from each other permitted accurate measurement of HA in each compartment, verifying that epidermis does contain HA. Hyaluronan is most prominent in the upper spinous and granular layers of the epidermis, where most of it is extracellular. The basal layer has HA, but it is predominantly intracellular, and is not easily leeched out during aqueous fixation. Presumably, basal keratinocyte HA is involved in cell cycling events, while the secreted HA in the upper outer layers of the epidermis are mechanisms for disassociation and eventual sloughing of cells [14]. Cultures of isolated keratinocytes have facilitated the study of epithelial HA metabolism [18]. Basal keratinocytes synthesize copious quantities of HA. When Ca++ of the culture medium is increased from 0.05 to 1.20 mM, these cells begin to differentiate, HA synthesis levels drop, and there is an onset of hyaluronidase activity.
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This increase in calcium that appears to simulate in culture the natural in situ differentiation of basal keratinocytes parallels the increasing calcium gradient observed in the epidermis. There may be intracellular stores of calcium that are released as keratinocytes mature. Alternatively, the calcium stores may be concentrated by lamellar bodies from the intercellular fluids released during terminal differentiation. The lamellar bodies are thought to be modified lysosomes containing hydrolytic enzymes, and a potential source of the hyaluronidase activity. The lamellar bodies fuse with the plasma membranes of the terminally differentiating keratinocytes, increasing the plasma membrane surface area. Lamellar bodies are also associated with proton pumps that enhance acidity. The lamellar bodies also acidify, and their polar lipids become partially converted to neutral lipids, thereby participating in skin barrier function. Diffusion of aqueous material through the epidermis is blocked by these lipids synthesized by keratinocytes in the stratum granulosum, the boundary corresponding to the level at which HA-staining ends. This constitutes part of the barrier function of skin. The HA-rich area inferior to this layer may obtain water from the moisture-rich dermis. And the water contained therein cannot penetrate beyond the lipid-rich stratum granulosum. The HA-bound water in both the dermis and in the vital area of the epidermis is critical for skin hydration. The stratum granulosum is essential for maintenance of that hydration, not only for the skin, but also for the body in general. Profound dehydration is a serious clinical problem in burn patients with extensive losses of the stratum granulosum.
Dermal Hyaluronan The HA content of the dermis is far greater than that of the epidermis, and accounts for most of the 50% of total body HA present in skin. The papillary dermis has the more prominent levels of HA than does reticular dermis. The HA of the dermis is in continuity with both the lymphatic and vascular systems, which epidermal HA is not. Exogenous HA is cleared from the dermis and rapidly degraded. The dermal fibroblast provides the synthetic machinery for dermal HA, and should be the target for pharmacological attempts to enhance skin hydration, and age-related changes. The fibroblasts of the body, the most banal of cells from a histological perspective, are probably the most diverse of all vertebrate cells with the broadest repertoire of biochemical reactions and potential pathways for differentiation. Much of this diversity is site specific. What makes
the papillary dermal fibroblast different from other fibroblasts is not known. However, these cells have an HA synthetic capacity similar to that of the fibroblasts that line joint synovium, responsible for the HA-rich synovial fluid.
Changes in Skin Hyaluronan with Aging The HA levels are high in a fetal circulation and fall shortly after birth. After maintaining a steady level for several decades, circulating levels of HA then begin to increase again in old age. Elevated levels of circulating HA are also found in the syndromes of premature aging, in progeria, and in Werner’s Syndrome. This increase in HA with age is counter-intuitive, and not understood. Increased HA levels in the bloodstream decrease immune competence. Various mechanisms have been invoked. An HA coating around circulating lymphocytes may prevent ligand access to lymphocyte surface receptors. The increased HA may represent one of the mechanisms for the immunosuppression in the fetus. The reappearance of high levels of HA in old age may, similarly, be one of the mechanisms of the deterioration of the immune system in the elderly. The increasing levels of HA with aging may be a reflection of the deterioration of hydrolytic reactions, including the hyaluronidases that maintain the steady state of HA. This is a far more likely mechanism than an increase in HA synthetic activity. Though dermal HA is responsible for most skin HA, epidermal cells are also able to synthesize HA. The most dramatic histochemical change observed in senescent skin is the marked decrease in epidermal HA. In senile skin, HA is still present in the dermis, while the HA of the epidermis has disappeared entirely. The proportion of total GAG synthesis devoted to HA is greater in epidermis than in dermis, and the reasons for the precipitous fall with aging is unknown. The synthesis of epidermal HA is influenced both by the underlying dermis, as well as by topical treatments, such as with retinoic acids, indicating that epidermal HA is under separate controls from dermal HA. In contrast with previous in vitro and in vivo observations, recent studies document that the total level of HA remains constant in skin with aging [14]. The major agerelated change is the increasing avidity of HA with tissue structures with the concomitant loss of HA extractability. Such intercalated HA may have diminished ability to take on water of hydration. This decreased volume of water of hydration HA is obviously a loss in skin moisture. An important study for the future would be to define precisely the hyaldherins, the HA-binding proteins, that decorate
Hyaluronan and the Process of Aging in Skin
the HA in senile skin, and to compare that profile with that of young skin, in both the dermal and epidermal compartments. Progressive loss in the size of the HA polymer in skin as a function of age has also been reported. The increased binding of HA with tissue as a function of age parallels the progressive crosslinking of collagen and the steady loss of collagen extractability with age. Each of these phenomena contributes to the apparent dehydration, atrophy, and loss of elasticity that characterizes aged skin.
Photo-Aging Repeated exposure to UV radiation from the sun causes premature aging of skin. UV damage causes initially a mild form of wound healing, and is associated first with elevated dermal HA. As little as 5 min of UV exposure in nude mice causes enhanced deposition, indicating that UV-induced skin damage is an extremely rapid event. The initial ‘‘glow’’ after sun exposure may be a mild edematous reaction induced by the enhanced HA deposition. But the transient sense of well being in the long run extracts a high price, particularly with prolonged exposure. Repeated exposures ultimately simulate a typical wound healing response with deposition of scar-like type I collagen, rather than the usual types I and III collagen mixture that gives skin resilience and pliability. The biochemical changes that distinguish photo-aging and chronological aging have not been identified. The abnormal GAGs of photo aging are those also found in scars, in association with the changes found late in the wound healing response, with diminished HA and increased levels of chondroitin sulfate proteoglycans. There is also an abnormal pattern of distribution. The GAGs appear to be deposited on the elastotic material that comprises ‘‘elastosis’’ and diffusely associated with the actinic damaged collagen fibers. These appear as ‘‘smudges’’ on H&E sections of sun-damaged skin, rather than between the collagen and elastin fibers as would be observed in normal skin.
Oxidative Stress Reactive oxygen species or free radicals are a necessary component of the oxygen combustion that drives the metabolism of living things. Though they are important for generating the life force, they simultaneously are extraordinarily harmful. Organisms had to evolve protective mechanisms against oxidative stress. Over the course of
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evolution, different enzymatic and non-enzymatic antioxidative mechanisms were developed, such as various vitamins, ubiquinone, glutathione, and circulating proteins, for example, hemopexin. Hyaluronan may also be one such mechanism, acting also as a free radical scavenger. Sunlight (UV light) is an additional generator of harmful oxygen-derived species such as hydroxyl radicals. Such radicals have the ability to oxidize and damage other molecules such as DNA causing cross-linking and chain scission. These hydroxyl radicals may also be destructive for proteins and lipid structures, as well as ECM components such as HA. After a very few minutes of UV exposure, disturbance in HA deposition can be detected. An anomalous situation exists, therefore, that HA can both be protective as a free radical scavenger, and at the same time a target of free radical stress. This paradox may be understood by a hypothetical model in which HA protects the organism from the free radical stress generated by the oxygen-generated internal combustion, but is itself harmed by the more toxic free radicals generated by the external world, by UV irradiation. The generation of HA fragments by UV may underlie some of the irritation and inflammation that often accompanies long term or intense sun exposure. As discussed above, HA fragments are themselves highly angiogenic and inflammatory, inducing the production of a cascade of inflammatory cytokines. Further complications have occurred in this assembly of metabolic attack and counter-attack reactions that have been compiled in the selective forces of evolution. Unusually high levels of antioxidants are present in skin, such as Vitamins C and E, as well as ubiquinone and glutathione. However, these precious compounds are depleted by exposure to sunlight. To prevent this sun-induced cascade of oxidative injuries, topical preparations containing antioxidants have been developed in the past several decades. Initially, such antioxidants were added as stabilizers to various dermatologic and cosmetic preparations. In particular, lipophilic Vitamin E has been the favorite as a stabilizing agent. However, following oxidation, Vitamin E is degraded into particularly harmful pro-oxidative metabolites. In the past several years, increasing concentrations of antioxidants have been used in such skin preparations, in an attempt to create complementary combinations, or to create constant recycling pairs that alternatingly oxidize and reduce each other. Finally, molecules such as HA should be protected by topical antioxidants, to prevent degradation. Topical antioxidants, protecting against free radical damage as well as maintaining HA integrity may have major effects against natural aging and photo-aging.
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Inflammation Chronic inflammation also causes premature aging of the skin, as observed in patients with atopic dermatitis. The constant inflammatory process leads to decreased function of the skin barrier, accompanied by loss of skin moisture. Presumably, the skin of such patients contains decreased levels of HA. Alternatively, the HA may reflect that found in chronological aging, with a change in the ability to take on water of hydration with enhanced association with tissue structures and loss of extractability. Demonstration of such changes and the precise histolocalization of this decreased HA deposition would be of intrinsic interest, a study that has not yet been performed. The acute inflammatory process is associated initially with increased HA levels, the result of the cytokines released by the polymorphonuclear leukocytes, the predominant cells of the acute inflammatory process. The erythema, swelling, and warmth of the acute process are followed later by the characteristic dry appearance and the formation of wrinkles. The precise mechanisms are unknown, but may relate to the differences between acute and chronic inflammatory cells and the attendant chemical mediators released by such cells. Alternatively, initiation of a wound healing response, with collagen deposition, may be a mechanism invoked for the premature aged appearance of the skin in chronic inflammation.
Hyaluronan Synthases A single enzyme is now recognized as being able to synthesize HA, dual-headed transferases that utilize alternately the two UDP-sugar substrates, UDP-glucuronic acid, and UDP-N-acetylglucosamine [19]. The HA cytoplasmic product is extruded through the plasma membrane into the extracellular space by means of an ABC transporter system that permits unconstrained polymer growth. Such growth could not occur in the Golgi or on the endoplasmic reticulum where most sugar polymers are synthesized, without destruction of the cell. There are three synthase genes in the mammalian genome, coding for HAS-1, -2, and -3. They are differentially regulated, with each producing a different size polymer. Sequence data of the HAS isoforms suggest that they contain seven membrane-associated regions and a central cytoplasmic domain possessing several consensus sequences that are substrates for phosphorylation by protein kinases. The ABC transporter system proteins required for HA transport through the plasma membrane are encoded at a
chromosomal region immediately adjacent to the HA synthase genes. In situ expression of the HAS-1 and -2 genes are upregulated in skin by TGF-b, in both dermis and epidermis, but there are major differences in the kinetics of the TGF-b response between HAS-1 and -2, and between the two compartments, suggesting that the two genes are independently regulated. This also suggests that HA has a different function in dermis and epidermis. Stimulation of HA synthesis also occurs following phorbol ester (PMA) and PDGF treatment, although a direct effect on HAS has not been demonstrated. Glucocorticoids induce a nearly total inhibition of HAS mRNA in dermal fibroblasts and osteoblasts. Extracts of dermal fibroblasts indicate that HAS-2 is the predominant HA synthase therein. This may be the molecular basis of the decreased HA in glucocortcoid-treated skin. However, an additional effect on rates of HA degradation has not been examined.
Hyaluronidases Hyaluronan is very metabolically active, with a half-life of 3 to 5 min in the circulation, less than 1 day in skin, and even in an inert a tissue as cartilage, the HA turns over with a half-life of 1 to 3 weeks. This catabolic activity is primarily the result of hyaluronidases (HYALs), endoglycolytic enzymes with a specificity in most cases for the b 1–4 glycosidic bond. The hyaluronidase family of enzymes has, until recently, been neglected, in part because of the great difficulty in measuring their activity. They are difficult to purify and characterize, are present at exceedingly low concentrations, have very high specific activities that are unstable in the absence of detergents and protease inhibitors during the purification procedures. Once purified, these enzymes appear to be perfectly stable. New assay procedures have now facilitated their isolation and characterization. The human genome project has also promoted explication at the genetic level, and a virtual explosion of information has ensued [20]. Six hyaluronidase-like sequences are present in the mammalian genome, resulting probably from two duplication events, resulting in three genes, followed by en masse block duplication, generating six hyaluronidase genes. All are transcriptionally active with unique tissue distributions. In the human, three genes (HYAL1, HYAL2, and HYAL3) are found tightly clustered on chromosome
Hyaluronan and the Process of Aging in Skin
3p21.3, coding for HYAL1, HYAL2, and HYAL3. Another three genes (HYAL4, PHYAL1 (a pseudogene), and sperm adhesion molecule1 (SPAM1)) are clustered similarly on chromosome 7q31.3. They code respectively for HYAL4, a pseudogene transcribed but not translated in a human, and PH-20, the sperm enzyme. The enzymes HYAL1 and HYAL2 constitute the major hyaluronidases for HA degradation in somatic tissues. HYAL1, an acid-active lysosomal enzyme, was the first somatic hyaluronidase to be isolated and characterized. It is a 57 kDa single polypeptide glycoprotein that also occurs in a processed 45-kDa form, the result of two endoprotease reactions. The resulting two chains are bound by disulfide bonds. This is not a zymogen-active enzyme relationship, since the two isoforms have similar specific activities. Only the larger form is present in the circulation, while both isoforms occur in urine, in tissue extracts, and in cultured cells. Why an acid-active hyaluronidase should occur in plasma is not clear. Some species do not have detectable enzymatic activity in their circulation, but an inactive 70 kDa precursor form of the enzyme is present in such sera, detectable by Western blot. HYAL1 is able to utilize HA of any size as substrate, and generates predominantly tetrasaccharides. HYAL2 may also be acid-active, anchored to plasma membranes by a GPI (glycosyl-phosphatidylinositol)link. HYAL2 occurs also in a processed soluble form. Again, the difference in function between the two isoforms is not known. HYAL2 cleaves high molecular weight HA to a limit product of approximately 20 kDa, or about 50 disaccharide units, while HYAL1 is able to digest the high molecular weight polymer to a limit digestion product consisting predominantly of tetrasaccharides. HYAL1 and HYAL2 have similar structures, and the difference in their reaction products requires explanation. The biological properties of HA in aqueous solution is controlled by reversible tertiary structures, as defined by NMR spectroscopy. Evidence suggests a b-pleated sheetlike array stabilized by H- and hydrophobic bonds. Easy transitions between secondary and tertiary structures occur that are convenient mechanisms for switching between functions. The 20 kDa or 50-disaccharide unit is around the size at which such stable tertiary structures are expected to form. Polymers greater than 20 kDa provide the preferred substrate for HYAL2. The enzyme cleaves at a much slower rate once the HA substrate loses tertiary structure. The hyaladherins may also provide additional substrate specificity. The array of hyaladherins that bind
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to tertiary HA structures may differ from those that bind to HA chains with exclusively secondary structure. The specificity of HYAL2 may depend on a combination of differences in bound hyaladherins and on secondary versus tertiary structure.
Hyaluronidase Inhibitors Macromolecular Inhibitors The extraordinarily rapid turnover of HA in tissues suggests that tightly controlled modes exist for modulating steady state levels of HA. The HA of the vertebrate body is of unique importance, and rapid increases are required in situations of extreme stress. Rapid turnover of HA in the normal state indicates constant synthesis and degradation. Inhibition of degradation would provide a far swifter response to the sudden demand for increased HA levels than increasing the rate of HA synthesis. The ability to provide immediate high HA levels is a survival mechanism for the organism. This may explain the rapid rates of HA turnover that occur in the vertebrate animal under basal conditions. It can be compared to the need to suddenly drive an automobile much faster in the case of an emergency, not by stepping on the accelerator, but by taking a foot off the break. If inhibition of HA degradation by hyaluronidase occurs, then a class of molecules that have not been explored, the hyaluronidase inhibitors, are very important. It can be postulated that with extreme stress, hyaluronidase inhibitors would be found in the circulation as acute phase proteins, the stress response products synthesized by the liver. These would prevent the everpresent rapid destruction and allow levels of HA to quickly increase. Circulating hyaluronidase inhibitor activity has been identified in human serum over half a century ago. Modifications in levels of inhibitor activity have been observed in the serum of patients with cancer, liver disease, and with certain dermatological disorders. This area of biology is unexplored, and though some early attempts were made, these hyaluronidase inhibitors have never been thoroughly characterized at a molecular level. Cultured cells secrete hyaluronidases into the culture media, away from the cells. Such a phenomenon does not occur within tissues. The production of unopposed hyaluronidase activity would cause great havoc in tissues. Simultaneous deposition of hyaluronidases and their inhibitors is a reasonable scenario, one that parallels
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control of the matrix metalloproteinases by their TIMPs (tissue inhibitors of MMPs). An important corollary of such observations would be to examine the nature and changes of hyaluronidase inhibitors that occur as a result of aging.
Low Molecular Weight Inhibitors Classes of lower molecular weight inhibitors of hyaluronidase have been identified, some of which come from folk medicines, from the growing field of ethnopharmacology. Some anti-inflammatories as well as some of the ancient beauty aids and practices for freshening of the skin may have some of these compounds as the basis of their mechanism of action. Clinically, heparin used as an anticoagulant, has potent anti-hyaluronidase activity, as does indomethacin, a classic nonsteroidal anti-inflammatory agent, and salicylates. More recently, dextran sulfate and Vitamin C and some of its derivatives, have been shown to be potent inhibitors of vertebrate hyaluronidases.
Non-Enzymatic Degradation The HA polymer can be degraded nonenzymatically by a free radical mechanism, particularly in the presence of reducing agents such as thiols, ascorbic acid, ferrous, or cuprous ions. This mechanism of depolymerization requires the participation of molecular oxygen. The use of chelating agents in pharmaceutical preparations to retard free radical catalyzed scission of HA chains has validity. However, a carefully monitored effect of such agents on HA chain length in human epidermis has not been attempted. Whether such agents can also affect the integrity of dermal HA in protecting them from free radical damage, and whether these agents have any substantial effect on the moisturizing properties of skin HA remain important questions to be answered. These have major implications for the mechanisms of skin aging.
A Scheme for Hyaluronan Metabolism It is well established that HA is taken up by cells for degradation through the CD44 receptor. The high molecular weight extracellular polymer is tethered to the cell surface by the combined efforts of CD44 and the GPIanchored enzyme HYAL2. The HA-CD44-HYAL2
complex is enriched in specialized microdomains. These are invaginations of the plasma membranes termed lipid rafts, significant because they also recruit a large number of key signaling molecules. One category of lipid rafts is caveolae, structures rich in the proteins caveolin and flotillin. HYAL2 interacts with CD44 and with a Na+-H+ exchanger termed NHE1 that creates an acidic microenvironment for the acidactive hyaluronidase enzyme [21]. The HA is cleaved to the 20 kDa limit products corresponding to about 50 disaccharide units. The CD44, a multifunctional transmembrane glycoprotein that is the predominant HA receptor, is expressed in a number of different isoforms. The variant exons of CD44 specifically involved in the interaction with HYAL2 and NHE1 in the process of HA binding, uptake, and degradation have not been determined. The HYAL2-generated HA fragments are internalized, delivered to endosomes, and ultimately to lysosomes, where HYAL1 degrades the 20 kDa fragments to small disaccharides. Two lysosomal b-exoglycosidases, b-glucuronidase and b-N -acetylglucosaminidase, participate in this degradation.
The Hyaluronasome, a New Mini Organelle Based on the observations described above, it is possible to invoke a new mini-organelle specific for HA metabolism, termed the hyaluronasome. Parallels between glycogen and HA metabolism are the basis of this formulation. A glycogen mini-organelle occurs in both liver and muscle tissues. The hyaluronasome may resemble the glycogen granule, each involved in the metabolism of large carbohydrate structures, glycogen being a branched chain polymer of a-linked sugars, and HA, a straight chain polymer of b-linked sugars. Readily visualized by the electron microscope, glycogen granules appear as bead-like structures localized to specific subcellular locales. Each glycogen granule is a functional unit, not only containing carbohydrate, but also enzymes and other proteins needed for its metabolism. These proteins are not static, but rather associate and dissociate depending on the carbohydrate balance in the tissue. Regulation takes place not only by allosteric regulation of enzymes, but also due to other factors, such as subcellular location, granule size, and association with various related proteins. Such observations may be applicable to the hyaluronasome. A multiprotein membrane-associated
Hyaluronan and the Process of Aging in Skin
complex that contains HA synthetic activity has been described. This hyaluronate synthase complex may be a component of the hyaluronasome, containing synthetic as well as catabolic activities, a functional unit that could provide response mechanisms dependent on the metabolic state of the cell. Suggestive evidence comes from several sources. Cultured cells treated with low concentrations of hyaluronidase increase their levels of HA synthesis. Treatment of isolated membrane preparations with low concentrations of hyaluronidase has a similar effect. This is compatible with a feedback mechanism enabling cells to sense levels of HA that have been synthesized. Exogenously added hyaluronidase cleaves newly synthesized HA chains as they are being extruded through the plasma membrane, informing the cell that inadequate amounts of HA have been synthesized. The hyaluronasome, lying just under and partially embedded within the plasma membrane, could rely on a servomechanism using a receptor such as CD44 for relaying such feedback messages. Hyaluronidase treatment of culture cells modulates the profile of expression of CD44 variant exons, thus providing the exquisite controls necessary for such regulatory mechanisms. Levels of HA that cells deposit must respond to various physiological states including growth phase, confluence, inversely related to cell density in both fibroblasts, and keratinocytes, mitosis and cell detachment from the substratum, calcium concentrations, anoxia and lactate, viral transformation, and serum stimulation. Immunolocalization data indicate that some of the HAS enzymes and hyaluronidases colocalize. All of this evidence supports, the existence of the hyaluronasome minirganelle. The hyaluronasome, because of its ability to respond to extracellular events as well as to the intracellular metabolic state of the cell may contain HA receptors, RHAMM and CD44, HA synthase enzymes, the hyaluronidases, and hyaluronidase inhibitors, and HA-binding proteins. The hyaluronasome can regulate levels of HA deposition with great precision by allosteric regulation of the enzymes contained therein utilizing not only hyaladherins and related proteins, but perhaps by posttranslational modifications such as phosphorylation and sulfation. Levels of specific phosphorylated proteins are utilized in the analysis of signaling transduction pathways. However it was the phosphorylases that degrade glycogen and related proteins of glycogen catabolism that provided the paradigm for protein phosphorylation as a control mechanism. Similar modifications applied to the control of HA catabolism would be in that tradition.
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Enhancing Hyaluronan Deposition in Order to Counteract the Aging Process The Lactate Effect Markedly enhanced levels of HA occur in the stroma surrounding malignant tumors. The HA stimulates cell motility and hydrates tissues, creating spaces into which tumor cells can move in the process of invasion and metastatic spread. Lactate is usually the product of anaerobic metabolism. However, cancer cells produce lactate even when oxygen is abundant. The ability of malignant cells to generate lactate, even in the presence of sufficient quantities of oxygen is known as the Warburg effect. Application of lactate to skin is already a pharmacological technique thought to enhance HA deposition. Such preparations are available at both low and high doses, the later requiring a prescription.
Alpha-hydroxy Acids Fruit compresses have been applied to the face as beauty aids for millennia. The alpha-hydroxy acids contained in fruit extracts, tartaric acid in grapes, citric acid in citrus fruits, malic acid in apples, mandelic acid in almond blossoms and apricots are active principles for skin rejuvenation. Such alpha-hydroxy acids stimulate HA production in cultured dermal fibroblasts. The results of such alkaline preparations may depend more on their peeling effects, rather than on the ability of alpha-hydroxy acids to stimulate HA deposition. Lactic acid, citric acid, and glycolic acid, in particular, though frequent ingredients in alpha-hydroxy acidcontaining cosmetic preparations, have widely varying HA-stimulating activity in dermal fibroblasts. Some of these preparations may owe their effectiveness to their traumatic peeling, astringent properties, with constant wounding of the skin. The cosmetic effects of these preparations of alpha-hydroxy acids, including lactic acid, involve increased skin smoothness with the disappearance of lines and fine wrinkles, all of which counteract the aging process. Long-term use, however, results in thickening of the skin, in both the epidermal and papillary dermal layers, because of a mild fibrous reaction. This results from a reaction similar to diffuse wound healing, and explains the increased thickness and firmness of both dermis and epidermis. The increased collagen deposition documented in skin after prolonged use is consistent with a
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wound healing effect. The normal aging process in skin, particularly the coarseness and thickening may resemble some of these reactions.
Vitamin C The structure of ascorbic acid resembles an alpha-hydroxy acid, which is generally not appreciated. Ascorbic acid is present in most fruits, and may underlie some of the effects attributed to fruit extracts. Vitamin C has pronounced HA-stimulating effects in the fibroblast assay. But its antioxidant activity confounds the effects it may induce. The deposition of HA is stimulated when Vitamin C is added to cultured fibroblasts. The most profound changes occur in the compartmentalization of HA. The preponderance of the enhanced HA becomes cell-layer instead of being secreted into the medium. The chemical reactions catalyzed by ascorbic acid that bind HA to cell or matrix components are not known. As aforementioned, derivatives of Vitamin C and its derivatives and analogs can function as hyaluronidase inhibitors. Thus, some of the ability of Vitamin C to enhance HA deposition may be attributed to its inhibition of hyaluronidase activity. Skin preparations containing high concentrations of Vitamin C, as currently in use, may have some validity as anti-aging therapies.
Vitamin A Hyaluronan hinders the onset of differentiation, as discussed earlier. Retinoic acid retards the differentiation of epidermal keratinocytes, as shown in skin organ cultures, a result of the ability of retinoic acid to stimulate HA deposition. Retinoic acid leads to the accumulation of HA in the superficial layers of the epidermis by stimulating HA synthesis specifically in keratinocytes. Some of this accumulation occurs as expanded intercellular HA, which may account for the weakened cohesion of keratinocytes observed both in vivo and in vitro. Topical applications of retinoic acid derivatives reduce the visible signs of aging and of photodamage, though there is little correlation between the histologic changes and the clinical appearance of the skin. Initial improvement in fine wrinkling and skin texture correlates with the deposition of HA in the epidermis. While Vitamin D is considered the ‘‘sunshine vitamin,’’ Vitamin A has been accepted as an apparent antidote for the adverse effects of sun exposure, and assumed to prevent and repair cutaneous photodamage. Application of
Vitamin A derivatives does reverse some of the sun damage to skin, particularly the roughness, wrinkling, and irregular pigmentation. For the over-40 generation, brought up in an era of ‘‘suntan chic,’’ appropriate preparations to restore or to prevent further deterioration of skin are critically important. Impairment of the retinoid signal transduction pathways occurs as a result of prolonged UV exposure. Down regulation of nuclear receptors for Vitamin A occurs, resulting in a functional deficiency of Vitamin A. Application of Vitamin A derivatives would appear to be an obvious treatment modality. Topical application of Vitamin A does increase the HA in the epidermal layer, increasing the thickness of the HA meshwork after prolonged treatment. Vitamin A thus enhances repair, as can be demonstrated in photo-aged hairless mouse model. The decline in GAG, and in particular HA deposition that occurs with UVB irradiation, can be prevented by retinoic acid treatment.
Vitamin E Radical scavengers such as a-tocopherols prevent oxidative degradation of HA. In tissue culture systems, the addition of Vitamin E to the medium prevents spontaneous degradation of HA, as does superoxide dismutase. In Vitamin-E-deficient animals, there is a decrease in GAGs in tissues, including HA. This could be reversed by the addition of Vitamin E to diets, suggesting that tocopherol supplements can enhance HA in human skin, and counteract the aging process.
Vitamin D Vitamin D, and in particular, the hormonally active di-hydroxy form, is a regulator of the proliferation and differentiation of skin cells, including not only epidermal kertainocytes, but also dermal fibroblasts and adipocytes. A result of prolonged UV exposure is dermal fibrosis, the excessive deposition of collagen and other ECM components within the dermis. The commandeering of mesenchymal cells to become fibroblasts, and the conversion of adipocytes to fibroblasts are thought to be the underlying mechanism. Pretreatment of skin with Vitamin D prevents the disappearance of adipocytes and the accumulation of fibroblasts. The appearance of HA, the first step in the wound healing response that initiates the cascade that leads to accumulation of the fibrous reaction, can be prevented by such treatment.
Hyaluronan and the Process of Aging in Skin
Steroids Topical and systemic treatment with glucocorticoids induces atrophy of skin causing premature aging, with a concomitant decrease in HA. In human skin organ cultures, hydrocortisone has a bimodal effect. At low physiological concentrations, 109 M, hydrocortisone maintains active synthesis and turnover of HA in the epidermis, while at high concentrations, 105 M, hydrocortisone reduces epidermal HA content. The effect is achieved through both decreased synthesis as well as decreased rates of degradation. The high concentrations of cortisone also enhance terminal differentiation of keratinocytes and reduce rates of cell proliferation. Hydrocortisone is also a potent inhibitor of HA synthesis in fibroblasts. HAS-2 is the predominant synthase of dermal fibroblasts, of the three HA synthase genes. Glucocorticoids induce a rapid and near total suppression of HAS-2 mRNA levels. The inhibition of HA deposition thus appears to occur at the transcriptional level. Progesterone inhibits HA synthesis in fibroblasts cultured from the human uterine cervix. Thus, the steroid effect on HA appears to be system-wide. Hydrocortisone, as well as dexamethasone suppresses the ability of TGF-beta to stimulate HA synthesis through the p38 MAP kinase induced activation of the HAS genes. Edema is one of the four cardinal signs of acute inflammation. The ability of glucocorticoids to suppress inflammation occurs in part by their ability to suppress the deposition of HA, the primary mechanism of edematous swelling that occurs during the inflammatory response. Skin is also an important target organ for estrogens. The estrogenic effect on skin is well characterized, as well as the effect of estrogen withdrawal. A major effect of estrogen is the increased levels of HA deposition and the associated water of hydration. Topical estrogens are also able to enhance HA deposition in skin, as documented in the hairless mouse skin model. The isoflavones found in soy bean extracts, such as genistein and daidzein, that are phytoestrogens, are also able to enhance HA deposition. Their estrogen-like structures may account for their ability to enhance HA deposition. Withdrawal of estrogen production, which occurs following menopause, explains some of the age-related changes in women. Women with increased levels of body fat have skin that ages more slowly following menopause. This may occur because the fat depots function as slow release capsules of estrogens. Such steroids are fat soluble, and are stored during periods of active estrogen production.
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Ethnopharmacology Agents Used to Retard Skin Aging Ethno-pharmaceuticals have long provided Western medicine with a wide variety of drugs that enhance the appearance of skin, and retard the aging process. Many of these function though effect HA metabolism. Fruit compresses have been applied to the skin for thousands of years as a traditional beauty aid. The alpha-hydroxy acids contained in fruit extracts enhance HA deposition through hyaluronidase inhibition. Other examples are the ginsenosides, major active ingredients of ginseng, which when applied topically, induce expression of the HAS-2 gene and increase skin content of HA. A myriad of other such anti-aging agents applied to skin from folk medicines await identification.
Conclusion and Future Perspectives The biology of HA and its metabolic cycles are in their infancy. The enzymatic steps that constitute extracellular and intracellular HA cycles are beginning to be sorted out. The goals that lie ahead are to identify all the reactions involved, and to devise mechanisms for modulating these reactions, with the ultimate goal of enhancing skin appearance and increasing the moisture content of damaged and aging skin.
References 1. Laurent UB, Dahl LB, Reed RK. Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver. Exp Physiol. 1991;76:695–703. 2. Reed RK, Lilja K, Laurent TC. Hyaluronan in the rat with special reference to the skin. Acta Physiol Scand. 1988;134:405–411. 3. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem. 1934;107:629–634. 4. Lee JY, Spicer AP. Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curr Opin Cell Biol. 2000;12:581–586. 5. Toole BP. Hyaluronan is not just ‘‘goo.’’ J Clin Invest. 2000;106: 335–336. 6. Evanko SP, Wight TN. Intracellular localization of hyaluronan in proliferating cells. J Histochem Cytochem. 1999;47:1331–1342. 7. Locci P, Marinucci L, Lilli C, Martinese D, Becchetti E. Transforming growth factor beta 1 hyaluronic acid interaction. Cell Tissue Res. 1995;281:317–324. 8. Kumar S, West DC. Psoriasis, angiogenesis and hyaluronic acid. Lab Invest. 1990;62:664–665. 9. Engstroem-Laurent A, Hellstroem S. The role of liver and kidneys in the removal of circulating hyaluronan. An experimental study in the rat, Connect. Tissue Res. 1990;24:219–224.
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10. Stern R, Asari AR, Sugahara KN. Size- specific fragments of hyaluronan: an information rich system. Eur J Cell Biol. 2006;85:699–715. 11. Day AJ, Prestwich GD. Hyaluronan-binding proteins: tying up the giant. J Biol Chem. 2002;277:4585–4588. 12. Lesley J, Hascall VC, Tammi M, Hyman R. Hyaluronan binding by cell surface CD44. J Biol Chem. 2000;275:26967–26975. 13. Cheung WF, Cruz TF, Turley EA. Receptor for hyaluronan-mediated motility (RHAMM), a hyaladherin that regulates cell responses to growth factors. Biochem Soc Trans. 1999;27:135–142. 14. Meyer LJM, Stern R. Age-dependent changes of hyaluronan in human skin. J Invest Dermatol. 1994;102:385–389. 15. Nedvetzki S, Gonen E, Assayag N, Reich R, Williams RO, Thurmond RL, Huang JF, Neudecker BA, Wang FS, Turley EA, Naor D. RHAMM, a receptor for hyaluronan mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy. Proc Natl Acad Sci USA. 2004;101:18081–18086.
16. Spicer AP, Tien JY. Hyaluronan and morphogenesis. Birth Defects Res Part C Embryo Today. 2004;72:89–108. 17. Lin W, Shuster S, Maibach HI, Stern R. Patterns of hyaluronan staining are modified by fixation techniques. J Histochem Cytochem. 1997;45:1157–1163. 18. Passi A, Sadeghi P, Kawamura H, Anand S, Sato N, White LE, Hascall VC, Maytin EV. Hyaluronan suppresses epidermal differentiation in organotypic cultures of rat keratinocytes. Exp Cell Res. 2004;296: 123–134. 19. Weigel PH, Hascall VC, Tammi M. Hyaluronan synthases, J Biol Chem. 1997;272:13997–14000. 20. Stern R, Jedrzejas MJ. The hyaluronidases; their genomics,structures, and mechanisms of action. Chem Reviews. 2006;106:818–829. 21. Stern R. A new metabolic pathway: hyaluronan catabolism. Eur J Cell Biol. 2004;83(1–9):1–9.
Pigmentation
51 Hyperpigmentation in Aging Skin Tomohiro Hakozaki . Cheri L. Swanson . Donald L. Bissett
Introduction Human skin color varies greatly around the globe, from very pale Celtic skin to very darkly pigmented skin in subSaharan African populations. Yet, all of these skin types can develop hyperpigmentary problems with aging, for example, postinflammatory hyperpigmentation (PIH), solar lentigos, and melasma. These problems occur widely in the human population, and the methods used to control them are of great interest, with particular desire to achieve uniformity of skin color. Several proven targets for pigmentation control are known, but recent genomic and proteomic understanding of melanogenesis, the melanocyte, melanocyte–keratinocyte interaction, and melanocyte–fibroblast interaction has revealed potentially hundreds of proteins and other effectors involved in the pigmentation process. This body of knowledge, while complex, should provide the basis for understanding specific aberrations that lead to hyperpigmentary problems. Advanced laboratory screening models and tools for skin color quantification are also available. These are increasing the pace of screening of materials and clinical evaluation for their effectiveness. This brief review will focus on problems of hyperpigmentation (particularly as they apply to aging skin), investigative methods to measure and understand the problems, and topical cosmetic treatment approaches.
Pigmentation Process The pigmentation process has been extensively described in many other documents [1] and so will not be discussed in detail here. Briefly, melanocytes are specialized dendritic cells interspersed among basal keratinocytes and serve the primary function of producing melanin in intracellular organelles called melanosomes that are then distributed to surrounding keratinocytes. Each melanocyte is in contact with and distributes melanosomes to many keratinocytes via their dendritic processes. Melanins are complex polymers derived from tyrosine and other
intermediates, which are converted through a multistep process of oxidative and complexation reactions to brownblack eumelanins and yellow-red pheomelanins, which create the diversity of coloration observed across the human population. The regulation of melanin production is very complex and involves more than 80 genes [2, 3]. The synthesis is regulated by various extracellular signaling components that trigger a signal transduction cascade. There is also evidence that fibroblasts participate in this signaling [4]. While the baseline state of melanin in each individual’s skin is dictated by genetic composition, internal and external triggers such as aging and UV exposure can lead to significant alterations in net synthesis of the melanins [5].
Skin Changes with Aging Relevant to Pigmentation Over the course of an individual’s life, skin undergoes many changes [6], and there are many theories regarding the causes of the changes. While there is still much to be learned about these and probably other causes, it is clear that key influencers in hyperpigmentation are environmental effects and hormonal changes, which will be discussed in the following section in the context of specific hyperpigmentary disorders (see Section on Hyperpigmentary Disorders and Their Causes). In general, the number of active melanocytes per unit area of skin decreases with age (10–20% decline per decade), and there are more active melanocytes in chronically sun-exposed skin than in nonexposed skin [6]. This increased number of active melanocytes in sun-damaged skin indicates the influence of chronic UV exposure (e.g., on face, hands, and arms) in stimulating melanogenic potential. Since chronic UV exposure also alters dermal fibroblast function in aging skin and since fibroblasts appear to play a regulatory role in melanin production [4], dermal damage from sunlight may contribute to the production of hyperpigmentation in exposed aging skin.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_51, # Springer-Verlag Berlin Heidelberg 2010
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Hyperpigmentary Disorders and Their Causes Postinflammatory Hyperpigmentation (PIH) Skin insults that result in inflammation can induce postinflammatory hyperpigmentation [7], which is particularly evident in people with darker skin. Among such insults are acne lesions, ingrown hairs, scratches, insect bites, and surfactant damage. As an example of the latter, exposure of human forearm skin to the harsh surfactant sodium lauryl sulfate (SLS) under patch for a few hours will produce erythema within a day. Over the course of 1–2 weeks after this SLS exposure, hyperpigmentation will result, particularly in darker skin, but it will occur even in Caucasian skin. Topical treatment with antiinflammatory agents such as phytosterol will prevent this (> Table 51.1). The most common cause of hyperpigmentation (sunlight exposure of skin) is probably a postinflammatory response to UV damage to skin [8]. That response may be the result of an obvious acute inflammatory event such as sunburn or of repeated sub-erythemal exposures to UV. While in the latter, there may not be visible erythema, histologically, such exposed skin has elevated inflammatory cell content, yielding a ‘‘subclinical’’ inflammatory process. It is supported by the fact that topical treatment with anti-inflammatory agents immediately after UVB exposure prevents induction of delayed tanning [9]. Inflammation may result in hyperpigmentation through several mechanisms. Among them is direct stimulation of melanocytes by inflammatory mediators such as IL-1-alpha or ET-1 [10]. Reactive oxygen species such . Table 51.1 Postinflammatory hyperpigmentation (PIH) on the forearm Erythema grade (day 2)
PIH grade (day 11)
Vehicle
2.09
0.93
5% Phytosterola
1.71*
0.55*
Test agent
A 20% solution of sodium lauryl sulfate (SLS) was applied to the forearm skin of Caucasian subjects (n = 19) under occlusive patch (0.2 mL solution in a 19-mm diameter chamber patch). The patch was removed after 1–4 h, depending on the individual subject responsiveness. After washing the site to remove surface SLS, the skin was treated topically twice daily for 5 days with test agent. The skin was graded (0–4 grading scales) daily for erythema and pigmentation (postinflammatory hyperpigmentation; PIH) for 11 days (D. L. Bissett, unpublished work) a Phytosterol is a plant oil-derived mixture of stigmasterol, sitosterol, campesterol, and brassicasterol * Statistically significantly different (p < 0.05) versus vehicle
as superoxide and nitric oxide generated in damaged skin (e.g., from UV exposure) or released as by-products from inflammatory cells are also known stimulators of melanocytes. Additionally, damage induced to epidermal cells can lead to release of endocrine inducers of pigmentation such as alpha-MSH [11]. The resulting hyperpigmentation induced by all these effects provides some measures of protection against subsequent insult since melanin has both UV absorption and reactive oxygen species scavenging capacity. The melanin produced during an inflammatory event also can enter the dermis where it is engulfed by macrophages, producing ‘‘melanophages.’’ These cells are often retained in the upper dermis for prolonged periods since removal of dermal melanin apparently is a very slow process. Thus, postinflammatory hyperpigmentation can be a very long-lived problem for the skin [1].
Solar (Actinic) Lentigos These hyperpigmented spots are also known as lentigines, age spots, and liver spots. They occur on sun-exposed parts of the body (in particular, the hands, arms, face, upper chest, and shoulders) and thus occur due to chronic exposure of skin to UV and the resultant chronic inflammation, such as the epidermal endothelin cascade [10]. Their dark appearance certainly results from excessive melanin in the region, and may result from overproduction of melanin in the hyperactive melanocytes [12], longer retention of melanin in aging epidermis due to the slower turnover of this tissue layer [6], longer retention of melanin in keratinocytes within rete ridges [13], and dermal melanin-containing melanophages, which have been observed histologically to lie beneath the lentigines [1]. Since with aging, there is reduced wound healing [6, 14] and reduced clearance of materials from dermis apparently due to vascular and lymphatic changes [6], the residence time of melanophages in dermis may be very long. Within lesional lentigo skin, the rete ridges are greatly exaggerated, extending deeper into the dermis [12]. This deep penetration runs counter to the general observation of flattening of the convoluted dermal–epidermal junction with aging, evidenced by the diminution of the rete ridges [6]. In solar lentigenes, the basement membrane is also perturbed [12], which likely contributes to melanin entering the dermis to result in melanophage formation. These observations suggest that there has been a change in the genetic and phenotypic expressions of cells (perhaps both epidermal and dermal) within the spot area as compared to cells in the surrounding non-spot skin.
Hyperpigmentation in Aging Skin
The expression levels of several melanogenesis-associated genes are increased in actinic lentigos [15, 16]. There is also an accentuation of the epidermal endothelin inflammatory cascade [10], together with decreased proliferation and differentiation of lesional keratinocytes [17]. Many of these changes appear to be permanent since these spots persist even when further UV exposure is avoided. The details of these apparent genomic expression changes have not been defined. While lentigos appear to be permanent, their melanin content and thus their intensity will vary seasonally. For example, in evaluation of women with facial hyperpigmented spots in October versus December (in Kobe, Japan, or Cincinnati, Ohio, USA), there is a marked reduction in the size of spots over that time period, suggesting that the lack of continued exposure to sunlight in winter leads to gradual reduction in melanin production (seasonal fading) even in hyperpigmented spots [18, 19]. Additionally, in a separate examination of facial spots in March versus May (in Cincinnati, Ohio, USA), there was a marked increase in the size of spots [20], consistent with the expected increased pigmentation due to increased sun exposure in spring (seasonal darkening). From a consumer appearance standpoint, hyperpigmented spots and uneven pigmentation are important in the perception of age. In a series of studies [21], facial images were digitally modified to remove all age-defining textural features (e.g., facial furrows, folds, lines, wrinkles), leaving only pigmentation as the variable. Naı¨ve judge evaluation and computer image analysis of the images revealed that pigmentation features can contribute to up to 20 years in perceived age of individuals. So pigmentation is an important component of age perception.
Melasma The hyperpigmentary disorder melasma is not well understood [1]. It occurs typically as symmetrical lesions on the face, primarily in darker skin type females at puberty or later in life. Sunlight exposure is likely a factor in the development of melasma since it occurs on the face (a sun-exposed body site) and since the condition worsens in the summer. Most melasma sufferers have a hypersensitivity to ultraviolet radiation, i.e., they display a lower minimum erythemal dose, and even brief exposures to sunlight can stimulate hyperpigmentation. There is also a hormonal component, likely progesterone, since episodes of melasma are often associated with pregnancy and the use of hormonal birth control. There may also be an estrogen component since estrogen receptor expression is increased in melasma [22].
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In melasma lesions, there is excess melanin present in both the epidermis and upper dermis, associated with extravascular macrophages [1]. Since there is only a slight increase in the number of melanocytes, the abnormality appears to be in function of the skin cells, in particular, increased expression of factors in keratinocytes, fibroblasts, and melanocytes of the involved skin [23]. In contrast to PIH, there is no apparent inflammatory phase involved in its development. Additionally, there is likely a genetic component predisposing individuals to melasma, although the specific genetic basis for it is not defined.
Genomics and Proteomics of Pigmentation The pigmentation process is complex as evidenced particularly by recent genomic and proteomic analysis. There are approximately 1,500 gene products (proteins) expressed in melanosomes of all developmental stages, with 600 of them being expressed at any given time, and with 100 of them apparently unique to the melanosome [24]. Added to this are many other proteins (membrane-associated, cytoskeletal, transport, etc.) involved in pigmentation in both the melanocyte and the keratinocyte, indicating the complexity of the pigmentary process. While the basic process (e.g., stimulation of melanocytes and conversion of tyrosine to melanin) is well studied, there are many regulatory elements that have emerged from recent research involved in signaling, in the transport of melanosomes within the melanocyte, and the transfer of melanosomes to the keratinocyte [25]. This complexity merely offers a plethora of opportunities to understand the pigmentation process and to control it. Less well studied are the events that occur in the keratinocyte once melanosomes have been transferred there. In addition to the melanosome engulfment process itself, presumably there are intracellular signals, regulatory elements, and transport mechanisms to distribute the melanosomes within keratinocyte. There is a process of melanin degradation to produce ‘‘melanin dust,’’ an apparently enzymatic process that is more active in lighter skin versus darker skin individuals [26]. This is an area ripe for further study.
Pigmentation Control Agents As noted above, since there are many processes and proteins involved in the pigmentary process, there is a wide array of targets against which to screen for pigmentation control agents. Among the many targets [1] are inhibitors
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of melanocyte stimulation (e.g., antioxidants, anti-inflammatory agents), cell receptor antagonists (e.g., alpha-MSH antagonists), inhibitors of melanin synthesis enzymes (e.g., tyrosinase, TRP-1, TRP-2), inhibitors of melanosome transport within the melanocyte and transfer to the keratinocyte (e.g., PAR-2 antagonists), and activators of melanin degradation within the keratinocyte. While there are several potent drug, over the counter, and surgical approaches to control of pigmentation (e.g., hydroquinone, trans-retinoic acid, corticosteroid, chemical peel surgery, laser surgery, and combinations of these therapies), the discussion here will focus on agents used in cosmetic formulations [27]. A classic target is inhibition of tyrosinase, the first enzyme in the conversion of tyrosine to melanin. A wide array of compounds, such as kojic acid, arbutin, ascorbic acid, ellagic acid, sulfhydryl compounds, and resorcinols, are effective tyrosinase inhibitors, as is a more recently discussed deoxyarbutin [28]. However, since several of these materials also have other effects, it is difficult to directly connect a specific mechanism to the observed effect on pigmentation. For example, sulfhydryl compounds are also effective antioxidants. > Table 51.2 overviews a short list of the many possible targets and a few agents effective against them. In a recent work, niacinamide and glucosamine (in particular, its derivative N-acetyl glucosamine [NAG]) have been determined to be effective in reducing melanin production in culture. In vitro, glucosamine reduces production of melanin by inhibiting activation of tyrosinase [19], while niacinamide inhibits melanosome transfer from melanocytes to keratinocytes [18]. Cosmetic moisturizer formulations containing niacinamide alone are effective in reducing the appearance of hyperpigmented spots in vivo [18, 29] and the addition of NAG to the formula yields greater effectiveness [19] (> Fig. 51.1). Another new addition to the array of pigmentation control agents is N-undecylenoyl-L-phenylalanine, which has been reported to inhibit binding of alpha-MSH to the melanocyte in vitro and is effective as a component of cosmetic moisturizer formulations in clinical testing [30], as shown in > Fig. 51.2. Sunscreen is also effective in reducing the appearance of hyperpigmentation by preventing the entrance of UV into skin to stimulate melanocytes. Clinical testing among Japanese females in late summer-fall season (in Kobe, Japan) using SPF 15 sunscreen alone demonstrated acceleration of fading of facial tanning compared to the control [18]. However, even relatively high sunscreen dose (SPF 15) is not completely protective, such as against incidental sunlight exposure. In clinical testing involving daily use of
. Table 51.2 Pigmentation control targets and some reported effective agents Pigmentation control target examples
Effective agent examples
Tyrosinase inhibition
Hydroquinone, resorcinols, kojic acid, arbutin, deoxy-arbutin, ascorbic acid (vitamin C)
Tyrosinase copper chelation
Ellagic acid, kojic acid
Inhibition of tyrosinase glycosylation
Glucosamine, N-acetyl glucosamine, tunicamycin
Melanosome transfer
Niacinamide, protease inhibitors
Inhibit binding of alpha- N-undecylenoyl-phenylalanine MSH to melanocyte Down regulation of tyrosinase
Retinoid (trans-retinoic acid, retinol and its esters, retinaldehyde)
Antioxidant
Vitamin C compounds, vitamin E, sulfhydryl compounds
Anti-inflammatory agent
Hydrocortisone, phytosterol, glycyrrhetinic acid, tranexamic acid, chamomile extract
Increase epidermal turnover
Retinoids, salicylic acid, alpha-hydroxy acids, alphaketo acids, adenosine monophosphate
SPF 15 sunscreen [20], there was still a marked increase in the size of spots in March versus May (in Cincinnati, Ohio, USA), consistent with increased pigmentation due to increased sun exposure in spring (seasonal darkening). There was greater protection against this seasonal darkening when subjects used a three-way combination of SPF 15 sunscreen, niacinamide, and NAG, thus indicating the opportunity for greater effectiveness by combining sunscreen with non-sunscreen technologies.
Other Skin Chromophores While the focus of this review is on hyperpigmentation, it is informative to mention briefly that the appearance of pigmentation also involves other chromophores in the skin. For example, increased vascular content (hemoglobin) associated with hyperpigmentation has been described for melasma [31], and the same may be the case for hyperpigmented spots. Additionally, there is the spontaneous Maillard reaction (glycation; reaction between
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. Figure 51.1 Hyperpigmented spot reduction by topical treatment with formulas containing niacinamide and N-acetyl glucosamine. Spot area fraction was determined by algorithm-based computer image analysis of Caucasian facial digital images (n = 35). More negative numbers indicate reduction in hyperpigmentation (improvement). N = niacinamide. NAG = N-acetyl glucosamine. The indicated p value for N + NAG is versus N
. Figure 51.2 Hyperpigmented spot reduction by topical treatment with formulas containing niacinamide and N-undecyloyl-Lphenylalanine. Spot area fraction was determined by algorithm-based computer image analysis of Japanese facial digital images (n = 40). More negative numbers indicate reduction in hyperpigmentation (improvement). N = niacinamide. NUP = N-undecylenoyl-L-phenylalanine. The indicated p value for N + NUP is versus N
protein and sugar) that produces yellow-brown chromophores [29, 32]. When this occurs in proteins with long biological half-lives (e.g., structural proteins), the glycation end products accumulate with aging, and the yellow-brown color increases and is persistent. Thus, hyperpigmented areas may appear to be darker in part due to other chromophores, opening the opportunity for further understanding of the problem and for additional approaches to treatment.
Investigative Tools Many of the targets noted above can be investigated in simple mechanism-specific solution assays, melanocyte cell culture, or melanocyte–keratinocyte coculture systems in the laboratory [18]. These methods permit screening of potentially large numbers of compounds for their inhibitory and stimulatory effects on the specific processes being evaluated. For example, one screening
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assay involves a simple mixture of tyrosinase and tyrosine in which a brown product (melanin) quickly appears and can be quantified colorimetrically. Tyrosinase inhibitors, of course, reduce the melanin produced. A simple assay of this type can be readily performed in a 96-well plate, allowing rapid robotic high-throughput screening of thousands of compounds. Assays involving cells are more complex, but even those can be constructed in a multi-well-plate format for moderate-throughput screening of potentially hundreds of compounds. Establishing an array of such simple assays permits the screening of a substantial library of compounds through all these assays to identify promising candidates quickly. Another useful laboratory model that has emerged over the past few years is the skin equivalent culture [4]. These three-dimensional cultures can contain both dermal and epidermal compartments, with fibroblasts, keratinocytes, melanocytes, and potentially other cell types. When they are raised to the air–liquid interface, the keratinocytes will be differentiated to form a stratum corneum structure. Also, live human skin organ culture has potential as a useful model [33]. Versus submerged culture of cells, an advantage of both skin equivalent and organ cultures is that they can be treated topically with simple solutions or even complex emulsion formulations or commercial skin care products. Another particular advantage is that they are not mechanism-specific – since most or all of the pigmentation machineries are present in the cultures, they potentially can be responsive to materials that affect any of the pigmentation targets. These cultures are available from commercial suppliers. While these are useful tools for material evaluation, they are relatively low throughput due particularly to the high cost of the cultures, and also to the time involved in manipulating the cultures over the course of the multiday experiments. There are some in vivo laboratory models that have been used to evaluate pigmentation inhibitory materials. As a recent example, zebra fish have been used in testing topical compounds [34]. Also, a mouse strain develops hyperpigmented spots in response to UV exposure [35], so has potential use as a lentigo model. Other models [1] include live human skin transplanted onto nude mice, Yucatan mini-pig, pigmented SKH-hr/2 mouse, and pigmented guinea pig. Like the skin-equivalent cultures, these models will not be mechanism-specific and thus are likely to be responsive to a wide range of material mechanisms. While all these models may not be broadly available, there is certainly an opportunity to pursue in vivo modeling as a tool in addressing pigmentation problems.
The final proof of value of a technology, of course, requires progressing materials from the laboratory into clinical testing to demonstrate on-skin activity. Clinical methods include live expert grading, chromameter [36], and color image capture and analysis [29]. A new useful clinical measurement tool in assessing effectiveness is based on the principles of noncontact SIAscopyTM, a recently described method to measure skin melanin content and distribution [37]. It rapidly captures facial maps of skin chromophores, permitting determination of the content and distribution of melanin in any spot or any area of the skin. It will also capture maps for any chromophore, and as long as the absorption spectra of the chromophores are known (e.g., hemoglobin, collagen), it can differentiate them to yield chromophore-specific distribution maps. Thus, there is also potential to determine spectra for other chromophores (glycated protein) and obtain distribution maps for them. Clinical testing on various body sites such as forearm, face, chest, and back [29, 38] have been reported, and all have utility in evaluating technology. Any thoroughly controlled clinical evaluation is expensive and therefore practicality limits testing to only the most promising candidates, several of which (as noted above) have proven to be effective in hyperpigmentation control.
Conclusion The continually increasing understanding of the pigmentation process and the underlying problems in hyperpigmentary conditions provide bases for establishing targets against which to screen new compounds to identify those that may be effective pigmentation control agents. With the advances in laboratory and clinical methodology, the screening process can occur much faster than in the past. In addition, the consumer desires effective pigmentation control technology, particularly in the cosmetic arena, since hyperpigmentation problems increase perceived aged. Advanced pigmentary system understanding and new research capabilities are setting the stage for future technological advancements.
Cross-references > Pigmentation > The
in Ethnic Groups New Face of Pigmentation and Aging
Hyperpigmentation in Aging Skin
References 1. Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne JP. The Pigmentary System. New York: Oxford University Press, 1998. 2. Hearing VJ. Biochemical control of melanogenesis and melanosomal organization. J Invest Dermatol Symp Proc. 1999;4:24–28. 3. Schallreuter KU. Advances in melanocyte basic science research. Dermatol Clin. 2007;25:283–291. 4. Cario-Andre M, Pain C, Gauthier Y, et al. In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation. Pig Cell Res. 2006;19:434–442. 5. Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 2007;21:976–994. 6. Gilchrest BA. Skin and Aging Processes. Boca Raton: CRC Press, 1984. 7. Taylor SC. Cosmetic problems in skin of color. Skin Pharmacol Appl Skin Physiol. 1999;12:139–143. 8. Hachiya A, Kobayashi T, Takema Y, et al. Biochemical characterization of endothelin-converting enzyme-1 alpha in cultured skinderived cells and its postulated role in the stimulation of melanogenesis in human epidermis. J Biol Chem. 2002;277:5395–5403. 9. Takiwaki H, Shirai S, Kohno H, et al. The degrees of UVB-induced erythema and pigmentation correlate linearly and are reduced in a parallel manner by topical anti-inflammatory agents. J Invest Dermatol. 1994;103:642–646. 10. Kadono S, Manaka I, Kawashima M, et al. The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol. 2001;116:571–577. 11. Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pig Cell Res. 2004;17: 96–110. 12. Noblesse E, Nizard C, Cario-Andre M, et al. Skin ultrastructure in senile lentigo. Skin Pharmacol Physiol. 2006;19:95–100. 13. Cario-Andre M, Lepreux S, Pain C, et al. Perilesional vs. lesional skin changes in senile lentigo. J Cutan Pathol. 2004;31:441–447. 14. Makrantonaki E, Zouboulis CC. Molecular mechanisms of skin aging: state of the art. Ann N Y Acad Sci. 2007;1119:40–50. 15. Motokawa T, Matsunaga J, Tomita Y. Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions. J Dermatol Sci. 2005;37:120–123. 16. Unver N, Freyschmidt-Paul P, Horster S, et al. Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin. Br J Dermatol. 2006;155:119–128. 17. Aoki H, Moro O, Tagami H, et al. Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics. Br J Dermatol. 2007;156:1214–1223. 18. Hakozaki T, Minwalla L, Zhuang J, et al. The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer. Br J Dermatol. 2002;147:20–31. 19. Bissett DL, Robinson LR, Raleigh PS, et al. Reduction in the appearance of facial hyperpigmentation by topical N-acetyl glucosamine. J Cosmet Dermatol. 2007;6:20–26. 20. Kimball AB, Kaczvinsky JR, Bissett DL, et al. Reduction in the appearance of facial hyperpigmentation by a combination of
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49 In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model Akira Date . Tomohiro Hakozaki
Introduction When the skin is exposed to ultraviolet (UV) light consisting of UVA (320–400 nm) and UVB (290–320 nm), reactive oxygen species (ROS) such as superoxide anion radical (O2–), hydrogen peroxide (H2O2), hydroxyl radical (OH), singlet oxygen (1O2), as well as lipid peroxides, and their radicals (LOOH and LOO) are formed [1, 2]. It is well documented that these free radicals and ROS cause oxidative cellular stress, cell injury, and DNA damage in the epidermis [3, 4], and eventually induce inflammation, skin photoaging, phototoxicity, or malignant tumors [5–8]. To protect skin from these radical species, there are multiple natural defense mechanisms in the skin. For instance, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) play important roles in protecting the skin against degenerative changes by free radicals or ROS [9, 10]. In order to examine the impact and role of UVinduced free radicals or ROS in the skin, it is essential to detect and visualize them in a real time. However, due to extremely short-lived and essentially nonemissive nature, free radials and ROS are difficult to detect directly. Several evaluation methods such as chemiluminescence (CL) probe [1, 11], photon emission detection [12–15], fluorescence detection [16, 17], and electron spin resonance (ESR) spectroscopy using spin probes [18–21] have been developed to detect ROS and investigate their species and behaviors. Meanwhile, in the past 2 decades, human skin equivalent models have been developed for multiple purposes, such as a replacement of animal models for compound safety evaluation. Now several models have become commercially available, and are used not only for safety assessment, but also to investigate biological functions of skin or responses against various stimulations such as compound treatment or UV exposure.
This chapter introduces recent research and methods to detect and visualize oxidative stress or ROS utilizing human skin equivalent models.
Oxidative Stress Measurement for the Evaluation of UV-Induced DNA Damage in a Human Skin Equivalent Model Several techniques were developed for assessing skin damages in human skin equivalent models. Among them, an oxidatively modified DNA base, 8-hydroxy-2’-deoxyguanosine (8-OHdG), is induced by hydroxyl radical (OH), singlet oxygen (1O2), photodynamic reaction, or peroxynitrite (ONOO ). It is also mutagenic when present during DNA replication [22]. In 2006, a combination of a human skin equivalent model and 8-OHdG immunohistochemistry was proposed by Toyokuni et al. [23]. UVBinduced DNA modification was evaluated by utilizing produced 8-OHdG level as a biomarker. Specifically, the human skin equivalent model was exposed to UVB radiation, and the induction of 8-OHdG was examined by immunohistochemical analysis with catalyzed signal amplification on formalin-fixed paraffin sections. The immunohistochemical images were processed with Image J (National Institutes of Health [NIH] image software) to qualify 8-OHdG by multiplying positively stained area and density (8-OHdG index) [4, 24]. Formation of 8-OHdG in the skin equivalent model by UVB exposure was demonstrated, and it is produced in a UV-dose-dependent manner (> Fig. 49.1). Interestingly, little nuclear staining of 8-OHdG was observed in the negative control samples without UVB exposure, but the increase of 8-OHdG staining was clearly observed in the UVB-exposed samples. The effect of pretreatment of several antioxidants was also examined. The formation of 8OHdG was effectively suppressed by the treatment of antioxidative compounds (> Fig. 49.2). It provides evidence to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_49, # Springer-Verlag Berlin Heidelberg 2010
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In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
. Figure 49.1 Localization of 8-OHdG in a human skin equivalent model. (a) Hematoxylin and eosin staining of an untreated specimen. Immunostaining of 8-OHdG after UVB exposure (2.25 mJ/cm2/min) at various time periods. (b) 0 min, (c) 12 min, (d) 18 min, (e) 24 min, (f) 36 min, (g) 48 min, and (h) 60 min. Bar = 100 mm
the fact that the combination of human skin equivalent model and 8-OHdG immunohistochemistry is considered as a distinct screening strategy for identifying active compounds, which prevent UV-induced skin damage with the following three characteristics: (1) alternative to animal experiments; (2) expensive instruments are not required; and (3) higher sensitivity compared with the animal model. More recently, new research was reported by Bernerd and Asselineau utilizing a skin reconstructed model containing a dermal equivalent and a fully differentiated epidermis [25]. The authors investigated the effects of UV light (UVB and UVA) on photo-damage by employing the sunburn cell formation, which is linked to the presence of DNA lesions, such as pyrimidine dimers and (6,4) photoproducts being a direct chromophore for UVB radiation. They found that UVB-induced damage was
essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA-induced damage was mostly located within the dermal compartment. The model and end points used for UVB- and UVA-induced damages appeared to be very useful for the in vitro evaluation of sunscreens or compounds, in particular to investigate their protective effects against the effects of UV radiation. It will also allow to distinguish the efficiency of UV absorbers depending on their absorption spectrum [25, 26]. Currently, there is an emerging concept that there are fragile genomic sites to oxidative stress as well as UVspecific DNA base modifications. In the future, human skin equivalent models will be further applied to the investigation of skin damages at molecular or cellular level by combining with the emerging new technologies such as laser capture micro-dissection (LCM), which is a
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. Figure 49.2 Amelioration of UVB-induced 8-OHdG in a human skin equivalent model by various agents. Immunohistochemistry of 8-OHdG after UVB exposure of 40.5 mJ/cm2 (18 min) (a) with or (b) without prior treatment of 10 mM ascorbate. (c) Quantitation of 8-OHdG immunostaining by NIH image freeware. Asc, ascorbate; bC, b-carotene; SOD, Cu, Zn-superoxide dismutase. Means SEM, N = 5; **p < 0.01 vs untreated control (No UV); #p < 0.05, ##p < 0.01 versus UVB exposure alone. (d) Cell viability determined by MTT assay after UVB exposure of 135 mJ/cm2 (60 min). Means SEM, N = 4 6; **p < 0.01 vs UVB exposure alone. Bar = 100 mm
new method for isolating specific cells of interest from microscopic regions of tissue that has been sectioned [27].
Photon Emission and Fluorescence Technique to Detect ROS in Human Skin Equivalent Models Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered ultraweak radiation emitted from the biological tissues. Several physical or chemical environmental stressors generate ROS, which trigger oxidative reactions in/around the cells or tissues and thereby induce a correlated ultraweak photon emission (UPE) signal. Several works on photon emission detection and imaging in the skin have been
documented just recently. In 2008, Khabiri and Hangen reported a highly sensitive method to assess oxidative processes in biological molecules using weak photon emission generated due to oxidation of proteins or amino acids. For instance, strong UPE signals are detected by the oxidation of Phe, Trp, His, and Cys, and weak UPE signals from Lys and Thr. They proposed the noninvasive method for monitoring of UVA-induced oxidative skin stress by UPE measurement to assess the potency of topical antioxidants in ex vivo porcine skin and in vivo human skin [12, 13]. Niggli et al. reported an improved UPE measurement from UVA laser-induced biophotonic emission of different cultured cells to detect biophysical changes between young and adult fibroblasts, as well as the changes between fibroblasts and keratinocytes [14]. Van Wijk et al. recently reviewed the current status of human photon emission
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techniques and the protocols for recording the human oxidative status. Systematic studies on human emission have presented information on: (a) procedures for reliable measurements and spectral analysis; (b) anatomic intensity of emission and left–right symmetries; (c) biological rhythms in emission; (d) physical and psychological influences on emission; (e) novel physical characteristics of emission; and (f) the identification of UPE with the staging of ROS-related damage and disease. It is concluded that both patterns and physical properties of UPE hold considerable promise as measure for the oxidative status [15]. While UPE detection is one emerging technique, fluorescence techniques with chemical probes for detection and visualization of ROS are also useful tools. Fluorescence as well as chemiluminescence can offer high detection sensitivity. There are several research studies on the detection and imaging of ROS in the UV-exposed human skin equivalent models by using fluorescent techniques. Hanson and Clegg reported the method to observe and quantify UV-induced ROS in ex vivo human skin [16]. Their proposed method consists of two-photon fluorescence microscopy to detect UV-induced ROS. They observed ROS by using a human skin equivalent model, and the epidermis and the dermis of ex vivo human skin. In their study, the human epidermal skin model was incubated with the nonfluorescent ROS probe dihydrorhodamine (DHR), which reacted with ROS such as O2 and H2O2 to form fluorescent rhodamine-123. They reported that the two-photon excitation provides a depth penetration through the skin unlike confocal microscopic techniques. Thus, this method can provide submicron spatial resolution such that subcellular areas where ROS are generated could be detected. This would enable the monitoring of UV-induced ROS at different depths within the skin. In the future, the method might be applicable to evaluate the ability of sunscreens or antioxidants to prevent ROS generation and photo-damage at targeted depth or the region in the skin [17].
Electron Spin Resonance Technique to Detect ROS in a Human Skin Equivalent Model Electron spin resonance (ESR) techniques have been widely used to study ROS and oxidative stress in biological systems in vitro. However, limited number of research studies has been documented by using human skin equivalent models, ex vivo human skin, and even less for in vivo human skin.
In 2000, Togachi et al. reported a spin-trapping detection of ROS using X-band ESR spectroscopy and described the detection of ROS such as singlet oxygen (1O2) and hydroxyl radical (OH) by the spin trap 5,5-dimethyl-1pyrroline-N-oxide (DMPO) and ESR spectroscopy in vitro. They also did in vivo ESR detection of ROS using a nitroxide spin probe, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), for noninvasive imaging of oxidative stress in living animals [18]. In 2003, Herrling et al. reported the detection of UVinduced free radicals and ROS generated in the ex vivo human skin (skin biopsies) by ESR spectroscopy using several nitroxides such as 2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-carboxy-2,2,5,5-tetramethyl pyrrolidine-1-oxyl (PCA) [19]. The authors found that the reduction rates were different among the nitroxides. TEMPO was decreased due to both UV radiation and enzymatic activity in the skin, while PCM and PCA were sufficiently stable in the skin, and solely reduced by UVgenerated free radicals/ROS. They also imaged the spatial distribution of UV-induced free radicals and ROS by using the PCA probe. By assuming the homogeneous distribution of PCA in the skin, they estimated the penetration profile of UVA and UVB irradiation, as the UV irradiation decreases the PCA intensity corresponding to its irradiance and penetration into the skin. Interestingly, this reduction was caused mainly by UVA radiation (320–400 nm), suggesting the importance of UVA protection. In 2006, they reported their further work on detection and imaging of UVA-induced ROS in the human skin biopsies by the combination of the nitroxide spin probe PCA and an L-band ESR spectrometer. The main parts of ROS were generated by UVA (320–400 nm) so that the spatial distribution of free radicals reaches up to the lower side of the dermis. In addition, they proposed a new radical sun protection factor (RSF) to assess antioxidant compounds and UV filters on how much they can protect against UV-induced ROS [20]. As another application of ESR techniques, Date and Hakozaki’s group recently reported ESR spin-trapping method with new probes to detect ROS in a human epidermal skin model [21]. 5,5-Dimethyl-1-pyrolline-1-oxide (DMPO) probe, which is known to detect superoxide anion radical (O2–) and hydroxyl radical (OH) selectively was used. The combination of 2,2,6,6-tetramethyl4-pyperidone (TMPD) and K3Fe(CN)6 for the detection of singlet oxygen (1O2) was also examined. The model was verified by testing the application of the antioxidants known to scavenge specific ROS such as mannitol (OH scavenger), SOD (O2– scavenger), ascorbate (O2–, OH,
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
and 1O2 scavenger), and b-carotene (1O2 quencher). It demonstrated diminishing of the ESR signal of DMPOOH (to detect O2– and OH) by mannitol (OH scavenger) or SOD (O2– scavenger), as well as suppression of the ESR signal of 4-oxo-TEMPO (TMPD-1O2) to detect 1 O2 by b-carotene (1O2 quencher) compared with UVBexposed control samples, as expected (> Figs. 49.3 and > 49.4). This technique will be a useful tool not only to predict UV-induced ROS-related responses in human skin, but also to find the protective compounds against UV-induced oxidative stress in the human skin.
Chemiluminescent Technique to Detect or Visualize ROS in a Human Skin Equivalent Model Chemiluminescent (CL) techniques have been widely used to study oxidative stress in biological systems in vitro; however, several methods are reported using human skin equivalent models, explant organ skin, or in vivo skin. In 2000, a unique technique using an in vivo real-time chemiluminescent (RT-CL) detection and two-dimensional ultra low-light imaging of endogenously generated ROS in the skin of living animals after UVA light exposure was proposed by Yasui and Sakurai [1]. They used a CL probe, cypridina luciferin analog (CLA) and an ultra-low-light
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imaging apparatus equipped with a charge-couple device (CCD) camera (NightOWL™). CLA reacts specifically with superoxide anion radical (O2–) or singlet oxygen (1O2), and emits chemiluminescence at 380 nm according to the consecutive reaction through the intermediate, dioxetane (> Fig. 49.5). The CL emissions are measured using the high-performance ultra-low-light imaging luminograph system. The authors applied this CL method to measure the rate constants of O2– or 1O2 and also demonstrated that it is useful not only in characterizing the ROS, but also in finding protective compounds against UV-induced skin damage and in characterizing ROS generated in the UV (UVA and UVB) light-exposed skin [2, 28]. With this method, they identified that superoxide anion radical was formed intrinsically, and superoxide anion radical and singlet oxygen were generated by UVA exposure to living animal skin. In addition, they indicated that antioxidative ability against ROS in the skin decreases by aging [2]. The former method is informative and useful; however, it uses live animals. In 2006, the combination of a human skin equivalent model (EpiDerm™ skin model EPI-200, MatTek) and the RT-CL method consisting of a sensitive CL probe CLA and an ultra-low-light imaging apparatus was proposed by Yasui et al. as a new novel tool to detect and visualize UVB-induced ROS generation [29]. With this system, CL emission due to the reaction
. Figure 49.3 Effect of antioxidants to suppress O2 and OH by ESR with DMPO. The human skin equivalent model was treated with 1 M DMPO, and exposed to UVB at a dose of 27 mJ/cm2. 10 mM Mannitol, 10 mM ascorbate, and 100 mM SOD pretreatments significantly scavenged the ESR signal of DMPO-OH, and 100 mM catalase (CAT), 10 mM glutathione (GSH), and 10 mM diethylenetriamine-N,N,N’,N’’,N’’-pentaacetic acid (DTPA) pretreatments slightly scavenged its ESR signal. While, 10 mMb carotene (BCA) and 10 bmg/mL chondroitin sulfate B (CSB) pretreatments did not scavenge its ESR signal **p < 0.01 vs UVB exposure (n = 3/each group)
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In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
. Figure 49.4 Effect of antioxidants to suppress 1O2 measured by ESR with TMPD/ K3Fe(CN)6. The human skin equivalent model was treated with 200 mM TMPD and exposed to UVB at a dose of 27 mJ/cm2, and then 100 mM K3Fe(CN)6 was added to the system. The ESR signal of 4-oxo-TEMPO due to the reaction between 1O2 and TMPD was considerably observed. 10 mM ascorbate (ASC), 10 mM b-carotene (BCR), and 10 mM glutathione pretreatments significantly scavenged the ESR signal of 4-oxo-TEMPO (TMPD-1O2), and 100 mM catalase (CAT), 10 mg/mL chondroitin sulfate B (CSB),10 mM diethylenetriamineN,N,N’,N’’,N’’-pentaacetic acid (DTPA), 10 mM mannitol, and 100 mM SOD pretreatments slightly scavenged its ESR signal **p < 0.01 vs UVB exposure (n = 3/each group)
. Figure 49.5 Principle of CLA probe. Cypridina luciferin analog (CLA) reacts specifically with superoxide anion radical (O2–) or singlet oxygen (1O2) and emits chemiluminescence at 380 nm according to the consecutive reaction through the intermediate, dioxetane
of CLA with endogenously generated ROS increased significantly in the UVB-exposed skin compared with that in the intact skin, maximum level being observed at a dose of 27 mJ/cm2. The treatment of SOD (O2– scavenger) and b-carotene (1O2 quencher) effectively suppressed UVBinduced CL intensities, indicating the generation of O2– and 1O2 in the skin equivalent model under UVB exposure. These results were consistent with those observed in the skin of living animals, thus supporting the relevancy of usage of skin equivalent model for the purpose.
Date et al. have further evaluated various antioxidative compounds such as ascorbate, b-carotene, SOD, and yeast ferment filtrate (YFF), which was reported to suppress superoxide anion and hydroxyl radical [21]. A typical visualization of CL from the human skin equivalent model with and without UVB exposure is shown in > Fig. 49.6 [30]. The UVB-exposed skin samples exhibited significantly higher CL levels (red color) than did the intact skin samples (green-yellow color), indicating the increased generation of ROS in the epidermal skin model. The tested
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
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. Figure 49.6 Typical visualization of the chemiluminescent signals in the human skin equivalent model due to ROS generation. The highest level (red color) of ROS generation in the skin was found 44.5 min after UVB exposure at the dose of 27 mJ/cm2. The low chemiluminescence (green-yellow color) was also observed in the human skin equivalent model without UVB exposure, suggesting that live epidermal cells produce ROS intrinsically. ROS reducing effects of antioxidative compounds such SOD, b-carotene, ascorbate, and YFF were clearly demonstrated
. Figure 49.7 Semiquantification of UVB-induced ROS and effect of antioxidants. Suppressive effects of SOD, b-carotene (BCR), ascorbate (ASC), and YFF on the UVB-induced ROS in the human skin equivalent model. Data are expressed as the means SD for 8–10 samples in each experiment. Significant suppressions versus control [UVB(+)] were observed in all treatments in UVB-exposed group (*p < 0.05), while no significant differences versus control [UVB( )] were found in all treatments in non-UVB-exposed group (p > 0.05)
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In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model
compounds greatly reduced UVB-induced CL intensities immediately after the measurement. For semiquantitative comparison, the areas under the curve of CL intensities (AUC) were calculated for all tested compounds as shown in > Fig. 49.7. Compared with the UVB-induced control group, the treatment with antioxidative compounds exhibited statistically significantly lower CL intensities. This unique method will be an effective tool for (1) investigating the impact of ROS in human skin damage and photoaging, and (2) screening protective compounds to suppress ROS generation against UVB-induced skin damage with a powerful visualization ability.
Conclusion With the advance of technology, more and more methods are available to detect and visualize free radicals and ROS in the skin and its equivalent models. In the future, these in vitro methods using human skin equivalent models will be considered to be a relevant and handy tool to identify protective compounds against oxidative stress and its aging effect, and predict ROS-related responses in human skin as a substitute for animal model.
Cross-references > Effect
of Ozone on Cutaneous Tissues and Genetic Factors in Facial Aging in
> Environmental
Twins > Global
Warming and its Dermatological Impact on Aging Skin > Skin Photodamage Prevention: State of the Art and New Prospects
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5. Kligman LH. The ultraviolet-irradiated hairless mouse: a model for photoaging. J Am Acad Dermatol. 1989;21:623–631. doi:10.1016/ 0738-081X(95)00154-8 6. Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T. UV-induced skin damage. Toxicology. 2003;189:21–39. doi:10.1016/S0300–483X(03)00150-1 7. Bech-Thomsen N, Wulf HC. Carcinogenic and melanogenic effects of a filtered metal halide UVA source and a tubular fluorescent UVA tanning source with or without additional solar-simulated UV radiation in hairless mice. Photochem Photobiol. 1995;62:773–779. doi:10.1111/j.1751-1097.1995.tb08729.x 8. Kripke ML, Cox PA, Alas LG, Yarosh DB. Pyrimidine dimmers in DNA initiate systemic immunosuppression in UV-irradiated mice. Proc Natl Acad Sci USA. 1992;89(16):7516–7520. PMID: 1502162 PMCID: PMC49741 9. Sasaki H, Akamatsu H, Horio T. Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT. J Invest Dermatol. 2000;114:502–507. doi:10.1046/j.1523-1747.2000.00914.x 10. Hellemans L, Corstjens H, Neven A, Declercq L, Maes D. Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery. J Invest Dermatol. 2003;120:434–439. doi:10.1046/j.1523–1747.2003.12056.x 11. Ou-Yang H, Stamatas G, Saliou C, Kollias N. A chemiluminescence study of UVA-induced oxidative stress in human skin in-vivo. J Invest Dermatol. 2004;122:1020–1029. doi:10.1111/j.0022-202X. 2004.22405.x 12. Khabiri F, Hagens R, Smuda C, Soltau A, Schreiner V, Wenck H, Wittern KP, Duchstein HJ, Mei W. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)-measurement. I: mechanisms of UPE of biological materials. Skin Res Technol. 2008;14(1):103–111. doi:10.1111/j.1600-0846.2007.00205.x 13. Hagens R, Khabiri F, Schreiner V, Wenck H, Wittern KP, Duchstein HJ, MeiW. Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin. Skin Res Technol. 2008;14 (1):112–120. doi:10.1111/j.1600-0846.2007.00207.x 14. Niggli HJ, Tudisco S, Lanzano` L, Applegate LA, Scordino A, Musumeci F. Laser-ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts. Indian J Exp Biol. 2008;46(5):358–363. PMID: 18697620 15. Van Wijk R, Van Wijk EP, Wiegant FA, Ives J. Free radicals and lowlevel photon emission in human pathogenesis: state of the art. Indian J Exp Biol. 2008;46(5):273–309. PMID: 18697612 16. Hanson KM, Clegg RM. Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin. Photochem Photobiol. 2002;76:7–63. doi:10.1562/0031-8655(2002) 0760057OAQOUI2.0.CO2 17. Hanson KM, Clegg RM. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol Biol. 2005;289:413–422. ISBN: 9781592598304 PMID: 15502202 18. Togashi H, Shinzawa H, Matsuo T, Takeda Y, Takahashi T, Aoyama M, Oikawa K, Kamada H. Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging. Free Radic Biol Med. 2000;28(6):846–853. doi:10.1016/S0891-5849(99)00280-4 19. Herrling T, Fuchs J, Rehberg J, Groth N. UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides. Free Radic Biol Med. 2003;35(1):59–67. doi:10.1016/S0891-5849(03) 00241-7
In vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model 20. Herrling T, Jung K, Fuchs J. Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin. Spectrochim Acta A: Mol Biomol Spectrosc. 2006;63(4):840–845. doi:10.1016/j. saa.2005.10.013 21. Date A, Hakozaki T, Yoshii T, Yasui H, Sakurai H. Detection and identification of reactive oxygen species and followed free radicals generated in the UVB-exposed three dimensional human epidermal cells- EpidermTM as measured by ESR spin-trapping method. In: The 126th annual meeting of pharmaceutical society of Japan, Sendai, Japan (2006), pp 28, [R] am-174. 22. Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2’deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res. 1997;387:147–163. PMID: 9439711 23. Toyokuni S, Yasui H, Date A, Hakozaki T, Akatsuka S, Kohda H, Yoshii T, Sakurai H. Novel screening method for ultraviolet protection: Combination of a human skin-equivalent model and 8-hydroxy-2’-deoxyguanosine. Pathol Int. 2006;56(12):760–762. doi: 10.1111/j.1440-1827.2006.02043.x 24. Toyokuni S, Tanaka T, Hattori T, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T. Quantitative immunohistochemical determination of 8-hydroxy-2’-deoxyguanosine by a monochlonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest. 1997;76:365–374. PMID: 9121119
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25. Bernerd F, Asselineau D. An organotypic model of skin to study photodamage and photoprotection in-vitro. J Am Acad Dermatol. 2008;58(5 Suppl 2):155–159. doi:10.1016/j.jaad.2007.08.050 26. Fourtanier A, Moyal D, Seite´ S. Sunscreens containing the broadspectrum UVA absorber, Mexoryl1 SX, prevent the cutaneous detrimental effects of UV exposure: a review of clinical study results. Photodermatol Photoimmunol Photomed. 2008;24(4):164–174. PMID 18717957 doi:10.1111/j.1600-0781.2008.00365.x 27. Espina V, Heiby M, Pierobon M, Liotta LA. Laser capture microdissection technology. Expert Rev Mol Diagn. 2007;7(5):647–657. doi:10.1586/14737159.7.5.647. PMID 17892370 28. Nishimura H, Yasui H, Sakurai H. Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in-vivo chemilumiminescent detection and tape stripping methods. Exp Dermatol. 2006;15(11):891–899. doi: 10.1111/j.16000625.2006.00484.x 29. Yasui H, Hakozaki T, Date A, Yoshii T, Sakurai H. Real-time chemiluminescent imaging and detection of reactive oxygen species in the UVB-exposed human skin equivalent model. Biochem Biophys Res Commun. 2006;347:83–88. doi:10.1016/j.bbrc.2006.06.046 30. Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H, Sakurai H. Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Arch Dermatol Res. 2008;300(Suppl 1):S51–56. doi: 10.1007/s00403-007-0804-3
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42 Infrared A-induced Skin Aging Peter Schroeder . Jean Krutmann
Introduction Extrinsic skin aging has, for many years, been mainly attributed to ultraviolet (UV) radiation. Recently, it has become evident that other parts of the solar electromagnetic spectrum contribute as well. Among these, infrared radiation, especially Infrared A, has received increasing attention. This chapter will summarize the current knowledge about the epidemiological evidence, molecular principles, and prevention/protection, as it concerns skin aging induced by Infrared A.
Infrared Radiation Physical Basics and Natural and Artificial Sources Solar radiation is filtered by the earth’s atmosphere; the part reaching the earth surface includes the wavelengths from 290 to 4,000 nm and is divided into three bands: ultraviolet radiation (UV, 290–400 nm), visible light (400–760 nm), and infrared radiation (IR, 760–4,000 nm). Infrared radiation is further subdivided into IRA (l = 760–1,440 nm), IRB (l = 1,440–3,000 nm), and IRC (l = 3,000 nm–1 mm). While the photon energy of IR is lower than that of UV, the total amount of solar energy reaching human skin contains approximately 54% IR, while UV only accounts for 7% [1]. Most of the IR radiation lies within the IRA band (30% of total solar energy), which deeply penetrates the human skin, while IRB and IRC only affect the upper skin layers (> Fig. 42.1). In comparison, IRA penetrates better than UV into the skin, with approximately 50% reaching the dermis [1–3]. The main source of IR radiation is the sun; the actual solar dose reaching the skin is influenced by several factors: ozone layer, position of the sun, latitude, altitude, cloud cover, and ground reflections. Based on these parameters, it should be noted that the overall composition of sunlight, e.g., in terms of the UV/IRA ratio is changing throughout the day. In addition to natural sunlight, artificial IR sources are constantly gaining importance; they are used
for therapeutic as well as for lifestyle purposes. While therapeutic use of IRA provides beneficial effects, for example, in wound healing, lifestyle-motivated applications of IRA, e.g., for ‘‘wellness’’ irradiations or for means of skin rejuvenation appear to be quite paradoxical [4].
Infrared Radiation and Skin Aging The role of IR radiation in premature skin aging was described over 20 years ago by L. Kligman [5]. She was the first to report that infrared radiation enhances UV-induced skin damage in guinea pigs. This prompted her to investigate the effect of IR alone; as a consequence, she could demonstrate that IR leads to elastosis, with ‘‘IR inducing the production of many fine, feathery fibers’’ and ‘‘a large increase in ground substance, a finding also seen in actinically damaged human skin.’’ From these observations, she has concluded that IR radiation contributes to skin aging. It took, however, almost 20 years until the underlying molecular mechanisms could be identified.
Molecular Mechanisms Schieke et al. reported in 2002 that low, physiologically relevant doses of IRA lead to a disturbance of the dermal extracellular matrix. IRA irradiation results in an induction of Matrixmetalloproteinase-1 (MMP-1) in vitro in human dermal fibroblasts, while expression of the respective tissue inhibitor TIMP-1 was not increased. This finding has, since then, been confirmed in independent studies by different workgroups in vitro and in vivo [6, 7]. Matrixmetalloproteinases (MMPs) are zinc-dependent endopeptidases responsible for the degradation of extracellular matrix components such as collagen and elastin. Under physiological conditions, MMPs are part of a coordinate network and are precisely regulated by their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs). The unbalanced activity of MMPs with excessive proteolysis is thought to be a major pathophysiological factor in extrinsic skin aging. The increased expression of MMPs without a respective increase in TIMP expression results in the
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_42, # Springer-Verlag Berlin Heidelberg 2010
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Infrared A-induced Skin Aging
. Figure 42.1 Skin penetration of infrared radiation. Different wavelengths of natural and artificial radiations have different penetration capabilities. IRA penetrates well into the skin, approximately 50% of IRA is absorbed in the dermis. IRB reaches the dermis as well, while IRC is nearly completely absorbed in the epidermis
cleavage of fibrillar collagen, and thus impairs the structural integrity of the dermis [8–10]. This impairment can be partially countered by an increased expression of collagen itself. It is therefore important to note that IRA has recently been found to decrease the expression of the dominant human collagen gene Col1a1 in vitro and in vivo [11, 6]. Taken together, IRA disturbs the collagen equilibrium of the skin in two ways: (1) by increasing the amount/ activity of MMP-1, which results in an increased collagen degradation and (2) by decreasing de novo synthesis of collagen. While the biological endpoints of IRA irradiation resemble those found after UV irradiation, the underlying cellular molecular processes are completely different. This is particularly evident if UVA and IRA are being compared: the primal event in both cases is an increased amount of reactive oxygen species (ROS), which on a first glare seems to indicate a similarity rather than a difference. More detailed analysis – however – revealed huge differences between UVA and IRA. UVA induces an increased production of ROS by NADPH-oxidases, which are located in the cytoplasma membrane [12] and in addition repetitive
UVA irradiation results in damage to the mitochondrial DNA (mtDNA) [13]. IRA, on the other hand acts via a disturbance of the mitochondrial electron transport chain (mtETC). This multiprotein facility, driven by reduction equivalents (NADH/H + and FADH2), is responsible for energy conservation by transferring electrons to oxygen, while building up an electrochemical proton gradient across the inner mitochondrial membrane, which in turn fuels the production of ATP from ADP and Pi. As this process is not error free, relatively small amounts of ROS are always generated. Upon IRA irradiation this amount is significantly increased [4]. ROS are often recognized only as damaging agent, but they are well known to function in terms of cellular signaling. Reactive oxygen species (ROS) can serve to trigger molecular signaling responses and several studies indicate that ROS cause an inactivation of protein-tyrosine phosphatases (PTPs) by oxidizing conserved cysteine residues in the active sites of PTPs and thereby lead to a net increase in kinase phosphorylation/activation [14]. After IRA irradiation, not only the mitochondrial levels, but also the cellular ROS levels are increased and a disturbance of the cellular glutathione (GSH) equilibrium is observed [15]. GSH is one of the most important endogenous antioxidants; it can prevent or repair oxidative damage, and as a consequence it is oxidized itself, forming the glutathione dimer (GSSG). In this regard, IRA irradiation leads to a significant shift of the GSH/ GSSG equilibrium towards the oxidized form [15]. IRA-induced ROS production is not just a by-product of the irradiation, but of functional relevance because boosting the cellular antioxidative defense by increasing the cellular GSH content abrogated the IRA-induced upregulation of MMP-1 [15]. In addition, use of specific antioxidants in cell culture has also been shown to decrease the IRA-induced effects [7]. Mitochondria are known to act as a hub for cellular signaling with disruption of the mtETC being a prominent inducer of such retrograde (i.e., from mitochondria to nucleus) signaling [16]. In contrast to anterograde signaling processes here the nuclear gene expression is regulated by events originating in the mitochondria. The IRA-induced increase in mitochondrial ROS was recently found to initiate such a retrograde signaling cascade (> Fig. 42.2). Downstream of mitochondrial ROS, the IRA radiationinduced signaling pathway relevant for MMP-1 induction has been found to involve the activation of MAPKinases. Three distinct MAPK pathways have been characterized: the extracellular signalregulated kinase 1/2 (ERK1/2) pathway (Raf-MEK1/2-ERK1/2), the c-Jun N-terminal kinase
Infrared A-induced Skin Aging
. Figure 42.2 Infrared A-induced signal transduction. IRA radiation leads to an increased amount of mitochondrial ROS, which in turn leads to initiation of retrograde signaling, finally resulting in an increased expression of MMP-1 mRNA and protein and a decreased expression of Col1a1
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subsequent study by the same group indicates that IRA influences cutaneous wound repair by altering the levels of transforming growth factor (TGF)-b1 and MMP-2 [21]. Yet another study showed an influence of IRA on protein expression of ferritin: an increased ferritin expression was detected after IRA irradiation of keratinocytes and fibroblasts [22]. Ferritin is involved in the cellular antioxidative defense and the induction of this putative defense system in human skin most likely reflects a cellular response to oxidative processes triggered by IRA. Frank et al. showed that IRA interferes with apoptotic pathways, namely the mitochondrial apoptosis pathway [23] and reported that IRA signals via p53 [24]. The abrogating effect of IRA on apoptosis induced by lethal doses of extrinsic factor has recently been confirmed by another study [25].
Dosimetry of IRA
pathway (MEKK1/3-MKK4/7-JNK1/2/3), and p38 (MEKKMKK3/6-p38 a–d) pathway also termed stress-activated protein kinases (SAPKs). The ERK1/2 pathway is primarily induced by mitogens such as growth factors, whereas the SAPK pathways are predominantly induced by inflammatory cytokines as well as environmental stress such as UV, heat, and osmotic shock. Activated MAPKs translocate to the nucleus, where they phosphorylate and activate transcription factors such as c-Jun, c-Fos, ATF-2, and ternary complex factors (TCF) leading to the formation and activation of homo- or heterodimeric forms of the transcription factor AP-1. The promoter region of MMP-1 carries multiple AP-1-binding sites. For IRA, it has been demonstrated that ERK1/ 2 and p38 are activated in dermal fibroblasts, but that only inhibition of ERK1/2 activation subdues the IRAinduced increase of MMP-1 (reviewed in [17]). Although up to now the main research focus has been on MMP-1 and Col1a1 it is very likely that the IRAinduced activation of MAPKinases affects the regulation of other genes as well. Indeed, several additional effects of IRA are known: Kim et al. reported that infrared exposure is involved in neoangiogenesis in human skin, because IRA induces an angiogenic switch by altering the balance between the angiogenic inducer VEGF and the angiogenic inhibitor TSP-2 [18]. Interestingly, increased neoangiogenesis is a prominent feature of photoaged human skin [19]. Others found that IRA irradiation led to a decrease in epidermal proliferation, Langerhans cell density, and contact hypersensitivity reaction in mice [20], and a
Human dermal fibroblasts withstand IRA doses up to at least 1,200 J/cm2 [26], but the gene regulatory effects can already be observed at much lower, physiologically relevant dosage, i.e., 54 [8], 240 [4], or 360 J/cm2 [15]. Increased levels of cytosolic and mitochondrial ROS were detected even after a treatment with 30 J/cm2 [15].
IRA Chromophores While the endogenous chromophores for IR are very likely to be part of the mtETC [27] and remain to be identified, several exogenous chromophores for IR are known. They are used for therapeutic purposes, e.g., in photodynamic therapy, and include palladium-bacteriopheophorbide and indocyanine green [28, 29].
Protection Against IRA Up to now, photoprotection of human skin has focused against UVB and/or UVA radiation. The studies discussed above indicate, however, that protection against IRA radiation has to be included in order to achieve complete protection. In this regard antioxidants appear to be promising. Based on the fact that mtROS are functionally relevant in the IRA-induced effects, antioxidants that target the mitochondria theoretically represent potential IRA protective substances. Indeed, it has been demonstrated in vitro and in vivo that such specific antioxidants protect against detrimental IRA effects, e.g., IRA-induced MMP-1 expression [7].
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In contrast, there are currently no chemical or physical UV filters available, which are suited for commercial suncare products, and which have been shown to provide IRA protection. The protective effect of textiles remains to be evaluated in terms of IRA protection. There is, however, data available showing that use of a black cloth at least partially provides IRA protection [18]. Finally, the topic of avoidance has to be discussed. Until now, there is no information source available that would provide a measure on the actual IRA load that would be comparable to the well-established UV index. Establishing a respective IRA index might be a considerable contribution.
10.
Conclusion
13.
As skin aging is a complex process, it is not surprising that ongoing research efforts uncover more and more environmental factors enfolding detrimental effects on the skin. Regarding natural sunlight or artificial sources of its components there is a major doubt that whether in addition to UV, IRA protection also has to be taken into account. IRA photoprotection requires specialized strategies with topical application of mitochondrially targeted antioxidants being a promising option.
References 1. Kochevar IE, Taylor CR, Krutmann J. Fundamentals of cutaneous photobiology and photoimmunology. In: Wolff K, Austen KF, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffel DJ (eds) Fitzpatrick’s Dermatology in General Medicine. New York: McGraw-Hill, 2007. 2. Cobarg CC. Physikalische Grundlagen der wassergefilterten InfrarotA-Strahlung. In: Vaupel P, Kru¨ger W (eds) Wa¨rmetherapie mit wassergefilterter Infrarot-A-Strahlung. Stuttgart: Hippokrates Verlag, 1995, pp. 19–28. 3. Hellige G, Becker G, Hahn G. Temperaturverteilung und Eindringtiefe wassergefilterter Infrarot-A-Strahlung. In: Vaupel P, Becker G (eds) Wa¨rmetherapie mit wassergefilterter Infrarot-A-Strahlung. Stuttgart: Hippokrates Verlag, 1995, pp. 63–80. 4. Schroeder P, Haendeler J, Krutmann J. The role of near infrared radiation in photoaging of the skin. Exp Gerontol. 2008; 43:629–632. 5. Kligman LH. Intensification of ultraviolet-induced dermal damage by infrared radiation. Arch Dermatol Res. 1982;272:229–238. 6. Kim MS, Kim YK, Cho KH, Chung JH. Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin. Mech Ageing Dev. 2006;127:875–882. 7. Schroeder P, Lademann J, Darvin ME, Stege H, Marks C, Bruhnke S, Krutmann J. Infrared radiation-induced matrix metalloproteinase in
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human skin: implications for protection. J Invest Dermatol. 2008;128:2491–2497. Brenneisen P, Sies H, Scharffetter-Kochanek K. Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann N Y Acad Sci. 2002;973:31–43. Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, Voorhees JJ. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138:1462–1470. Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997;337:1419–1428. Buechner N, Schroeder P, Kunze K, Maresch T, Calles C, Krutmann J, Haendeler J. Thioredoxin-1 protects from MMP-1 upregulation and collagen type Ia1 downregulation: implication for photoaging. Exp Gerontol. 2008 (this issue). Schauen M, Hornig-Do HT, Schomberg S, Herrmann G, Wiesner RJ. Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death. Free Radic Biol Med. 2007;42:499–509. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, Gers-Barlag H, Gefeller O, Krutmann J. Induction of the photoaging-associated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;122:1277–1283. Cross JV, Templeton DJ. Regulation of signal transduction through protein cysteine oxidation. Antioxid Redox Signal. 2006;8:1819–1827. Schroeder P, Pohl C, Calles C, Marks C, Wild S, Krutmann J. Cellular response to infrared radiation involves retrograde mitochondrial signaling. Free Radic Biol Med. 2007;43:128–135. Butow RA, Avadhani NG. Mitochondrial signaling: the retrograde response. Mol Cell. 2004;14:1–15. Schieke SM, Schroeder P, Krutmann J. Cutaneous effects of infrared radiation: from clinical observations to molecular response mechanisms. Photodermatol Photoimmunol Photomed. 2003;19:228–234. Kim MS, Kim YK, Cho KH, Chung JH. Infrared exposure induces an angiogenic switch in human skin that is partially mediated by heat. Br J Dermatol. 2006;155:1131–1138. Yaar M. Clinical and histological features of intrinsic versus extrinsic skin aging. In: Gilchrest BA, Krutmann J (eds) Skin Aging. New York: Springer, 2006, pp. 9–21. Danno K, Sugie N. Effects of near-infrared radiation on the epidermal proliferation and cutaneous immune function in mice. Photodermatol Photoimmunol Photomed. 1996;12:233–236. Danno K, Mori N, Toda K, Kobayashi T, Utani A. Near-infrared irradiation stimulates cutaneous wound repair: laboratory experiments on possible mechanisms. Photodermatol Photoimmunol Photomed. 2001;17:261–265. Applegate LA, Scaletta C, Panizzon R, Frenk E, Hohlfeld P, Schwarzkopf S. Induction of the putative protective protein ferritin by infrared radiation: implications in skin repair. Int J Mol Med. 2000;5:247–251. Frank S, Oliver L, Lebreton-De Coster C, Moreau C, Lecabellec MT, Michel L, Vallette FM, Dubertret L, Coulomb B. Infrared radiation affects the mitochondrial pathway of apoptosis in human fibroblasts. J Invest Dermatol. 2004;123:823–831. Frank S, Menezes S, Lebreton-De Coster C, Oster M, Dubertret L, Coulomb B. Infrared radiation induces the p53 signaling pathway: role in infrared prevention of ultraviolet B toxicity. Exp Dermatol. 2006;15:130–137. Jantschitsch C, Majewski S, Maeda A, Schwarz T, Schwarz A. Infrared radiation confers resistance to UV-induced apoptosis
Infrared A-induced Skin Aging via reduction of DNA damage and upregulation of antiapoptotic proteins. J Invest Dermatol. 2009;129(5):1271–1279, Epub 2008 Nov 27. 26. Schieke S, Stege H, Kurten V, Grether-Beck S, Sies H, Krutmann J. Infrared-A radiation-induced matrix metalloproteinase 1 expression is mediated through extracellular signal-regulated kinase 1/2 activation in human dermal fibroblasts. J Invest Dermatol. 2002;119:1323–1329. 27. Karu T. Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B. 1999;49:1–17.
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28. Koudinova NV, Pinthus JH, Brandis A, Brenner O, Bendel P, Ramon J, Eshhar Z, Scherz A, Salomon Y. Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): successful in vivo treatment of human prostatic small cell carcinoma xenografts. Int J Cancer. 2003;104:782–789. 29. Tseng WW, Saxton RE, Deganutti A, Liu CD. Infrared laser activation of indocyanine green inhibits growth in human pancreatic cancer. Pancreas. 2003;27:e42–e45.
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15 Neurotrophins and Skin Aging Mohamed A. Adly . Hanan Assaf . Mahmoud R. Hussein
Introduction Cutaneous aging is a complex biological phenomenon that consists of two superimposed components: intrinsic (true aging) and extrinsic (photoaging) aging. Intrinsic aging is largely genetically determined and represents an inevitable change attributable to the passage of time alone. It resembles aging that is seen in most internal organs and its underlying mechanisms probably involve decreased proliferative capacity, leading to cellular senescence and altered biosynthetic activity of skin-derived cells. Intrinsic aging is manifested primarily by physiologic alterations with subtle but undoubtedly important consequences for both healthy and diseased skin. The morphologic changes of intrinsic aging include smoothing and thinning of the skin with exaggeration of the expression lines. The intrinsic rate of skin aging in any individual is dramatically influenced by personal and environmental factors, particularly the amount of exposure to ultraviolet light (UV), that is, intrinsic and extrinsic aging are superimposed processes. Extrinsic aging is caused by environmental exposure, primarily to UV. It is observed in the sun-exposed areas (photoaging) and is manifested by the presence of skin wrinkles, pigmented lesions, patchy hypopigmentations, and actinic keratoses. It involves changes in the cellular biosynthetic activity and usually leads to gross disorganization of the dermal matrix. Photodamage, which considerably accelerates the visible aging of skin, also greatly increases the risk of cutaneous neoplasia. The molecular mechanisms underlying skin aging are poorly understood. They seem to be a multifaceted process influenced by various factors affecting different body sites at variable degrees. This chapter discusses the possible roles of some molecules involved in cutaneous aging, namely neurotrophins (NTs).
Overview of Neurotrophins Neurotrophins (NTs) are a family of structurally and functionally related polypeptides, which show about 50% amino acid sequence homology. NTs belong to a
family of growth factors, which control the development, maintenance, and apoptotic death of neurons. They also have multiple regulatory functions outside the peripheral and central nervous systems [1–3]. The NTs family consists of four structurally and functionally related proteins known as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). It is well known that all four members of the NTs family are synthesized as precursors which are cleaved by intracellular proteases to release the C-terminal mature proteins [4]. Mature NTs proteins are approximately 13 kDa in size and share about 50% of amino acid sequence homology. They exert their biological effects as dimers interacting with specific receptors. High-affinity receptors for NTs belong to the tyrosine kinase family. Tyrosine kinase receptor A (TrkA) is the highaffinity receptor for NGF, tyrosine kinase receptor B (TrkB) is the high-affinity receptor for BDNF and NT-4, and tyrosine kinase receptor C (TrkC) is the high-affinity receptor for NT-3 [5]. However, NT-3 may also bind to TrkA and TrkB receptors, but with low affinity. All four NTs interact with the low-affinity p75 kDa NT receptor (p75NTR), which is a member of the tumor necrosis factor family of receptors containing the cytoplasmic ‘‘death’’ domain, involved in mediating a number of responses independently or in association with Trk receptors [4, 6]. By interacting with Trk receptors and/or p75NTR, NTs induce a variety of biological responses in neurons as well as in non-neuronal cells. They control proliferation, differentiation, and survival, whereby these interactions occur. The signals promoting survival or differentiation are generated by NT interaction with Trk receptors and require receptor dimerization, autophosphorylation, and the subsequent involvement of a number of adaptor molecules coupling Trk receptors to the distinct intracellular signal transduction pathways [5]. NTs modulate synaptic transmission via Trk-associated regulation of intracellular Ca2+, promote survival via phosphorylation and inactivation of several proapoptotic substrates including Bad. They promote differentiation via activation of the Ras/Raf/ERK kinase/mitogen-activated protein kinase cascade [5]. p75NTR performs distinct functions depending on whether it is coexpressed with Trk receptors and/or
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_15, # Springer-Verlag Berlin Heidelberg 2010
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selected other growth factor receptors (sortilin, Nogo receptor complex), or whether it is expressed alone. The coexpression of p75NTR with Trk receptors increases high-affinity NT binding, enhances Trk ability to discriminate a preferred ligand from the other NTs, and promotes survival effects of the NTs [4, 6]. When p75NTR is coexpressed with sortilin (a non-G-protein-coupled neurotensin receptor), NT precursor proteins (pro-NTs) interacting with p75NTR–sortilin complex induce apoptotic death [7]. In case of coexpression of p75NTR with the Nogo receptor complex, Nogo induces growth inhibition [4]. When p75NTR is expressed alone on the cell surface, mature NT peptides or selected non-NT ligands (beta-amyloid or a fragment of the prion protein) are capable of inducing apoptosis or promote survival depending on the intracellular adaptor molecules present in target cells [4, 8]. Apoptotic signaling via p75NTR requires the presence of intracellular adaptor molecules (NT receptor-interacting factors 1 and 2, NT receptorinteracting MAGE homolog, and NT-associated death executor) that link p75NTR signaling with the JNK-p53Bax proapoptotic pathway. However, signaling through p75NTR expressed alone – besides inducing apoptosis – may also promote cell survival. Intracellular adaptor molecules interacting with the C-terminus of p75NTR (TNF receptor-associated factor 6, Fas-associated phosphatase-1, and receptor-interacting protein-2) link p75NTR with the NF-kb pathway and can thus promote survival. However, mechanisms involved in controlling the expression and preferential engagement of adaptor molecules in distinct cell types remain to be clarified.
NT Family Members are Expressed in the Mammalian Skin NTs were originally discovered in the nervous system where they were found to be involved in the differentiation and survival of the neurons. However, they were found to be expressed in a variety of tissues outside the nervous system where they have non-neuronal targets in the skin [3, 9–11], kidney, tooth, muscle, and heart [12]. In mice, NTs are expressed very early during embryonic development (E9.5–E10.5) in both the skin epithelium and the cutaneous mesenchyme [13]. The onset of NT expression in embryonic skin coincides with the time point of appearance of K5 and K14 in the epidermis (E9.5), whereas maximal NT synthesis coincides with the beginning of vibrissa development in facial skin (E12.5), and with the initiation of tylotrich hair follicle (HF) induction in dorsal murine skin (E14.5) [13]. This raises
the hypothesis that NTs fulfill multiple non-neurotrophic functions during skin development. In murine postnatal skin, NTs and their receptors are differentially distributed in distinct cell populations. NGF and NT-3 are expressed by basal epidermal keratinocytes in mice and humans [12, 14–21] (> Fig. 15.1). They are also produced by fibroblasts in vitro, and NGF stimulates fibroblast migration [14, 15]. In situ, BDNF and NT-3 are expressed in cutaneous nerve fibers and myocytes of the arrector pili and panniculus carnosus muscles of mice [16, 17]. In adult human skin, NGF and NT-3 are also expressed by fibroblasts, arrector pili muscle, sebaceous and sweat glands, and hair follicles [15, 16] (> Figs. 15.2 and > 15.3). NT receptors (TrkA, TrkC, and p75NTR) have been detected on human epidermal keratinocytes [12, 14–19]. In murine skin, only TrkA and TrkB isoforms are seen in epidermal keratinocytes, whereas TrkC and p75NTR are expressed in cutaneous nerves and in the HF [16, 17].
NTs and Epidermal Homeostasis Work over the past 10 years has indicated that NTs possess a range of functions outside the nervous system [12, 18, 19] and can be considered as growth factors in epithelial tissue homeostasis. It was demonstrated that normal human keratinocytes synthesize and secrete biologically active NGF [14, 20, 21]. In human skin, NGF is released in increasing amounts by proliferating keratinocytes, whereas secretion ends in more differentiated cells [22, 23]. Both exogenous and endogenous NGF are capable of inducing keratinocyte proliferation [12, 14–19]. On the other hand, in the presence of their normal mesenchymal environment, exogenous NGF can indeed either stimulate or inhibit murine epidermal and HF keratinocyte proliferation in situ, depending on whether the keratinocytes are in a state of relative quiescence or are already maximally proliferating [24]. The proliferative effects of autocrine NGF on human keratinocytes are also confirmed by the use of the natural alkaloid K252a, an inhibitor of TrkA phosphorylation. Indeed, K252 blocks keratinocyte proliferation, in the absence of exogenous NGF [18]. Moreover, human keratinocytes transfected with NGF proliferate to a significantly greater extent than mocktransfected cells [25]. Keratinocytes express and release NTs, other than NGF [16, 17, 26], and BDNF, NT-3, and NT-4 stimulate murine epidermal keratinocyte proliferation in situ [16, 17]. NGF is secreted at highest levels as compared to the other NTs, whereas NT-3 and NGF upregulate each other’s secretion in human keratinocytes. NGF expression
Neurotrophins and Skin Aging
15
. Figure 15.1 Expression of NTs and their receptors in normal human skin shown in red color with tyramide signal amplification (TSA) and avidin–biotin complex (ABC) immunostaining techniques. (A) Nerve growth factor (NGF) (200¥ ), (B) NT3 (200¥ ), (C) Tyrosine Kinase A (TrkA) (200¥ ), (D) TrkC (200¥ ), (E): p75NTR (200¥ ), F, G, H, I and J refer to as the expressions of NGF, NT-3, TrK A, TrK C and p75NTR, respectively, confirmed with ABC immunostaining. (INF) Infundibulum. Adapted from Adly et al. [17, 21] ß 2005, 2006, Wiley Blackwell and Reprinted with permission from Adly et al. [32] ß 2009, Elsevier
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. Figure 15.2 Immunoreactivity of nerve growth factor (NGF) protein in adult human scalp anagen VI HF (hair Follicle), shown in red color with avidin–biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A, B, F, and G show IR in the distal region of HF. C, D, and H show immunoreactivity (IR) in the mid region of hair follicle HF. E and I show IR in the proximal bulb region of HF. J is a schematic summary representation of IR in the whole HF shown in red color. K shows IR in catagen HF. l shows IR in telogen HF. CTS connective tissue sheath; DP dermal papilla; HCo hair cortex; HMe hair medulla; HMC hair matrix cells; HS hair shaft; INF infundibulum; IRS inner root sheath; ORS outer roof sheath. (Reprinted with permission from Adly et al. [21]) ß 2006, Wiley-Blackwell
Neurotrophins and Skin Aging
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. Figure 15.3 Immunoreactivity of NT-3 in human scalp skin and HF, shown in red color with avidin–biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A and G show the epidermis. B–F (panel 1) and H–L (panel 2) show IR in anagen VI HF. C and J show IR in the sebaceous gland (SG). N and R show IR in the sweat gland. O shows IR in the early anagen, some fibroblasts in the dermis, and adipocytes in subcutis. M and P show IR in telogen HF. Q shows IR in catagen HF. S is a schematic summary representation of IR in anagen VI HF shown in red color. APM arrector pili muscle. (Reprinted with permission from Adly et al. [17]) ß 2005, Wiley-Blackwell
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is downregulated by UVB irradiation [23, 25], whereas NT-3 release is augmented by UVA. At the skin level, TrkA and TrkC mediate NGF- and NT-3-induced keratinocyte proliferation, respectively. Indeed, keratinocytes overexpressing TrkA proliferate significantly better than controls [27], and increasing concentrations of anti-NT-3 antibody inhibit keratinocyte proliferation [26]. In mouse skin, epidermal keratinocytes express TrkA and TrkB, and all NTs are capable of stimulating their proliferation in ex vivo-cultured skin explants [16, 17, 24]. In human skin, epidermal keratinocytes express TrkA, TrkB, and TrkC proteins in different intensities [21] and NTs can stimulate keratinocyte proliferation and epidermal homeostasis. Apoptosis plays a fundamental role in epidermal homeostasis by counterbalancing cell proliferation, and apoptotic cells are consistently present in normal human epidermis [28–30]. In vitro, NGF, but not the other NTs, can rescue human epidermal keratinocytes from spontaneous and UVB-induced apoptosis via TrkA [25, 28, 29]. Although UVB downregulates NGF and TrkA in human keratinocytes, NGF-overexpressing keratinocytes are protected from UVB-induced apoptosis [25]. NGF protects keratinocytes from cell death via the Bcl-2 family of apoptosis inhibitors. Indeed, K252a fails to induce apoptosis in keratinocytes overexpressing Bcl-2, and UVB causes a decrease in Bcl-2 and Bcl-xL expression in mock-transfected keratinocytes, but not in NGFoverexpressing cells. NGF prevents the cleavage of the enzyme poly (ADP-ribose) polymerase, a substrate for caspases, which is induced in human keratinocytes by UVB [25]. These observations are consistent with a model whereby autocrine NGF protects human keratinocytes from apoptosis through its high-affinity receptor TrkA by maintaining constant levels of Bcl-2 and Bcl-xL, which in turn block caspase activation [25]. The above-mentioned observations clearly show that NTs mediate proliferative and survival signals in epidermal keratinocytes through their high-affinity Trk receptors. Still, the role of the low-affinity p75NTR in NGF signaling in keratinocytes remains to be clarified. Although TrkA is evenly distributed in the basal keratinocyte layer (> Fig. 15.1c), p75NTR is expressed in basal keratinocytes with an irregular pattern (> Fig. 15.1e). As human keratinocytes lack functional TrkB [25], BDNF and NT-4 obviously signal through p75NTR in these cells. Indeed, BDNF and NT-4 induce apoptosis in cultured human keratinocytes. This is in agreement with the observation of a similar function of p75NTR in the catagen phase of the hair cycle [31] and the strong
expression of p75NTR protein in the anagen–catagen transition and early catagen stages of the human hair follicle cycle [32]. Therefore, a balance between the lowand the high-affinity NT receptors exists in keratinocytes. However, the exact stimuli and conditions whereby NGF and other NTs signal life or death in keratinocytes are yet to be defined. In addition, it should also be determined whether NTs and their receptors could play a role in the development of non-melanoma skin cancers by stimulating proliferation and inhibiting apoptosis, in a manner similar to what has been shown for prostate [33] and breast neoplasia [34].
NTs and Melanocytes During skin development, neural crest-derived melanoblasts migrate into the skin and differentiate into melanocytes, which populate the basal layer of the epidermis and the HFs. Together with other paracrine signaling molecules (fibroblast growth factor [FGF], bone morphogenetic proteins, noelin-1, stem cell factor, hepatocyte growth factor, endothelins), NTs play an important role in the control of melanoblast migration, viability, and differentiation [14, 29]. Normal human melanocytes also express p75NTR [32] and its expression level is upregulated by a variety of stimuli including UV irradiation [35]. Keratinocyte-derived NGF, whose expression is also upregulated by UV irradiation [20, 36], may influence epidermal melanocytes in a paracrine manner. In vitro, NGF is chemotactic for melanocytes and stimulates melanocyte dendrite formation [14]. Although under optimal basal culture conditions, there is no effect of NGF on melanocyte cell yields or melanogenesis, both NGF and NT-3, the latter expressed by dermal fibroblasts [15], increase melanocyte survival when the cells are maintained in medium depleted of growth factors [15, 37]. Interestingly, phorbol 12-tetra decanoate 13 acetate, a strong activator of protein kinase C, upregulates the expression of p75NTR and induces the expression of TrkA in melanocytes [15]. Although the exact mechanism that regulates phorbol 12-tetra decanoate 13 acetate-induced p75NTR and TrkA upregulation is not known, phorbol 12-tetra decanoate 13 acetate is recognized to have a striking effect also on melanocyte dendricity. It is possible that this differentiated morphology of melanocytes is part of an integrated complex of differentiated functions that includes induction of receptors to NGF. In contrast with TrkA expression that requires induction, melanocytes constitutively express TrkC, albeit the
Neurotrophins and Skin Aging
expression is likely to be low as it was detected by the sensitive reverse transcriptase-PCR methodology [15]. Also, in contrast with TrkA expression, TrkC expression is decreased after phorbol 12-tetra decanoate 13 acetate, suggesting that although melanocytes can bind both NGF and NT-3, different signals that preferentially induce a specific high-affinity receptor determine which NT would exert its effect. Thus, the effects of NGF and NT-3 on melanocytes may be influenced by outside signals through modulations of their high-affinity receptor expression. Indeed, using UV-irradiated cultured melanocytes and human melanoma cells, NGF supplementation enhances cell survival, markedly reduces apoptotic cell death, and increases the level of the antiapoptotic Bcl-2 protein, which is expressed strongly by melanocytes in vivo even in the absence of UV irradiation [23, 37]. The data suggest that NGF, which is constitutively produced by neighboring epidermal keratinocytes, may preserve the population of cutaneous melanocytes that would otherwise be depleted by sun exposure. In contrast, NT-3, which is strongly expressed by nonproliferating fibroblasts [15], like those in the dermal compartment of nondamaged human skin, could help in melanocyte maintenance during steady state conditions.
NTs Expression in Human Skin Decreases with Aging Recent studies have revealed that the expression of NTs and Trk receptors within the human skin decreases with aging. NGF is a member of a family of structurally and functionally related polypeptides known as the neurotrophins (NTs) [38]. Since its discovery, NGF is known to guide and sustain neuronal development and differentiation within peripheral neural networks. NGF can regulate tissue morphogenesis, remodeling, proliferation, and apoptosis [3]. The common neuroectodermal origin of the cutaneous epithelium and the nervous system makes it reasonable to hypothesize that the same growth factors, which govern the development, and maintenance, of neurons are also involved in skin morphogenesis [3]. The NGF is established locally in the skin by glia cells, epithelial cells, fibroblasts and Merkel cells. The skin is a rich source of NGF and the epidermis is recognized as a site of NGF expression. Shortly thereafter, it became clear that epidermal keratinocytes are not only important NGF sources, but also are NT targets expressing NT receptors. In murine skin organ cultures, NGF is produced by keratinocytes. Also, NGF
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stimulates the proliferation and inhibits the apoptosis in cultured human epidermal keratinocytes. Interestingly, NGF is critical for proper innervation of this peripheral sensory organ. Thus, defects in NTs singling are associated with severe sensory skin disorders that inhibit wound healing [3].
Downregulation of NGF Protein Expression with Aging The expression pattern of NGF was examined previously in different age groups. NGF protein expression exhibited striking age-associated changes within the human epidermis [3]. In sun-protected skin specimens derived from young individuals ( Fig. 15.4). In contrast, in the skin derived from old donors (>60 years), no or only a weak NGF expression was detected (> Fig. 15.4). Semiquantitative analysis of NGF immunostaining revealed that NGF protein expression values were significantly higher in young ages than in old ages (> Tables 15.1 and > 15.2). NGF immunoreactivity was strongest in the ages 6, 15, and 18 years and decreased gradually in the ages 33 and 39 years (> Fig. 15.4). In the young ages, the expression was detected in all layers of the epidermis except for the stratum corneum (> Fig. 15.4a–c), whereas in the middle ages (33 and 39 years) the expression was confined to the Malpighian layer and stratum spinosum (> Fig. 15.4d, e). In the old ages (>60 years), NGF expression was dramatically reduced and the expression was detected mainly in the stratum basale (> Fig. 15.4f–j). Among old ages (60–81 years), the expression was strongest in the age of 60 years (> Fig. 15.4f ), whereas it was greatly diminished in older ages (> Fig. 15.4g–i) until it became completely negative in the age of 81 years (> Fig. 15.4j). Although NGF expression was seen in the stratum granulosum of the skin derived from certain old ages (68- and 78-year-old donors), its immunoreactivity was weak (> Fig. 15.4g, i). The level of NGF protein in the sweat glands does not apparently differ with aging (> Fig. 15.5). The age-related decrease of NGF protein expression in the human epidermis is parallel with its level in the nervous system. There was a gradual reduction of NGF levels with aging in the brain and thymus of rats; and a low amount of NGF protein in the plasma of old subjects. Also, the administration of NGF in rats can reduce agerelated atrophy of the neurons. Similarly in human,
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. Figure 15.4 Nerve growth factor protein expression in human skin derived from different aged donors shown in red color with tyramide signal amplification (TSA) technique. (A–E) Young age group (5–39 years). (A) 6Y, (B) 15Y, (C) 18Y, (D) 33Y, and (E) 39Y. (F and G) Old age group (60–81 years). (F) 60Y, (G) 68Y, (H) 71Y, (I) 78Y, and (J) 81Y. Magnification: 200¥ (Reprinted with permission from Adly et al. [3] ) ß 2006, Elsevier
administration of NGF can delay retinal degeneration in patients with retinitis pigmentosa [39, 40]. The strong expression of NGF protein in the skin of young individuals may be due to an increased receptormediated internalization of NGF proteins released by nerve endings [41, 42], or altered expression of certain cytokines, such as tumor necrosis factor alpha (TNF-a), that influence the synthesis of NGF. In transgenic mice, the basal level of brain NGF can be influenced negatively or positively by local expression of TNF-a [19, 43]. The decrease of NGF protein expression in aged skin may reflect impairment of these mechanisms or a reduction in the number of high-affinity NGF binding sites. Indeed,
as women age, they become hypo-estrogenic; therefore, a hypothesis to be tested is that NGF changes with aging might by related to hormonal change, and not as much with aging. To test this hypothesis, NGF protein expression in skin of male subjects need to be examined in this type of studies.
Downregulation of NT-3 Protein Expression with Aging Similarly, NT-3 protein expression in human skin underwent age-associated decrease (Adly MA, 2009). In young
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Neurotrophins and Skin Aging
. Table 15.1 Nerve growth factor protein expression in human skin of different ages. The staining results were examined by the authors and were scored as (–) for absent, (+) for weak, (++) for medium, and (+++) for intense nerve growth factor (NGF) protein expression Age (years)
Stratum basale
Stratum spinosum
Stratum granulosum
Stratum corneum
6
+++
+++
+++
–
8
+++
+++
+++
–
11
+++
+++
+++
–
13
+++
+++
+++
–
15
+++
+++
+++
–
18
+++
+++
+++
–
32
+++
++
++
–
33
+++
++
++
– –
34
+++
++
++
36
+++
++
++
–
37
+++
++
++
–
39
+++
++
++
–
60
++
+
–
–
64
+
+
–
–
68
+
–
–
–
71
+
–
–
–
74
+
–
–
–
76
+
–
–
–
78
+
–
–
–
81
+
–
–
–
. Table 15.2 Expression values of nerve growth factor in human skin of different ages. The immunoreactivity score (IR score) was evaluated by multiplying the percentage of positive cells (PP%) and the staining intensity (SI). First, the PP% was scored as 0 for 75%. Second, the SI was scored as 1 for weak, 2 for medium, and 3 for intense staining, following other groups. Values between brackets represent the standard errors of mean Basal layer Age groups (years)
SI
Spinous layer
Granular layer
PP
IRS
SI
PP
IRS
SI
PP
IRS
6–18
2.8 {0.2}
3.8 {0.2}
11.0 {1.0}
2.8 {0.1}
3.8 {0.2}
11.0 {1.0}
2.8 {0.2}
3.7 {0.2}
10.5 {1.0}
19–50
2.8 {0.1}
3.7 {0.3}
6.8 {1.1}
1.8 {0.1}
3.1 {0.1}
5.7 {0.3}
1.7 {0.3}
2.6 {0.4}
5.1 {0.8}
51–81
1.0 {0.0}
1.8 {0.1}
1.8 {0.1}
1.1 {0.1}
1.4 {0.4}
1.4 {0.4}
0.75 {0.1}
0.70 {0.1}
0.9 {0.1}
ages, NT-3 expression was very strong and immunoreactivity was detected in almost all layers of the epidermis, including the stratum corneum in the ages below 20 years. Alternatively, in old ages, the NT-3 expression was very weak or completely absent.
Conclusion The neurotrophins are a family of polypeptide growth factors. These proteins are critical not only for the development, but also for the maintenance of the vertebrate
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. Figure 15.5 Nerve growth factor protein expression in sweat glands derived from different aged donors. The reactivity appeared as red color (tyramide signal amplification [TSA] technique). Nerve growth factor (NGF) expression in the following ages in years (Y). (a) 6Y, (b)33Y, (c) 39Y, (d) 71Y, (e) 78Y, and (f) 81Y(Reprinted with permission from Adly et al. [3] ) ß 2006, Elsevier
nervous system. Recently, several leads indicate that these factors could have a broader role than their name might suggest, in particular, the putative role of NGF, and its receptor TrkA and NT-3 in cutaneous homeostasis and in skin aging. To date knowledge about the expression pattern of neurotrophins in skin
remains rudimentary. The chapter discussed the expression of neurotrophins and their receptors in different cutaneous structures based on the data obtained from the studies of the human scalp skin. The clinical and therapeutic ramifications of these studies are open for further investigations.
Neurotrophins and Skin Aging
References 1. Botchkareva NV, Botchkarev VA, Welker P, Airaksinen M, Roth W, Suvanto P, Muller-Rover S, Hadshiew IM, Peters C, Paus R. New roles for glial cell line-derived neurotrophic factor and neurturin: involvement in hair cycle control. Am J Pathol. 2000;156(3): 1041–1053. 2. Botchkarev VA, Botchkareva NV, Peters EM, Paus R. Epithelial growth control by neurotrophins: leads and lessons from the hair follicle. Prog Brain Res. 2004;146:493–513. 3. Adly MA, Assaf H, Hussein MR. Age-associated decrease of the nerve growth factor protein expression in the human skin: preliminary findings. J Dermatol Sci. 2006;42(3):268–271. 4. Teng KK, Hempstead BL. Neurotrophins and their receptors: signaling trios in complex biological systems. Cell Mol Life Sci. 2004; 61(1):35–48. 5. Segal RA. Selectivity in neurotrophin signaling: theme and variations. Annu Rev Neurosci. 2003;26:299–330. 6. Dechant G, Barde YA. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat Neurosci. 2002;5(11):1131–1136. 7. Nykjaer A, Willnow TE, Petersen CM. p75NTR–live or let die. Curr Opin Neurobiol. 2005;15(1):49–57. 8. Yaar M, Zhai S, Fine RE, Eisenhauer PB, Arble BL, Stewart KB, Gilchrest BA. Amyloid beta binds trimers as well as monomers of the 75-kDa neurotrophin receptor and activates receptor signaling. J Biol Chem. 2002;277(10):7720–7725. 9. Peters EM, Hansen MG, Overall RW, Nakamura M, Pertile P, Klapp BF, Arck PC, Paus R. Control of human hair growth by neurotrophins: brain-derived neurotrophic factor inhibits hair shaft elongation, induces catagen, and stimulates follicular transforming growth factor beta2 expression. J Invest Dermatol. 2005;124 (4):675–685. 10. Peters EM, Hendrix S, Golz G, Klapp BF, Arck PC, Paus R. Nerve growth factor and its precursor differentially regulate hair cycle progression in mice. J Histochem Cytochem. 2006;54 (3):275–288. 11. Botchkareva NV, Botchkarev VA, Albers KM, Metz M, Paus R. Distinct roles for nerve growth factor and brain-derived neurotrophic factor in controlling the rate of hair follicle morphogenesis. J Invest Dermatol. 2000;114(2):314–320. 12. Sariola H. The neurotrophic factors in non-neuronal tissues. Cell Mol Life Sci. 2001;58(8):1061–1066. 13. Ernfors P, Lee KF, Jaenisch R. Target derived and putative local actions of neurotrophins in the peripheral nervous system. Prog Brain Res. 1994;103:43–54. 14. Yaar M, Grossman K, Eller M, Gilchrest BA. Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J Cell Biol. 1991;115(3):821–828. 15. Yaar M, Eller MS, DiBenedetto P, Reenstra WR, Zhai S, McQuaid T, Archambault M, Gilchrest BA. The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes. J Clin Invest. 1994;94(4):1550–1562. 16. Botchkarev VA, Metz M, Botchkareva NV, Welker P, Lommatzsch M, Renz H, Paus R. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as ‘‘epitheliotrophins’’ in murine skin. Lab Invest. 1999;79(5):557–572. 17. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M, Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity
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receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression. Br J Dermatol. 2005;153(3):514–520. Bonini S, Rasi G, Bracci-Laudiero ML, Procoli A, Aloe L. Nerve growth factor: neurotrophin or cytokine? Int Arch Allergy Immunol. 2003;131(2):80–84. Aloe L. Nerve growth factor, human skin ulcers and vascularization. Our experience. Prog Brain Res. 2004;146:515–522. Di Marco E, Marchisio PC, Bondanza S, Franzi AT, Cancedda R, De Luca M. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes. J Biol Chem. 1991;266(32):21718–21722. Adly MA, Assaf HA, Nada EA, Soliman M, Hussein M. Expression of nerve growth factor and its high-affinity receptor, tyrosine kinase A proteins, in the human scalp skin. J Cutan Pathol. 2006;33 (8):559–568. Pincelli C, Sevignani C, Manfredini R, Grande A, Fantini F, BracciLaudiero L, Aloe L, Ferrari S, Cossarizza A, Giannetti A. Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes. J Invest Dermatol. 1994;103(1):13–18. Stefanato CM, Yaar M, Bhawan J, Phillips TJ, Kosmadaki MG, Botchkarev V, Gilchrest BA. Modulations of nerve growth factor and Bcl-2 in ultraviolet-irradiated human epidermis. J Cutan Pathol. 2003;30(6):351–357. Paus R, Luftl M, Czarnetzki BM. Nerve growth factor modulates keratinocyte proliferation in murine skin organ culture. Br J Dermatol. 1994;130(2):174–180. Marconi A, Vaschieri C, Zanoli S, Giannetti A, Pincelli C. Nerve growth factor protects human keratinocytes from ultraviolet-Binduced apoptosis. J Invest Dermatol. 1999;113(6):920–927. Marconi A, Terracina M, Fila C, Franchi J, Bonte F, Romagnoli G, Maurelli R, Failla CM, Dumas M, Pincelli C. Expression and function of neurotrophins and their receptors in cultured human keratinocytes. J Invest Dermatol. 2003;121(6):1515–1521. Pincelli C. Nerve growth factor and keratinocytes: a role in psoriasis. Eur J Dermatol. 2000;10(2):85–90. Pincelli C, Haake AR, Benassi L, Grassilli E, Magnoni C, Ottani D, Polakowska R, Franceschi C, Giannetti A. Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2. J Invest Dermatol. 1997;109(6):757–764. Pincelli C, Yaar M. Nerve growth factor: its significance in cutaneous biology. J Invest Dermatol Symp Proc. 1997;2(1):31–36. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. J Invest Dermatol. 2000;115 (2):141–148. Botchkarev VA, Botchkareva NV, Albers KM, Chen LH, Welker P, Paus R. A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression. FASEB J. 2000;14(13): 1931–1942. Adly MA, Assaf HA, Hussein MR. Expression pattern of p75 neurotrophin receptor protein in the human scalp skin and hair follice: Hair cycle-dependent expression. J Am Acad Dermatol. 2009;60 (1):99–109. Krygier S, Djakiew D. The neurotrophin receptor p75NTR is a tumor suppressor in human prostate cancer. Anticancer Res. 2001;21(6A):3749–3755. Nakagawara A. Trk receptor tyrosine kinases: a bridge between cancer and neural development. Cancer Lett. 2001;169 (2):107–114.
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35. Peacocke M, Yaar M, Mansur CP, Chao MV, Gilchrest BA. Induction of nerve growth factor receptors on cultured human melanocytes. Proc Natl Acad Sci USA. 1988;85(14):5282–5286. 36. Di Marco E, Mathor M, Bondanza S, Cutuli N, Marchisio PC, Cancedda R, De Luca M. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop. J Biol Chem. 1993;268(30):22838–22846. 37. Zhai S, Yaar M, Doyle SM, Gilchrest BA. Nerve growth factor rescues pigment cells from ultraviolet-induced apoptosis by upregulating BCL-2 levels. Exp Cell Res. 1996;224(2):335–343. 38. Alleva E, Petruzzi S, Cirulli F, Aloe L. NGF regulatory role in stress and coping of rodents and humans. Pharmacol Biochem Behav. 1996;54(1):65–72. 39. Alberch J, Perez-Navarro E, Arenas E, Marsal J. Involvement of nerve growth factor and its receptor in the regulation of the cholinergic function in aged rats. J Neurochem. 1991;57(5):1483–1487.
40. Garcia-Suarez O, Germana A, Hannestad J, Perez-Perez M, Esteban I, Naves FJ, Vega JA. Changes in the expression of the nerve growth factor receptors TrkA and p75LNGR in the rat thymus with ageing and increased nerve growth factor plasma levels. Cell Tissue Res. 2000;301(2):225–234. 41. Backman C, Rose GM, Hoffer BJ, Henry MA, Bartus RT, Friden P, Granholm AC. Systemic administration of a nerve growth factor conjugate reverses age-related cognitive dysfunction and prevents cholinergic neuron atrophy. J Neurosci. 1996;16(17):5437–5442. 42. Amendola T, Aloe L. Developmental expression of nerve growth factor in the eye of rats affected by inherited retinopathy: correlative aspects with retinal structural degeneration. Arch Ital Biol. 2002; 140(2):81–90. 43. Aloe L. Rita Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology. Trends Cell Biol. 2004;14(7):395–399.
Pathology
28 Pathology of Aging Skin Qunshan Jia . J. Frank Nash
Introduction Human skin is the largest and the most complex organ functioning as a physical and biochemical barrier to protect the human body from water loss as well as environmental insults including pathogens, chemicals, physical agents and solar ultraviolet radiation (UVR) throughout life. More than that, the skin provides crucial physiological functions including immune defense, thermoregulation, sensoring, endocrine as well as metabolism. Aging is a chronological process accompanied by a progressive loss of physiological function in multiple organs. Skin undergoes an aging process accompanied by physical changes, clinical manifestations and significant psychological consequences. According to recent statistics, around 25% of Americans are expected to be 65 years or older by the year 2030 [1]. Therefore, it is important to understand the chronological skin aging process and its accompanying physiological consequences for medical reasons and for the personal care industries.
Skin Development and Anatomic Structure To better understand the pathology and physiology of the aging skin, it is crucial to understand the development, anatomical structure and physiological function of normal skin.
The Structure and Function of Skin Barrier The skin is structurally divided into epidermis and dermis separated by the basal lamina. Epidermis is the major protective outer layer, with keratinocytes as the major cell population. The epidermis is a stratified epithelium derived from a single layer of ectoderm after gastrulation. Wnt/BMP signaling enables the ectoderm cells to adopt an epidermal fate instead of neurogensis by inhibiting fibroblast growth factor (FGF) signaling pathways. Over time, the embryonic epidermis will differentiate
into epidermal cells under the influences of BMP, Notch signaling, the hair placode and eventually the hair follicle induced by Wnt signaling and its downstream signaling including SHH, GLI1, PTC. The sebaceous glands are appendages of hair follicles, the origin of which remains unknown [2]. The stratified epidermis is around 100–150 m thick, which can be further divided into four distinct layers: stratum basale, stratum spinosum, stratum granulosum and stratum corneum, as illustrated in > Fig. 28.1. The stratum basale consists of epidermal stem cells, a single layer of columnar cells attached to the basal lamina via hemidesmosomes and expressing keratin(K)14 and K5. Once the cell leaves the basal layer toward the skin surface, it starts to express K1 and K10 in the stratum spinosum. The stratum spinosum, which contains lipid-enriched lamellar bodies, becomes progressively larger due to keratin synthesis and lipogenesis and finally reaches a stage called stratum granulosum with unique lamellar bodies and is ready to differentiate into corneocytes. Finally, intracellular organelles in the corneocyte undergo selfdestruction and the lipid packaged in lamellar granules (LG) is released into the intercellular space. The LGs are small organelles full of stacks of lipid lamelle consisting of phospholipids, cholesterol, glucosylceramides and several enzymes important for the lipid processing including acid hydrolases, ß-glucocerebrosidase, phospholipid A2 and lysosomal acid lipase [3]. Once released from the LGs, the short stacks of lipid membranes will reorganize and transform into an edge-to-edge fusion catalyzed by enzymes released at the same time. In the end, the stratum corneum will form the outermost seal with 18–21 cell layers 20–40 micrometer thick in human skin mainly consisting of dead corneocytes and the secreted lipids [3]. The brick and mortar structure is a classic model for the organization of the stratum corneum (> Fig. 28.2). The most important function of the stratum corneum includes prevention of water loss and percutaneous absorption of xenobiotics (> Table 28.1) [4]. During the final stages of keratinocyte differentiation, the intracellular keratins and filaggrin will interact with each other to form a condensed protein complex, transforming the epidermal
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_28, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 28.1 The structure of the epidermis. The epidermis contains stratum corneum, stratum granulosum, stratum spinosum and stratum basale form outside to inside. The stratum basale consists of epidermal stem cells, a single layer of columnar cells attached to the basal lamina via hemidesmosomes. The stratum spinosum is rich with lipid-enriched lamellar bodies, become progressively larger due to the keratin synthesis and lipogenesis and finally reach a stage called stratum granulosum which has unique lamellar bodies and is ready to differentiate into corneocyte. Finally, corneocyte intracellular organelles undergo self destruction and the lipid packaged in lamellar granules (LG) is released to the intercellular space. The stratum corneum forms the outermost seal with 18–21 cell layers 20–40 micrometer thick in human skin mainly consisting of dead corneocytes and the secreted lipids. Filaggrin is a key protein required for the formation of the stratum corneum (SC) barrier which is also essential for SC hydration, as it acts as a source of hygroscopic amino acids and their derivatives, known as natural moisturizing factor (NMF)
. Figure 28.2 The simple ‘‘brick’’ and ‘‘mortar’’ model for stratum corneum. The dead corneocytes were embedded in the intercellular lipid matrix, which forms the primary barrier for the skin
Pathology of Aging Skin
28
. Table 28.1 Multiple protective functions of mammalian stratum corneum (Adapted from Elias PM. [4]) Function
Principal compartment
Structure basis
Chemical basis
Permeability
Extracellular matrix
Lamellar bilayers
Ceramides, cholesterol, nonessential fatty acids in proper ratio
Antimicrobial
Extracellular matrix
Lamellar bilayers
Antimicrobial peptides, FFA, sph
Antioxidant
Extracellular matrix
Lamellar bilayers
Chol, FFA; secreted vit. E, redox gradient
Cohesion (integrity)
Extracellular matrix
Comeodesmosomes(CD)
Intercellular DSG1/DSC1 homodimers
Mechanical/ rheological
Corneocyte
Cornified envelope; keratin filaments
g-Glutamyl isopeptide bonds
Chemical (antigen exclusion)
Extracellular matrix
Extracellular lacunae
Hydrophilic products of CD
Psychosensory interface
Extracellar matrix
Lamellar bilayers
Barrier lipids
Hydration
Corneocyte
Cytosol
Filaggrin proteolytic product; glycerol
Electromagnetic radiation
Corneocyte
Cytosol
Cis-urocanic acid (histidase activity)
Initiation of inflammation
Cornocyte
Cytosol
Proteolytic activation of pro IL-1a/b
cell into a flatten corneocyte. The ‘‘bricks’’ are the multiple layers of protein-enriched dead corneocytes tightly packed and surrounded by a very dense cross-linked protein structure called the crucified envelope. This protein envelope is cross-linked with the surrounding lipid envelope, the ‘‘mortar’’, which is made up of hydrophobic lipid lamellae in the intercellular regions and gives the skin physical protection against water and other molecules [5]. Multiple genes have been identified which play important roles in skin barrier function. For example, aberrant expression of filaggrin will result in multiple barrier defects from atopic dermatitis to flaky skin [6]. Involucrin, loricrin and trihohyalin are major protein components on the surface of the corneocyte forming a protein envelope cross-linked by transglutaminases. Excessive loss of water and neonatal death has been observed in transglutaminase-deficient mice, illustrating the importance of the stratum corneum’s barrier function during development [7]. The specific cell–cell junction between stratum corneum and stratum granulosum, named desmosomes, is also critical, the loss of which disrupts the stratum cell adhesion resulting in barrier function defects [8]. The lipid composition of the stratum corneum is critical for barrier function. Studies have shown that the
stratum corneum mainly consists of equimolar ratios of ceramides, cholesterol and free fatty acids. Ceramides are crucial in the formation of the lipid envelope and a deficiency is associated with atopic dermatitis [9]. Cholesterol is synthesized by the epidermis and is important for intermixing different lipids. The cholesterol efflux is regulated by a membrane transporter named ATP binding cassette subgroup 1 member transporter or ABCA12. Failure of different enzymes involved in cholesterol synthesis results in significant epidermal barrier defects [6]. Finally, essential fatty acids deficiency will also result in a red, rough skin with significant transepidermal water loss [10, 11] and disruption of gene coding for fatty acid transport protein 4 (FatP4) has been found to result in neonatal death due to disturbed skin permeability [12]. The nucleated lower layers of the epidermis are important for mediating barrier function. The entire loss of the epidermis will lead to life-threatening water loss and rampant microbiological infection, compared to the relatively mild consequences associated with stratum corneum damage [6]. Multiple cell–cell tight junction proteins such as claudins, zonula occludens protein-1, and multi-PDZ protein-1 have been found in the stratum granulosum and the upper stratum spinosum. The
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importance of these tight junctions is illustrated by extreme water loss in mice with claudin-1 and E-cadherin deficiency. Adherens and desmosomes are a group of junction proteins essential for stabilizing the cell–cell adhesion in the living layer of the epidermis. Reducing desmoglein-3 and E-caldherin will result in a barrier defect leading to either leaky tight junctions or a lethal dehydration [6]. The intercellular gap junctions are important channels for cell–cell communication. Disturbing such gap junctions, found in connexin43 loss of function mouse studies, results in barrier defects [6]. Keratins are the major component of intra-keratinocyte filament web which interacts with the junction proteins, desmosomes and hemidesmosomes. Homozygous K10 knock-out mice will develop an extremely delicate epidermis while heterozygous K10 mice will show impaired barrier function repair and skin hydration defects, demonstrating the importance of this differentiation-related protein’s effect on epidermal barrier function [6].
Epidermis Homeostasis and Turnover In healthy skin, the epidermis replenishes itself and maintains a status of homeostasis in response to physical and chemical challenges. In humans, the turnover rate for the epidermis is approximately 4 weeks. During this process, cells in the stratum basale divide and move upward while undergoing differentiation during this progression. Both symmetric and asymmetric models have been used to describe this process. In the symmetric model, the stratum basale daughter cells with lower levels of integrin after mitosis will detach and begin the journey to becoming part of the stratum corneum. Daughter cells with higher levels of integrin will keep their proliferating potential and stay attached to the basement membrane. In the asymmetric model, the mitotic epidermal cells divide disproportionately so that the attached daughter cells receive more integrin and growth factors to keep them attached to the basement membrane and proliferating while the detached suprabasal daughter cells receive less or dissimilar signals such as Notch, leading to differentiation and commitment to stratum corneum replacement [2]. Proliferation and growth of cells in the stratum basale must be tightly controlled to ensure the normal function of the stratum corneum. Recent studies have shown that the extracellular matrix (ECM) and growth factors residing in the basement membrane play a crucial role in controlling cell proliferation in the stratum basale. Two
types of junctions are responsible for the cell–ECM connection. The hemidesmosomes containing a6b4 integrin and the focal adhesions containing a3b1 integrin control growth and migration through a physical interaction with kinases such as Ras/MAPK signaling. The E-caldherin in cell–cell junctions also contributes to basal cell proliferation and migration through its association with regulatory proteins such as Rho-GTPase. Although the exact mechanisms are unclear, it is accepted that the adherens junction can serve as a signaling center and sense the epidermal cell density and thus provide a feedback loop through specific signaling kinase pathways to control cell activity and proliferation [2]. The epidermal barrier function is also maintained by multiple signaling pathways as indicated by studies of acute skin disruption. For example, proinflammatory cytokines including interleukin-1, interleukin-6 as well as tumor necrosis factors have been observed following acute barrier damage. As well, knocking down these genes will delay barrier formation after acute disruption [6]. Recently, a calcium gradient was identified in the epidermis with the highest calcium concentration in stratum granulosum and the lowest concentration in stratum corneum. Calcium is crucial for lamellar body exocytosis and epidermal protein synthesis at the later stage of keratinocyte differentiation and migration in response to barrier permeability damage. In addition, calcium plays important roles for transglutaminase I activity and cell–cell adhesion during epidermal differentiation. The relationship between the calcium level and the transmembrane water loss has been observed in Darier’s and Hailey-Hailey diseases caused by the defects of a gene encoding a calcium transporter [6]. Another signaling pathway important in maintaining the barrier is 3’5’-cyclic adenosine monophosphate (cAMP), a secondary messenger in multiple systems. Biochemistry studies have revealed a reverse relationship between the cAMP level and the barrier recovery after acute epidermal damage. Vascular endothelial growth factor (VEGF) generated by the epidermis is also required for the integrity of the barrier function with homozygous VEGF knock-out mice showing impaired permeability barrier homeostasis after acute repeated tape striping. Finally, a new mechanism has been suggested for maintaining epidermal homeostasis in which both the lipid matrix and corneocytes are involved. In general, the disruption of stratum corneum will inevitably increase cytokines and calcium concentrations within the epidermis, which in turn will promote lipid synthesis and secretion into the intercellular matrix to facilitate barrier recovery. At the same time, epidermal cornification will be enhanced by serine protease and caspase-14
Pathology of Aging Skin
signaling turned on by protease activated receptor type 2 (PAR2) in response to acute barrier defects [13]. In addition to the keratinocytes, several other cellular populations have also been found in the epidermis including Langerhans cells, Merkel cells, and melanocytes. Langerhans cells are dendritic cells residing in the epidermis involved in antigen presentation and immunoserveillance [14]. Melanocytes are cells derived from neural crest responsible for skin pigment and hair color through melanin production. They are usually found in the basal layer of epidermis and in hair follicle. The Merkel cells are also derived from the neural crest cells and are associated with the sensory nerve endings. The dermis, located below the epidermis basement membrane, is rich with blood supply providing nutrients and circulatory support to the epidermis. In humans, the boundary between the epidermis and dermis undulates due to epidermal protrusions into the dermis resulting in the ‘‘rete pegs’’. The cellular populations account for 10% of the dermal content including the fibrocyte, monocyte, histiocyte, Langerhans cells, lymphocytes, and eosinophils, along with the vascular- and lymphatic-associated cells while the remaining 90% is mainly connective tissue matrix made up of Type I collagen, elastic fibers, and blood vessels. The collagen in the dermis provides mechanical protection to the body as well as the shape and form by holding all structures together. The blood plexus provides oxygen and nutrients to the living part of the epidermis and removes waste products of metabolism from the epidermis. Body temperature regulation through control of blood flow and sweating is also achieved by dermis as well as the sensations of touch, pain, heat and cold through the neural fiber embedded in the dermis [15].
Skin Physiology Immune Function While the skin acts as the principle barrier to protect the body from the water loss, it also serves as a non-specific defense against infections. Phagocytic cells such as neutrophils, macrophages and natural killer cells (NK) present in the skin play a significant role in defense against pathogens. Meanwhile the complement system and cytokines turned on by components of pathogens, e.g., LPS, also contribute to the innate immune response of the skin. Both the innate and adaptive immune responses are equally critical to the host defense against foreign invaders. The skin possesses important peripheral components such as Langerhans cells (LC) and extravasated lymphocytes from
28
circulation for adaptive immune responses. The dendritic LC will process and present an antigen to the resident T lymphocytes which will initiate a cell-mediated acquired immune response [16–19]. Generally, the skin will initiate a rapid cytokine-driven cutaneous inflammation in response to keratinocyte injury followed by a specific adapted immune response to foreign antigens with high specification and immune memory. Recent studies have shown the epidermal keratinocyte cell–cell junctions will promote the immune response after the skin is injured. It has been found that b-catenins in cell–cell junctions function not only as adhesion molecules but also as transcription regulators. Through unknown mechanisms, p120 catenin and a-catenin can affect the transcriptional activity of NF-kB to induce cytokines. It is hypothesized that keratinocyte injury will trigger the catenin/NF-kB signaling cascade, inducing cytokines, chemokines which will recruit immune cells to initiate the innate immune response first, and adapted immune response later. Once the epidermis is repaired and the cell–cell junction returns to normal, the inflammation will diminish. This is supported by the findings that chronic inflammation will develop after a-catenin and p120 are mutated [2, 18]. It has also been found that acute barrier disruption not only increased hapten infiltration but also promoted skin immune function by promoting the production of cytokines on epidermal cells and upregulation of co-stimulatory molecules on Langerhans cells. Thus, the intact skin barrier function is of importance for skin immune function.
Skin Metabolism In addition to its physical barrier, the skin also provides a biochemical barrier by virtue of enzymatic activities in epidermis and dermis. This enzymatic and biochemical barrier enable the skin to biotransform topically applied xenobiotics and to function as an important extrahepatic organ. Most of the metabolizing enzymes are located in the epidermis [20]. For example, cytochrome P-450 is present in the skin and responsible for the oxidative metabolism of steroid hormones including androgens, estrogens, progesterone and glucocorticoids, as well as fatty acids. As the major Phase-I enzyme, P-450 plays a critical role in metabolic detoxification or, in some cases, activation. The presence of Phase-II conjugating enzymes such as glucoronyltransferase, sulfotransferase and glutathioneS- transferase enable the skin to further detoxify and eliminate the metabolites generated from Phase-1 reactions. The major enzymes found in the skin and involved in metabolism are presented in > Table 28.2 [21]. An
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. Table 28.2 Classes of enzymes detected in skin (Adapted from Smith et al. [21]) Phase I enzymes activaton/ functionalization mechanisms
Function
Microsomal mixed function oxidase
Hydroxylation
Cytochrome P450
Alcohol oxidation
NADPH dehydrogenase
Epoxidation N-, O- and S-dealkylation oxidative deamination N- and S-oxidation Dehalogenation Reduction of azo and nitro compounds Ring cleavage of heterocyclic ring systems
Alcohol dehydrogenases
Interconversion of alcohols and aldehydes
Aldeyde dehydrogenases
Conversion of aldehydes to acids
Flavin-containing monooxygenases
Oxidation of secondary and tertiary amines Oxidation of imines and arylamines
Esterases
Ester hydrolysis to yield acid and alcohol
Amidases
Amide hydrolysis
Phase II enzymes and conjugation reactions UDP-glucuronosyltransferase
Glucuronidation
Sulphotransferase
Sulphation
Methyltransferase
Methylation
Acetyltransferase
Acetylation
Glutathione S-transferase
Glutathione conjugation at epoxides and halides
Miscellaneous reactions condensation
Amino acid conjugation
example of metabolic activation is the enzyme alcohol dehydrogenase which is present in keratinocytes and involved in the transformation of the innocuous chemical cinnamic alcohol into cinnamic aldehyde, a potent skin sensitizer. Finally, a recent study reported that the metabolic rate in the skin affects dermal penetration of lipophilic compounds with a higher metabolic rate generally correlated with greater penetration [22].
Skin Sensory and Thermoregulation Advances in immunochemical staining have revealed the presence of neuronal fibers in the epidermis. More than 90% of these are small-diameter, unmyelinated C-fiber and/or thinly myelinated A-d fibers located in the border between of the basement membrane and epidermis. The major functions of cutaneous neuronal fibers are sensory and integration of incoming signals for pain, itch and other stimuli. The epidermal nervous system possesses important efferent paracrine and trophic functions that affect cutaneous cells, immune cells, and other axons. This is supported by findings that nociceptive nerve endings have close contacts with LC, mast cells and even keratinocytes. The trophic effects of this innervation were identified in studies where nerve endings were cut resulting in a reduction of the keratinocyte mitotic rate and a ‘‘shiny atrophic’’ skin. In addition, most dermal vessels, eccrine sweat as well as arrector pili muscles are densely innervated by the neuron fibers [23]. Thermal regulation of the human body is controlled by receptors located in the skin called transient receptor potential (TRP) superfamily which are cation-selective ion channels consisting of six transmembrane subunits. TRPs are activated within a specific temperature range in response to the environmental temperature changes. With the cooperation of central nerve system, healthy individuals will get rid of excess heat by sweating and vasodilation when it is too hot and minimize heat loss through vasoconstriction, and increase heat generation by shivering when it is too cold. The sweat glands dispersed within the skin control body temperature by energy demanding sweat evaporation. Up to 2 L of sweat can be evaporated in an hour mainly by the eccrine glands. This control for body temperature through sweat production is important for thermal maintenance [24].
The Structural and Physiological Changes in Aged Skin Chronological aging is an inevitable biological process leading to structural and functional changes during the lifespan of all organisms. This programmed route is inherently determined by genetics and is significantly affected by multiple environmental factors. Dryness, wrinkles, irregular pigmentation are the primary visual findings of aging skin in human beings accompanied by histological changes ranging form impaired stratum corneum replenishment to a flattening of the dermal–epidermal junction, a marked elasticity loss and atrophy of the dermal
Pathology of Aging Skin
. Table 28.3 Characteristic of intrinsic aging (Adapted from Farage et al. [88]) Characteristic
Intrinsic aging
Overall Metabolic processes
Slow down
Clinical appearance
Smooth unblemished, loss of elasticity, fine wrinkles
Skin color
Pigment diminishes to pallor
Skin surface marking
Maintains youthful geometric patterns
Onset
Typically 50s–60s (woman earlier than men)
Severity
Only slightly associated to degree of pigmentation
Epidermis Thickness
Thins with aging (not consistent)
Proliferative rate
Lower than normal
Keratinocytes
Modest cellular irregularity
Dermoepidermal junction
Modest reduplication of lamina dense
Dermis Elastin
Elastogenesis followed by elastolysis
Elastin matrix
Gradual decline in production of dermal matrix, only modest increase in the number and thickness of elastic fibres in the reticular dermis
connective tissue due to a reduction and disorganization of its major extracellular matrix components [25–27]. The characteristic histological changes and its impacts on barrier function during aging will be reviewed (> Table 28.3).
Anatomic Changes in Aging Skin Epidermis The overall histology change in epidermal thickness is neither obvious nor consistent with the conclusion that this structure is ‘‘thinner’’ in aged skin [28]. There is general agreement, however, that the intersection of the epidermis and dermis is flattened in aged skin with a correspondingly diminished connecting surface area leading to increased fragility and reduced nutrient transfer between the dermal and epidermal layers. A significant, 30–50%, decrease in
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epidermal turnover rate has also been observed in the third to eighth decades of life. Such an impaired epidermal turnover rate might account for some of the pathological findings in aged people [15, 25, 26]. Another significant change in the epidermis of aging skin is in the amount of glycosaminoglycans (GAGs), the primary constituents of skin which hold water up to 1,000 times their weight. Hyaluronic acids (HAs) are a major type of GAGs and are produced by fibroblasts in the dermis and keratinocytes in the epidermis. In the epidermis, HAs are localized in the epidermal intercellular spaces at the middle spinous layer. The total amount of HAs is reduced significantly in the epidermis which may contribute to the reduced water binding and the visible changes of the aged skin, including wrinkling, altered elasticity, reduced turgidity and diminished capacity to support the microvasculature of the skin. However, the skin of older subjects has a comparable level of HAs in dermis compared to younger ones [15, 26].
Dermis In contrast to the epidermis, a consistent lost of 20% dermal thickness has been observed in aged skin, characterized by reduced cellular components and vascular networks. For example, in aging skin a reduction of collagen due to decreased synthesis by fibroblasts has been observed. As the primary structural component and the most abundant protein found in dermis, collagen is responsible for conferring strength and support to the structure of human skin. In aged skin, the collagen organization is characterized by disarrayed thickened fibrils in rope-like bundles in comparison to the more structured pattern observed in younger skin. The ratio of collagen types also changes with age due to the loss of collagen I in aged skin. Overall, the collagen content per unit area of skin surface is known to decline approximately 1% per year. The loss of collagen in intrinsically aged skin will lead to an epidermal and dermal atrophy characterized by flattening of the rete ridges and subsequent wrinkle formation (> Table 28.3). The accumulation of broken elastic fibers has been observed as well. Elastin is a connective tissue protein that allows many tissues in the body to resume their shape after stretching or contracting. An accumulation of amorphous elastin material has been associated with aging and attributed to the increased level of matrix metalloproteinases which are thought to play a role in elastin degradation. The aging dermis has a reduced vascular network in comparison to young skin which will result in reduced blood flow, impaired nutrient exchange,
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inhibited thermoregulation, and decreased skin surface temperature. Melanocytes which can synthesize melanin to protect the skin from UV light are also significantly reduced with age by a rate about 8–20% per year. For this reason, sun protection remains critically important for elderly patients [15, 25–27].
The Barrier Function Change of the Aged Skin Basal trans-epidermal water loss (TEWL) has been measured in human and mouse to assess the impact of aging on skin barrier function. The results have revealed no significant change in TEWL in aged subjects (>80 years) compared to the young adult (70 years old [36]. A delayed formation of mature lamellar membrane after 6 hours of disruption was found at the SG-SC boundary in the epidermis of moderately aged subjects accompanied by diminished BGC activity revealed by in situ zymography assay. In addition to the lipid processing defects in the epidermis, an increased pH has been proposed to activate a different family of enzymes, namely the serine proteases, which will degrade the cell–cell junction protein corneodemosomes (CD) disrupting the epidermal integrity in moderately-aged subjects. Histological studies in the epidermis of moderately-aged subjects revealed a significant correlation between a decrease of CD and the impaired SC integrity. The co-location of NHE1 with the BGC and serine proteases at the SC–GC junction and the decrease of NHE1 activity within the moderately aged epidermis
Pathology of Aging Skin
suggested that NHE1 might be responsible for the agingrelated pH increase and barrier defects in moderately-aged epidermis. NHE1 is the only sodium-proton exchanger class of non-energy-dependent transporters expressed in keratinocytes and in epidermis, which has been shown to affect the intracellular pH. Recent studies indicated that NHE1 is located at the SG–SC boundary and deletion of which will reduce SG–SC acidification and impair SC barrier function recovery as well after acute tape stripping. In addition, an altered SC lipid processing and defects of lamellar membrane maturation has been observed in NHE / epidermis [35, 37]. Taken together, these data suggest that the age-related NHE1 down-regulation may, in part, account for the pH abnormality found in aged epidermis.
Peroxisome Proliferator Activated Receptors (PPARs) The down regulation of PPARs with age might also contribute to a defective barrier in aging skin. PPARs are a group of nuclear receptors that heterodimerize with RXR and are activated by fatty acids, prostaglandins, eicosanoids as well as other lipid metabolites. Based on in situ hybridization studies, PPARa and g are expressed in epidermis. Topical application of PPARa activators accelerates SC acidification, which, in turn, significantly improves SC barrier function and epidermis integrity by enhancing LB secretion and lipid processing [38]. It has been reported that serine phospholipase A2 (sPLA2) was activated by PPARa activator, and simultaneous treatment of with PPARa and sPLA2 inhibitors will reverse PPARa induced SC acidification suggesting PPAR/sPLA2 might be important signaling pathways for SC pH regulation [38]. Very interestingly, PPARa mRNA level decreased about 30% in rat kidney (25 months) compared to their young controls (13 months). Similarly, significantly, 53% and 64% decreases of PPARa nuclear protein level and DNA binding activity in kidney extract, respectively, was observed in older rats. Although the epidermal expression of PPARa in older rats was not tested, a similar trend would be expected. If this is true, it might help to explain the increased pH level in the epidermis of aging skin. However, in one study, it was fount that PPARa knock-out did not affect the SC’s pH and its barrier function in mice. While knocking out PPARg would significantly impair the SC barrier function, no age-related decline of PPARg level was noticed in aged epidermis [38–40]. So it would be
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very interesting to clarify the relationship between the PPARs, SC pH and barrier function in future studies.
The Psychological Stress, Estrogens and Barrier Function in Aged Skin Epidermal barrier function can be affected by psychological stress-induced glucocorticoid (GC) secretion. Previous studies have shown that long-term treatment with GC will reduce epidermal proliferation resulting in an atrophy of the skin. Shorter duration of treatment with GCs will block epidermal lipid production which delays lipid secretion and lamellar body formation resulting in impaired epidermal barrier function. This effect was reversed by topical application of lipids [41, 42]. Indeed, when compared with healthy young subjects, it is conceivable that the elderly are more ‘‘stressed’’, accompanied by activation of the hypothalamus–pituitary–adrenal (HPA) axis and an increased GCs which might contribute to abnormalities of epidermal barrier function in older people [41]. It has been recognized that the serum estrogen level is also important for the maintenance of skin function in females. Age-related loss of estrogen will affect skin collagen content, dermal thickness and elasticity as well as water content and might result in an impaired skin barrier. Although a recent study showed estrogen will promote epidermal mitotic activities, the exact mechanism(s) by which estrogen influences skin barrier function is unclear [43–46].
Immune Function Change in Aged Skin The increased prevalence of skin infections in older subjects is suggestive that aging is associated with reduced peripheral immunity [19]. Physical barrier function defect, increased pH, skin dehydration, elevated GC level and accumulated oxidative stress products all contribute to the suboptimal function of the skin immune system in aged human beings. At a cellular level, no significant differences in Langerhans Cell abundance or localization between young and aged skin were noticed. However, the pan-haematopoietic cell antigen CD45 positive cells were found to be reduced and there was a significant loss of T cells and atypical dendritic epidermal T cells (DETCs) in murine aging skin suggesting an impaired immune response [47].
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Metabolism There have been few studies examining aging skin metabolism. In general, hepatic-mediated enzymatic activities decline during chronological aging. A similar trend might be expected in the skin. The effects of aging on skin metabolism are mainly affected by the physiological and pathological factors during the aging process. Chief among these is oxidative stress. Due to the attenuated ability to degrade oxidized proteins during aging, the accumulation of such products might further disturb cellular metabolism. At the same time, the functioning metabolic enzymes might also be attacked by free radicals [48]. Oxidative stress will also damage keratinocyte mitochondrial function, which forces the cell to switch the balance to anaerobic glycolysis for compensation [49]. In addition, cutaneous blood vessel networks have been found to be decreased due to the angiogenesis defects in the aging skin. Therefore a decrease of metabolic rate will be expected because of the suboptimal level of oxygen [50].
Sensory Function and Thermal Regulation in Aged Skin As mentioned, skin sensory innervation systems consist of free nerve endings located in the dermis and epidermis which play an important role in skin homeostasis. The function of these sensory nerves is associated with immune function, inflammation, wound healing, thermoregulation, and hair growth. Protein gene product 9.5 (PGP 9.5) immunostaining studies have shown an ageassociated decrease in skin sensory innervation networks. In chronological aging, a decrease in skin innervation is associated with the loss of neuronal networks around the sweat glands, and a decrease in perception of thermal stimuli and tactile sensation. However, painful sensation is relatively conserved [51]. Both the adrenergic vasoconstrictor system and an active vasodilator system work in concert to achieve the skin thermoregulatory by adjusting skin blood flow. Skin blood flow will increase as the core body temperature increases and, sweating and cutaneous active vasodilation (AVD) will occur to increase the evaporation once a threshold is reached. Reflex vasoconstriction (VC) of cutaneous blood vessels in response to cooling will effectively minimize heat loss. It has been found that there is a reduction in the ability to raise skin blood flow during heat stress in older subjects. Evidence from heat stress studies have shown that the attenuated cutaneous reflex vasodilation is mainly due to the attenuated NO-mediated pathways in older (71 6 year)
compared to the young (23 2 year) subjects [52, 53]. On the other side, the ability to reduce skin blood flow in response to cooling is also compromised in advanced age which pose a health risk for older in response to cooling situation. Recent studies have shown that the nonnoradrenergic-mediated VC mechanisms are mainly responsible for the aged related defects in reflex cutaneous VC [54, 55].
The Possible Molecular Mechanisms for Skin Aging Telomere and Skin Aging It is well recognized that cellular proliferation is accompanied by a progressive chromosome shortening of the telomere structure at the end of chromosome. Telomeres are the tandem repetitive DNA sequences at ends of mammal chromosomes which generally consist of several thousand base pairs with the 30 strand overhanging by 75–300 bases, i.e. (TTAGGG)n. The major function of the telomeric repeats is to protect the chromosome by shortening during cell proliferation. Recent studies have revealed telomere decrease with every cell division, which in all likelihood is inversely related to the individual’s physiologic age. At the same time, the single-stranded telomere overhang can also form a loop structure by telomeric repeat binding factor 2 (TRF2) to provide further protection of DNA integrity. Loop disruption will result in digestion of the overhanging sequence and various DNA damage responses including cellular apoptosis and senescence. This may occur naturally after critical telomere shortening or DNA damage. Dominant negative TRF2 transfection will disrupt the telomere loop structure and promote cell senescence. As the telomere reaches a threshold length after proliferation, the cells may become old and lose normal physiologic function. It has been reported that the telomere length plays an important role in controlling the age-associated transcript profile and cellular capacities during aging [56–59]. The length of telomeres is dynamic and maintained by ribonucleoprotein enzyme telomerase which can lengthen the terminal regions of telomeric DNA by addition of tandemly repeated telomeric sequences. The teleomeric balance between lengthening and shortening is influenced by genetic, developmental and physiological factors [60, 61]. For example, oxidative stress can accelerate shortening of the telomere in human fibroblasts probably through the accumulation of single-strand DNA breaks. An increase of the glutathione peroxidase (GPX-1) and copper–zinc
Pathology of Aging Skin
superoxide dismutase (CuZnSOD) mRNA in human fibroblasts will decrease the rate of telomere shortening. In an epidemiology study, a continuous loss of the telomere DNA during the aging process was observed in individuals ranging from 0 to 90 years old in peripheral hematopoietic cells suggesting a negative correlation between chronological aging and telomere shortening [62]. In addition, extensive studies in humans have revealed a correlation between telomere shortening and aging in multiple cell types including peripheral lymphocytes, fibroblasts, brain, esophageal mucosa, gastric mucosa, mixed large and small intestine mucosa, large bowel mucosa, kidney and liver [63, 64]. The relationship between telomere shortening and skin aging has also been observed in several studies. For example, analysis of DNA samples from sun protected epidermis obtained from 52 subjects ranging from 0 and 101 years old in Japan showed a rate of 36 bp reduction per year in epidermis with the estimated telomere lengths in the epidermis around 13.3 kb at birth. In another study, the length of the telomeric TTAGGG repeat sequences in skin sample from 21 human subjects between 0 and 92 years of age was measured. The results showed a statistical reduction of telomere length by a rate of 19.8 bp/year [65]. Telomere length was also measured in the skin of nine elderly patients (age range 73–95 years). The average length of telomere with this group is about 7–8 kb (7,792 596 bp). An inverse relationship between the age and telomere length was also detected in skin specimens with an average of reduction about 79 bp [65]. The yearly telomere reduction rate in the epidermis ranges from 19–75 bp a year. It did not match with the high turnover rate of the epidermis which is roughly every 4 weeks. So, in skin, the lengthening of teleomers may exist and possibly predominate. It has to be mentioned here that the epidermis telomere length measured in above studies never fell below the critical size as 5–6 kb identified in previous studies even in advanced age subjects. However, since the current technology can not measure the exact telomere length in single cells and for a single chromosome, it can not be excluded that the telomere either in a sub-population of the cells or a subgroup of the chromosome is shorter than the critical size. So, how the telomere shortening affects cell senescence remains to be investigated [63–65]. Other evidence supporting the telomere shortening and aging process comes from the telomerase activity studies [64]. Telomerase is a ribonucleoprotein DNA polymerase complex including the protein telomerase reverse transcriptase (TERT, or hTERT in humans) and a catalytic RNA (TERC). Generally, telomerase activity is
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very low in most normal human somatic cells because of the lack of expression of TERT. Previous studies have shown a decrease in telomerase activity in response to life stress and a lower telomerase activity in response to chronic emotional stress. A recent life style intervention study also revealed an increase in telomerase activity over a 3-month period after decrease of physiologic stress. In the same study, a relation between the diet associated LDL cholesterol and the telomerase activity has been observed. All these findings are suggesting a decrease of the telomerase activity in the epidermis as the age progresses [66]. Although the normal epidermis contained no or only slight telomerase activity determined by the TRAP assay, epidermal cells reserve the potential to increase telomerase activity in response to stressors such as UV light to maintain chromosome stability. For example, only one of seven specimens from sun-protected epidermis in adults showed detectable human telomerase RNA, whereas the epidermal basal cells in all samples obtained from sunexposed areas showed moderate human telomerase RNA signals. Thus, sun-exposed skin contains higher levels of telomerase activity than sun-protected skin which suggests telomerase is expressed by epidermal cells and can function as a protective mechanism in response to environmental stresses. Although age related decrease in telomerase activities has been observed in previous studies, it would very interesting to confirm this relationship in epidermis with more robust experimental data in the future. Generally, basal cells in normal epidermis have been reported to possess telomerase activity [63, 64]. In vitro studies have identified a telomere senescence characterized by enlarged cell morphology, activation of lysosomal b-galactosidase and increased inflammatory transcript profile. Dermal fibroblasts without telomerase will reach senescence after 90 population doubling (PD) cycles characterized by shorter telomere and reduced replicative potential. Gene array studies have shown an increase in pro-inflammatory genes such as p21, MCP1, IL-1, IL-15, ICAM-1, tPA, stromelysins, which might be responsible for the age-related cellular matrix degradation. However, dermal fibroblasts transfected with hTERT showed higher level of telomerase, normal length of telomere and a similar replicative potential as those from younger subjects at stage PD 20. At the same time the altered gene expression pattern was also rescued to the normal level. To identify the telomerase effects of senescent dermal fibroblast on the dermal integrity, a dermal reconstitution system was employed. The results showed that the senescent fibroblast will show a intermittent splitting between the dermis and epidermis junction in
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skin reconstitutions, while the transfected fibroblasts show a tight, intact junction, suggesting the function of telomerase for the integrity of the skin structure [63, 64]. In a recent study, a constitutively expressed telomerase reverse transcriptase (TERT) construct was introduced into a mice model to address telomerase activity on skin function during aging. The transgenic mice expressed a high level of TERT compared to their controls and at the same time they had a decreased incidence of degenerative inflammatory pathologies in skin, a better preservation of both the thickness of the epidermis and of the subcutaneous fat layer compared to their corresponding controls at the advanced age, suggesting telomerase’s anti-aging effects on the maintenance of the skin epithelia [67]. Recently, p53 and its down stream transcriptional target cycline-dependent kinase inhibitors p21/SD11 and p16 ink4a have been shown to be involved in the telomereinduced senescence in aged human fibroblast cells. At the same time, activated p21/SD11 and p16 ink4a will block the pRb phosphorylation which will lead to the programmed cell aging [68]. Disruption of cellular telomere structure will trigger on p53 and p21/SD11 pathways which in turn will induce a growth arrest and increase the intracellular level of reactive oxygen species as well. It has been found that the mouse embryonic fibroblasts show a higher superoxide anion and hydrogen peroxide production and lower catalase activity due to the deficiency of the telomerase activity which can be rescued by the telomerase restoration [69]. Although, more data are needed to clarify how the telomere/telomerase activity affect the skin barrier function during the progress of aging, all current data suggest a tight correlation between the normal skin function and the skin telomere integrity and telomerase activity.
DNA Repairing Potential in Aging Skin DNA repair capacity is impaired in primary dermal fibroblasts of older subjects. Cells possess DNA repair mechanisms to remove damaged segments mainly through nucleotide excision repair (NER) pathway or base excision repair (BER) pathway during the G1 and G2 phase, otherwise the cell will undergo apoptosis to protect the organism from potential cancerous transformation. In a UV-induced model, the cell repair capacity in the primary dermal fibroblast cell was compared between young and old subjects. The results showed that initially, UV exposure will induce similar levels of DNA damage in both groups and this will remain at 20% after
6 h in ‘‘young’’ group. In contrast, the residue level remains high, around 60–80%, in the aged subjects suggesting there is a significant difference in terms of the dermal DNA repair capacity between the young and old groups. At the same time, FACS assay revealed a significant decrease in S phase population in aged dermal fibroblast cells, suggesting that the cellular replicative potential is impaired in aged dermal fibroblast cells. However, it is surprising to find no changes in cellular antioxidant system in aged fibroblast compared to the young ones [70]. In a similar study, a lower DNA repair capacity for strand-breaks has also been observed in aged dermal fibroblasts in response to acute oxidative stress [70, 71]. Overall the lower repair capacity might account for accumulated DNA damage found in skin of older subjects and this might result in the chromosome unstability, cellular growth arrest, apoptosis as well as the chronic dermal inflammation induced by the oxidative stress.
Oxidative Stress and Skin Aging It has been very well accepted that the oxidative stress is one of the most important driving factors in the aging process [72]. Both endogenously and exogenously generated free radicals will produce oxidative damage to cellular components including DNA during the life time, which in turn will accumulate and disturb normal cellular function. To maintain normal cell function, cells are also equipped with antioxidant defenses including nonenzymatic antioxidants such as glutathione and several enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPx) to scavenge the ROS. Generally, reactive oxygen species (ROS), such as superoxide anion radicals can be formed during mitochondrial respiration and phagocytosis which will be transformed into H2O2 and hydroxyl radicals after a series of enzymatic reactions. Hydroxyl radical is capable of oxidizing any macromolecule such as lipid, DNA and protein resulting in oxidative damage. The hydroxyl radical is known to react with purine and pyrimidine bases as well as the deoxyribose backbone represented by the formation of 8-OH-G. Polyunsaturated fatty acids are extremely sensitive to oxidation with the formation intermediate peroxyl radicals (ROO.) which will finally form the malondialdehyde (MDA) and hydroxynonenal (HNE) as the major toxic end products of lipid peroxidation. Hydroxyl radicals can also react with the residues of proteins, in particular
Pathology of Aging Skin
cystine and methionine to form mixed disulphides. The concentration of carbonyl group and advanced glycation end products (AGEs) serve as a good measure of ROS-mediated protein oxidation. Recent studies have shown that oxidized proteins may lose their structure and aggregate to form protein complexes. Since the antioxidant capacity of tissues decreases during aging, the accumulation of oxidative damage to cells resulting from aerobic metabolism is the molecular basis for the free radical theory of aging process. This has been supported by experimental data showing an accumulation of oxidative damage such as 8-oxo-20 -deoxyguanosine (8-oxodG) residues from DNA, AGEs from proteins, and hydroperoxides and thiobarbituric acid-reacting substances (TBARS) from lipids in the tissues of aged animals [73–78]. Since skin is continuously exposed to oxidative stress from environmental factors and endogenous aerobic metabolism, the damage likely accumulates. It has been found that, in aged rat skin, the oxidized lipid phosphatidylcholine hydroperoxide (PCOOH) increases form 3.46 1.02 mmol/PC mol at 6 months to 7.14 1.63 mmol/PC mol at 24 months. The TBARS content increases from 4.71 1.53 nmol/mg protein at 6 months to 11.10 2.05 nmol/mg protein at 30 months. The free 7-hydroperoxycholesterol (ChOOH) content also increased form 22.83 3.97 at 6 month to 42.58 16.59 mmol/free Ch mol at 24 months. The oxidized DNA in rat skin also increase gradually with age and reach the level 2.04 0.27 8-oxodG/105 dG at 30 months compared to 1.67 0.16 8-oxodG/105 dG at 6 months of age. Although the skin possesses an efficient anti-oxidant activities the increased ROS products in aged skin suggests a chronic accumulation effects during the life time [79–81]. The mitochondria are the primary site for aerobic metabolism. Normal leakage of electron from the electron transport chain makes the mitochondria mtDNA as the primary target of radical oxygen due to the absence of repairing mechanisms within mitochondria. The accumulated mtDNA damage over time will shut down mitochondria function and promote the cell aging and apoptosis [82–85]. In addition, ROS can be produced from a wide range of normal cellular activities including lipoxygenase, COX, plasma membrane NADPH oxidase, NADH dehydrogenase, cytochrome P450, cytochrome b5, microsomal electron transport as well as peroxisome and xanthine oxidase. In addition to already mentioned antioxidant systems, cells also possess an anti-oxidative protein named thioredoxin, which was recently found to play an important role to maintain the ROS homeostasis.
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Experimental evidence showing a decreased level of antioxidant potential in aging cells suggested the redox imbalance at old age. The increased ROS is associated with elevation of MAPKs including ERK, p38 and JNK which in turn will trigger on the NFkB and its downstream pro-inflammatory chemokines and cytokines induction including IL1-b, IL-6, TNF-a, COX2 as well as the VCAM-1 and ICAM-1. So the imbalanced redox might link to its potential inflammatory responses found in aged skin [82, 86, 87].
Conclusion Without question, the skin is a magnificent organ and survival is not possible without it. It is the interface between external and internal worlds, which is nothing short of miraculous. Its beauty is the source of admiration. The primary function of the skin is to serve as a barrier. To this end, the structure of the epidermis is highly regulated in coordinated, dynamic balance between proliferation, differentiation and desquamation. An acidic pH is required to maintain the barrier function by providing an optimal environment for the function of enzymes involved in lipid production/processing, water content and cell–cell conjunction. In addition to its barrier function, the skin also has a role in immunosurveillance, metabolize and sensory reception/transmission. From birth, the skin, like every other organ, begins the journey of aging. The environmental insults which the skin protects against, exact a heavy toll over years. As well, changes in the internal structure, leading to change in the internal milieu further contribute to the decline in structure and function of the skin. It is quite probable that oxidative damage is the singular most damaging event in skin leading to DNA damage, telomere shortening, protein glycosylation and collegen/elastin degradation among several deleterious events. Maintenance of healthy skin throughout life includes moisturization and protection against environmental insults.
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27. Kappes UP. Skin ageing and wrinkles: clinical and photographic scoring. J Cosmet Dermatol. 2004;3:23–25. 28. Whitton JT, Everall JD. The thickness of the epidermis. Br J Dermatol. 1973;89:467–476. 29. Elias PM, Ghadially R. The aged epidermal permeability barrier: basis for functional abnormalities. Clin Geriatr Med. 2002;18:103–120, vii. 30. Ghadially R. Aging and the epidermal permeability barrier: implications for contact dermatitis. Am J Contact Dermat. 1998;9:162–169. 31. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95:2281–2290. 32. Behne MJ, Barry NP, Hanson KM, et al. Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function. J Invest Dermatol. 2003; 120:998–1006. 33. Fluhr JW, Mao-Qiang M, Brown BE, et al. Functional consequences of a neutral pH in neonatal rat stratum corneum. J Invest Dermatol. 2004;123:140–151. 34. Fluhr JW, Behne MJ, Brown BE, et al. Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum. J Invest Dermatol. 2004;122:320–329. 35. Behne MJ, Meyer JW, Hanson KM, et al. NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging. J Biol Chem. 2002;277:47399–47406. 36. Choi EH, Man MQ, Xu P, et al. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol. 2007;127:2847–2856. 37. Hachem JP, Behne M, Aronchik I, et al. Extracellular pH Controls NHE1 expression in epidermis and keratinocytes: implications for barrier repair. J Invest Dermatol. 2005;125:790–797. 38. Fluhr JW, Man MQ, Hachem JP, et al. Topical peroxisome proliferator activated receptor activators accelerate postnatal stratum corneum acidification. J Invest Dermatol. 2009;129:365–374. 39. Dubrac S, Schmuth M. (P)PARsing epidermal development. J Invest Dermatol. 2006;126:241–242. 40. Man MQ, Barish GD, Schmuth M, et al. Deficiency of PPARbeta/ delta in the epidermis results in defective cutaneous permeability barrier homeostasis and increased inflammation. J Invest Dermatol. 2008;128:370–377. 41. Choi EH, Brown BE, Crumrine D, et al. Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity. J Invest Dermatol. 2005;124: 587–595. 42. Kao JS, Fluhr JW, Man MQ, et al. Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities. J Invest Dermatol. 2003;120: 456–464. 43. Stevenson S, Thornton J. Effect of estrogens on skin aging and the potential role of SERMs. Clin Interv Aging. 2007;2:283–297. 44. Verdier-Sevrain S, Bonte F, Gilchrest B. Biology of estrogens in skin: implications for skin aging. Exp Dermatol. 2006;15:83–94. 45. Brincat MP, Baron YM, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123. 46. Thornton MJ. The biological actions of estrogens on skin. Exp Dermatol. 2002;11:487–502. 47. McCullough JL, Kelly KM. Prevention and treatment of skin aging. Ann N Y Acad Sci. 2006;1067:323–331.
Pathology of Aging Skin 48. Widmer R, Ziaja I, Grune T. Protein oxidation and degradation during aging: role in skin aging and neurodegeneration. Free Radic Res. 2006;40:1259–1268. 49. Prahl S, Kueper T, Biernoth T, et al. Aging skin is functionally anaerobic: importance of coenzyme Q10 for anti aging skin care. Biofactors. 2008;32:245–255. 50. Chung P, Yu T, Scheinfeld N. Using cellphones for teledermatology, a preliminary study. Dermatol Online J. 2007;13:2. 51. Besne I, Descombes C, Breton L. Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. 2002;138:1445–1450. 52. Holowatz LA, Thompson CS, Minson CT, Kenney WL. Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin. J Physiol. 2005;563:965–973. 53. Holowatz LA, Houghton BL, Wong BJ, et al. Nitric oxide and attenuated reflex cutaneous vasodilation in aged skin. Am J Physiol Heart Circ Physiol. 2003;284:H1662–H1667. 54. Thompson CS, Kenney WL. Altered neurotransmitter control of reflex vasoconstriction in aged human skin. J Physiol. 2004;558: 697–704. 55. Scremin G, Kenney WL. Aging and the skin blood flow response to the unloading of baroreceptors during heat and cold stress. J Appl Physiol. 2004;96:1019–1025. 56. Boukamp P. Skin aging: a role for telomerase and telomere dynamics? Curr Mol Med. 2005;5:171–177. 57. Kosmadaki MG, Gilchrest BA. The role of telomeres in skin aging/ photoaging. Micron. 2004;35:155–159. 58. Shariftabrizi A, Eller MS. Telomere homolog oligonucleotides and the skin: current status and future perspectives. Exp Dermatol. 2007;16:627–633. 59. Sugimoto M, Yamashita R, Ueda M. Telomere length of the skin in association with chronological aging and photoaging. J Dermatol Sci. 2006;43:43–47. 60. Smogorzewska A, de Lange T. Different telomere damage signaling pathways in human and mouse cells. EMBO J. 2002;21:4338–4348. 61. Blackburn EH. Switching and signaling at the telomere. Cell. 2001;106:661–673. 62. Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med. 1999;190:157–167. 63. Sugimoto M, Yamashita R, Ueda M. Telomere length of the skin in association with chronological aging and photoaging. J Dermatol Sci. 2006;43:43–47. 64. Nakamura K, Izumiyama-Shimomura N, Sawabe M, et al. Comparative analysis of telomere lengths and erosion with age in human epidermis and lingual epithelium. J Invest Dermatol. 2002;119: 1014–1019. 65. Lindsey J, McGill NI, Lindsey LA, Green DK, Cooke HJ. In vivo loss of telomeric repeats with age in humans. Mutat Res. 1991;256:45–48. 66. Ornish D, Lin J, Daubenmier J, et al. Increased telomerase activity and comprehensive lifestyle changes: a pilot study. Lancet Oncol. 2008;9:1048–1057. 67. Tomas-Loba A, Flores I, Fernandez-Marcos PJ, et al. Telomerase reverse transcriptase delays aging in cancer-resistant mice. Cell. 2008;135:609–622. 68. Li GZ, Eller MS, Firoozabadi R, Gilchrest BA. Evidence that exposure of the telomere 3’ overhang sequence induces senescence. Proc Natl Acad Sci USA. 2003;100:527–531.
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69. Perez-Rivero G, Ruiz-Torres MP, Diez-Marques ML, et al. Telomerase deficiency promotes oxidative stress by reducing catalase activity. Free Radic Biol Med. 2008;45:1243–1251. 70. Hazane F, Sauvaigo S, Douki T, Favier A, Beani JC. Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation. J Photochem Photobiol B. 2006;82: 214–223. 71. Sauvaigo S, Bonnet-Duquennoy M, Odin F, et al. DNA repair capacities of cutaneous fibroblasts: effect of sun exposure, age and smoking on response to an acute oxidative stress. Br J Dermatol. 2007;157:26–32. 72. Callaghan TM, Wilhelm KP. A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part I: cellular and molecular perspectives of skin ageing. Int J Cosmet Sci. 2008;30:313–322. 73. Bickers DR, Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol. 2006;126:2565–2575. 74. Sander CS, Chang H, Hamm F, Elsner P, Thiele JJ. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int J Dermatol. 2004;43:326–335. 75. Trouba KJ, Hamadeh HK, Amin RP, Germolec DR. Oxidative stress and its role in skin disease. Antioxid Redox Signal. 2002;4:665–673. 76. Kohen R. Skin antioxidants: their role in aging and in oxidative stress – new approaches for their evaluation. Biomed Pharmacother. 1999;53:181–192. 77. Kaneko T, Tahara S, Taguchi T, Kondo H. Accumulation of oxidative DNA damage, 8-oxo-2’-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts. Mutat Res. 2001;487:19–30. 78. Meyer F, Fiala E, Westendorf J. Induction of 8-oxo-dGTPase activity in human lymphoid cells and normal fibroblasts by oxidative stress. Toxicology. 2000;146:83–92. 79. Sivonova M, Tatarkova Z, Durackova Z, et al. Relationship between antioxidant potential and oxidative damage to lipids, proteins and DNA in aged rats. Physiol Res. 2007;56:757–764. 80. Tahara S, Matsuo M, Kaneko T. Age-related changes in oxidative damage to lipids and DNA in rat skin. Mech Ageing Dev. 2001;122: 415–426. 81. Lasch J, Schonfelder U, Walke M, Zellmer S, Beckert D. Oxidative damage of human skin lipids. Dependence of lipid peroxidation on sterol concentration. Biochim Biophys Acta. 1997;1349:171–181. 82. Wei YH, Ma YS, Lee HC, Lee CF, Lu CY. Mitochondrial theory of aging matures – roles of mtDNA mutation and oxidative stress in human aging. Zhonghua Yi Xue Za Zhi (Taipei). 2001;64: 259–270. 83. Birch-Machin MA. The role of mitochondria in ageing and carcinogenesis. Clin Exp Dermatol. 2006;31:548–552. 84. James AM, Cocheme HM, Murphy MP. Mitochondria-targeted redox probes as tools in the study of oxidative damage and ageing. Mech Ageing Dev. 2005;126:982–986. 85. Cottrell DA, Turnbull DM. Mitochondria and ageing. Curr Opin Clin Nutr Metab Care. 2000;3:473–478. 86. Chung HY, Cesari M, Anton S, et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009;8:18–30. 87. Chung HY, Sung B, Jung KJ, Zou Y, Yu BP. The molecular inflammatory process in aging. Antioxid Redox Signal. 2006;8:572–581. 88. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95.
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9 Pathomechanisms of Endogenously Aged Skin Evgenia Makrantonaki . Christos C. Zouboulis
Introduction There has been an unprecedented rapid expansion of the population of elderly people both in the developed and developing world [1]. Since 1840, life expectancy has increased at a rate of about 3 months/year [2], whereas the total worldwide aged population is expected to rise from 605 million in 2000 to 1.2 billion in 2025 and to nearly two billion in 2050 [3]. These demographic shifts mandate a better understanding of the aging process and better management strategies for age-associated diseases. Like all other organs, skin suffers progressive morphologic and physiologic decrement with increasing age and provides the first obvious evidence of the aging process. Skin aging can be classified into light-induced aging (photoaging, exogenous aging) and endogenous aging. The latter occurs in nonexposed areas, which are not in direct contact with environmental factors such as ultraviolet (UV) and infrared (IR) irradiation (e.g., the inner side of the upper arm) [4], and is mainly attributed to genetic factors and alterations of the endocrine environment. In contrast to photoaging, endogenously aged skin reflects degradation processes of the entire organism.
Pathomechanisms of Endogenously Aged Skin With advancing age the most pronounced changes in endogenously aged skin occur within the epidermis and affect mostly the basal cell layer. As a result, sun-protected aged skin appears thin, finely wrinkled, and dry (reviewed in > Table 9.1) [4]. Although the fundamental mechanisms are still poorly understood, a growing body of evidence points toward the involvement of multiple pathways in the generation of aged skin. Several theories have been proposed including the theory of cellular senescence [5], decrease in cellular DNA repair capacity and loss of telomeres [6–9], point mutations of extranuclear mitochondrial DNA (mtDNA) [10], oxidative stress [11], increased frequency of chromosomal abnormalities [12, 13],
and gene mutations. In addition, hormones have also been shown to play a distinct role.
Cellular Senescence The theory of cellular senescence describes the observed loss of the cell’s proliferative potential after a limited number of cell divisions [5]. According to this theory, cells possess a ‘‘biological clock,’’ which signals the end of their replicative life span, and as a consequence, they cannot be stimulated to enter the S1 phase by physiological mitogens, arresting at the G1 phase. This process can be partly explained by the selective repression of growth regulatory genes. Studies on keratinocytes [14], fibroblasts [15], and melanocytes [16] have revealed that they all show an age-associated decrease in cumulative population doublings. Fibroblasts, for instance, taken from a normal human tissue go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Towards the end of this time, proliferation slows down and finally stops, and the cells enter a state from which they never recover. The reduction in proliferative capacity of skin-derived cells in culture from old donors and patients with premature aging syndromes and the accumulation in vivo of senescent cells with altered patterns of gene expression also support the theory of cellular senescence.
The Free Radical Theory Oxygen radicals or reactive oxygen species (ROS) are increasingly considered as the major contributors to aging, and the protective mechanism against oxidative stress is observed as an indispensable function [17]. It has been shown that oxygen radical levels rise and antioxidant activity declines with advancing age [18]. Skin possesses many defensive mechanisms in order to reduce the production of ROS from internal sources.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_9, # Springer-Verlag Berlin Heidelberg 2010
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. Table 9.1 Morphological and functional changes in intrinsically aged skin [74] Thinning of epidermis by 10–50%
Increased vulnerability, fragility
Atrophy of the stratum spinosum
Increased vulnerability, fragility
Increased heterogeneity in size of basal cells
Increased vulnerability, fragility
Decreased mitotic activity, increased duration of cell cycle and migration time
Decreased desquamation, delayed wound healing
Slow replacement of lipids
Disturbed barrier function
Flattening of the dermoepidermal junction
Decrease in surface contact area, increased risk of separation by shearing forces
Decrease and heterogeneity of melanocytes
Graying of hair, guttate amelanosis, lentigines
Decrease of Langerhans cells
Diminished cutaneous immune function
Reduction of dermis thickness, decrease of fibroblasts
Reduced strength and resiliency
Atrophy of the extracellular matrix
Reduced strength and resiliency
Reduction and disintegration of collagen and elastic fibers, deposition of exogenous substances (e.g., amyloid P)
Sensitization to deformational forces, fine wrinkle formation
Reduction of cutaneous microvasculature
Reduction of cutaneous vascular responsiveness, disturbed thermoregulation and supply with nutrients
Decrease of skin appendages and their function (e.g., sebaceous Decreased lipid and sweat production, disturbed glands, sweat glands, apocrine glands) re-epithelization of deep cutaneous wounds Thinning of subcutaneous fat
Reduced insulation and energy production
Reduction of nerve endings
Disturbed sensory function
For example, the activity of enzymes that indirectly produce oxygen metabolites can be altered (xanthine oxidase modulation). There is a repair system consisting of enzymes and small molecules, antioxidant enzymes such as catalase and peroxidase, and low-molecular weight antioxidants such as tocopherols, ascorbic acid, NADH, and carnosine, which can donate an electron and then scavenge ROS. Excess ROS production leads to accumulation of cellular damage [19, 20], which includes oxidation of DNA resulting in mutations and oxidation of membrane lipids leading to reduced transport efficiency and altered transmembrane signaling, processes whose consequence is the aging phenotype. A disturbed stress response is also known to be associated with a defect in proteolytic systems such as lysosomal activity and ubiquitine-proteosome pathway in somatic cells [21]. As a consequence, altered proteins cannot be eliminated, and this results in accumulation of misfolded and damaged proteins in the cells. Moreover, cumulative evidence suggests that ROS play a crucial role by participating in multiple MAP kinase pathways, which induce AP-1 and in turn the signal cascade, already mentioned above ( > Fig. 9.1). The free radical theory has also been supported by the fact that strategies that reduce metabolism and the production of ROS, such as dietary caloric restriction (DCR),
can extend the life span of experimental animals. Studies conducted in animal models demonstrated that DCR can retard the aging process by influencing stress response and altering the expression of metabolic and biosynthetic genes [22]. Cancer prevention due to alterations of hormone metabolism, hormone-related cellular signaling, oxidation status, DNA repair, and apoptosis has been also associated with DCR [23, 24]. In skin tissues of mice with DCR weight control, a palette of genes showed a differential expression when compared to mice receiving normal diet [24]. DCR could show profound inhibitory impact on the expression of genes relevant to cancer risks (e.g., neuroblastoma ras oncogene, neuroblastoma mycrelated oncogene 1, Rab40c, myeloblastosis oncogene-like 2, lung carcinoma myc-related oncogene 1, myeloblastosis oncogene, RAB5B, RAP2B, RAB34).
The Telomere Hypothesis The telomere hypothesis of cellular aging [25] proposes that loss of telomeres due to incomplete DNA replication and absence of telomerase provides a mitotic clock that signals cycle exit, limiting the replicative capacity of the somatic cell [9]. Human telomeres consist of repeats of
Pathomechanisms of Endogenously Aged Skin
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. Figure 9.1 A schematic overview of major biochemical changes and signaling pathways involved in the generation of endogenously aged skin. In aged skin, mitogen-activated protein (MAP) kinase signal transduction pathways play an important role in regulating a variety of cellular functions. Downstream effectors of the MAP kinases include several transcription factors including the c-Jun and c-Fos, which heterodimerize in order to form the activator protein 1 (AP-1) complex. AP-1 is a key regulator of skin aging, as it induces the expression of the MMP family and has been shown to inhibit type I procollagen gene expression through interference with TGF-b signaling pathway. It has been postulated that MAP kinases may be activated by excess production of reactive oxygen species (ROS), which occurs with advanced age and may be superimposed by extrinsic factors (e.g., UV/IR irradiation). Excess ROS production also leads to accumulation of cellular damage, which includes oxidation of DNA resulting in mutations, oxidation of proteins leading to reduced function, and oxidation of membrane lipids resulting in reduced transport efficiency and altered transmembrane signaling. NF-kB: nuclear factor-kappa B; TGF-b: transforming growth factor-b; IL-1: interleukin-1; IL-6: interleukin-6; IL-8: interleukin-8
the sequence TTAGGG/CCCTAA at chromosome end, which are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase [9, 26, 27]. By mechanisms that remain unclear, telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Telomerase is active in germline cells and in humans, and telomeres in these cells are maintained at about 15 kilobase pairs (kbp). In contrast, telomerase is not expressed in most human somatic cells like skin cells [7, 28]. As a result, their telomeres become 50–100 nucleotides shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, after many cell generations, DNA damage occurs at chromosome ends. The damage activates a p53-dependent cell-cycle arrest that
resembles the arrest caused by other types of DNA damage. The lack of telomerase in most somatic cells has been proposed to help protect humans from the potentially damaging effects of runaway cell proliferation, as occurs in cancer. Telomere loss is thought to control entry into senescence [7, 29, 30].
Genes and Mutations The mechanisms which seem to be associated with aging are complex [31]. Recent studies on models such as the yeast Saccharomyces cerevisiae [32], the nematode Caenorhabditis elegans [33], the fly Drosophila melanogaster
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[34–36], the mouse Mus musculus [37], and humans [38] show that single gene mutations can contribute to the initiation of aging and induce premature aging syndromes. However, there are no special genes that can cause agingassociated damages. The manifestation of aging is mostly due to the failure of maintenance and repair mechanisms [39, 40]. Studies on human keratinocytes have demonstrated altered expression of growth-regulating molecules with age; there is an increase of the baseline expression of the differentiation-associated genes like SPR2 and interleukin 1 receptor antagonist [41] and EGF binding and receptor phosphorylation is reduced and thought to be the result of age-related changes in a critical downstream signaling element [42]. In senescent fibroblasts, genes like the c-fos protooncogene [43], the helix-loop-helix Id-1 and Id-2 genes [44], and components of the E2F transcription factor [45] have been shown to be downregulated, and negative growth regulators are overexpressed including the p21 and p16 inhibitors of cyclin-dependent protein kinases [46]. Other changes seen in senescent skin fibroblasts include increased expression of IL-1 and of the EGF-like cytokine heregulin that modulates the growth and differentiation [47]. Moreover, elastin gene expression is markedly reduced after the age of 40–50, as determined by mRNA steady state levels [48]. Furthermore, recent studies indicate that endogenous and exogenous aging may share some fundamental pathways, and may have some common mediators. Photoaging is thought to be the superposition of UV irradiation from the sun on intrinsic aging [49]. Some of the similarities are changes in the MAP kinase signaling pathways, like decreases in ERK-dependent MAP kinase activity and increases in stress-activated JNK and p38 kinase [50], which result in reduced cell proliferation, differentiation, and cell survival [51], and enhanced growth arrest, apoptosis, and stress-related responses [51, 52]. As a consequence of the stressactivated MAP kinase pathways, the expression of c-jun and c-Jun N terminal kinase – an upstream activator of c-jun, is elevated in aged compared with young skin [50]. As c-jun is a constituent of the transcription factor AP-1, AP-1 is also elevated and subsequently the AP-1 regulated connective tissue-degrading enzymes MMP-1 (interstitial collagenase), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). In parallel, there is an observed reduction in the expression of tissue inhibitors of metalloproteinases [53, 54]. Another common feature is the increased insoluble degraded collagen and the reduction of types I
and III procollagen synthesis, which may result from the impaired TGFb signaling pathway [53, 55]. In recent studies, researchers have been focusing on gene mutations accompanying known progeroid syndromes, e.g., Hutchinson–Gilford progeria, Werner’s syndrome (WS), Rothmund–Thomson syndrome, Cockayne syndrome, Ataxia teleangiectasia, and Down syndrome. The most common skin disorders of these syndromes, which are characterized by an acceleration of the aging phenotype, are alopecia, skin atrophy and sclerosis, teleangiectasia, poikiloderma, thinning and graying of hair, and several malignancies. Most of these syndromes are inherited in an autosomal recessive way and mostly display defects in DNA replication, recombination, repair, and transcription. Expression gene patterns of skin cells derived from Werner patients [56], old and young donors showed that 91% of the analyzed genes had similar expression changes in WS and in normal aging implying transcription alterations common to WS and normal aging represent general events in the aging process. Ly et al. measured mRNA levels in fibroblasts isolated from young, middle-aged, and elderly patients with progeria and found chromosomal pathologies that lead to misregulation of key structural, signaling, and metabolic genes associated with the aging phenotype [13]. Further studies conducted to investigate changes in gene expression during skin aging have been performed on naturally aged human foreskin obtained from children and elderly males. Some of the mechanisms proposed to be involved in the induction of aging comprise disturbed lipid metabolism, altered insulin and STAT3 signaling, upregulation of apoptotic genes partly due to the deregulation of FOXO1, dowregulation of members of the jun and fos family, differential expression of cytoskeletal proteins (e.g., keratin 2A, 6A, and 16A), extracellular matrix components (e.g., PI3, S100A2, A7, A9, SPRR2B), and proteins involved in cell-cycle control (e.g., CDKs, GOS2) [57].
The Mitochondrial DNA Theory Genetic damage and instability outside the nuclear genome has been also suggested to contribute to aging [58]. The mtDNA synthesis takes place near the inner mitochondrial membrane, which is the site of formation of ROS, and the fact that mtDNA lacks excision and recombination repair has made many investigators believe that cumulative damage of the mtDNA may play a key role in the pathogenesis of the aging phenotype [10, 11].
Pathomechanisms of Endogenously Aged Skin
Examination of human fibroblast mtDNA in aged individuals revealed point mutations at specific positions in the control region for replication. Notably, a T414G transversion was found in a significantly higher proportion of persons older than 65 years when compared with younger persons [11].
Hormone Decline and Skin Aging One of the further factors that may play a predominant role in the initiation of skin aging is the physiological hormone decline occurring with age. Over time important circulating hormones decline due to a reduced secretion of the pituitary, adrenal glands and the gonads, or due to an intercurrent disease. Among them, growth factors (i.e., growth hormone [GH] and insulin-like growth factor-I [IGF-I]) and sex steroids (e.g., androgens and estrogens) show significant changes in their blood levels. In animal models, such as in organisms as diverse as the nematode C. elegans, the fly D. melanogaster, and the mouse M. musculus, the importance of hormonal signals on the aging phenotype has already been documented. Suppression of hormones such as insulin-like peptides, growth hormone (GH), and sterols [59], or their receptors can increase life span and delay age-dependent functional decline. Conboy et al. [60] showed that the age-related decline of progenitor cell activity of mice could be reversed by exposure to young serum and that the cells could retain much of their intrinsic proliferative potential even when old, underlining the great importance of the systemic environment. In an in vitro model of human hormonal aging, human skin cells cultured under hormone-substituted conditions showed altered lipid synthesis and metabolism and affected expression of genes being involved in biological processes, such as DNA repair and stability, mitochondrial function, oxidative stress, cell cycle and apoptosis, ubiquitin-induced proteolysis, and transcriptional regulation indicating that these processes may be hormone-dependent [61]. These studies illustrate the importance of the hormone environment for deterioration of the human organism and the aging process. The growth hormone (GH)/insulin-like growth factorI (IGF-I) axis is considered to be one of the most important signaling pathways involved in aging. Serum levels of IGF-I have been reported to increase from birth to puberty, followed by a slow decline through adulthood. This reduction has been correlated with the progressive decline of GH with advancing age [62]. Patients with isolated GH
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deficiency (IGHD), multiple pituitary hormone deficiency (MPHD) including GH, as well as primary IGF-I deficiency (GH resistance, Laron syndrome) present signs of early skin aging such as dry, thin, and wrinkled skin. Other resulting characteristics of GH/IGF-I deficiency are obesity, hyperglycemia, reduced body lean mass, osteopenia, lowered venous access, hypercholesterolemia, cardiovascular diseases and, subsequently, premature mortality [63–65]. Treatment of normal elderly males with GH resulted in amelioration and reverse of the aging signs and symptoms [66]. However, recent reports of an association of GH substitution and increased risk of prostate, lung, colon, breast cancer, as well as a possible decrease of insulin insensitivity all make further investigations necessary regarding safety and efficacy of GH substitution in the aging population [67]. On the other hand, menopause, which is characterized by a rapid decline of sex steroids, has been associated with a worsening of skin structure and functions, which can be at least partially repaired by hormone replacement therapy or local estrogen treatment [68]. Improvement of epidermal skin moisture, elasticity and skin thickness [69], enhanced production of surface lipids [70], reduction of wrinkle depth, restoration of collagen fibers [71], and increase of the collagen III/I ratio [72] have all been reported under hormone replacement therapy. In vitro test that studied the effects of GH, IGF-I androgens, and estrogens at age-specific levels on human skin cells have been documented. IGF-I was shown to play an important role in the regulation of the lipid synthesis in human sebocytes, while 17b-estradiol showed no significant effects on the biological activity of the cells. Dermal fibroblasts showed to be more susceptible to 17b-estradiol treatment, while IGF-I could significantly stimulate fibroblast proliferation. Furthermore, an interplay between the 17b-estradiol and IGF-I signaling pathways was documented in both cell types [73]. These results indicate the importance of IGF-I in the reduction of skin surface lipids and thickness with advanced age.
Conclusion In summary, several factors may contribute to endogenous skin aging underlining the complexity of this phenomenon. Amongst these are excess production of reactive oxygen species and impaired scavenge mechanisms, increased frequency of chromosomal abnormalities, telomere loss and point mutations of extranuclear mitochondrial DNA due to reduced DNA repair capacity.
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In addition, several genes and their mutations have been correlated with the aging phenotype. Hormones and their physiological decline with time also play a distinct role as shown in several in vitro and in vivo studies. Like the entire organism, skin follows the pathway of aging with time. In addition to internal factors, several environmental factors contribute to this process and sometimes accelerate the onset of aging. Skin functions deteriorate and this results in the development of a palette of diseases, which may jeopardise life quality. Awareness of the pathophysiology of skin aging as well as of preventive measures to avoid skin damage is the first step for successful, healthy aging.
Cross-references > Pathomechanisms
of Photoaged Skin
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10 Pathomechanisms of Photoaged Skin Jean Krutmann
Introduction Among all environmental factors, solar UV radiation is the most important in premature skin aging, a process accordingly termed photoaging. Over recent years, substantial progress has been made in elucidating the underlying molecular mechanisms. From these studies, it is now clear that both UVB (290–320 nm) and UVA (320– 400 nm) radiations contribute to photoaging. UVinduced alterations at the level of the dermis are best studied and appear to be largely responsible for the phenotype of photoaged skin. It is also generally agreed that UVB acts preferentially on the epidermis where it not only damages DNA in keratinocytes and melanocytes, but also causes the production of soluble factors including proteolytic enzymes, which in a second step affect the dermis; in contrast, UVA radiation penetrates far more deeply on average and hence exerts direct effects on both the epidermal and the dermal compartments (> Fig. 10.1). UVA is also 10–100 times more abundant in sunlight than UVB, depending on the season and time of the day. Therefore, it has been proposed that, although UVA photons are individually far less biologically active than UVB photons, UVA radiation may be at least as important as UVB radiation in the pathogenesis of photoaging [1]. The exact mechanisms by which UV radiation causes premature skin aging is not yet clear, but a number of molecular pathways explaining one or more of the key features of photoaged skin have been described. Some of these models are based on irradiation protocols, which use single or few UV exposures, whereas others take into account the fact that photoaging results from chronic UV damage, and as a consequence employ chronic repetitive irradiation protocols. Still others rely on largely theoretical constructs rather than experimental observations.
Mechanisms of Photoaging All organ systems are affected by aging processes, many in organ-specific ways; however, aging uniformly has the effect of reducing maximal function and reserve
capacity, as well as the ability to compensate for injury and a hostile environment. Ultimately, such losses are incompatible with life. Of interest, most, if not all, age-accelerating environmental factors damage DNA either directly or indirectly, often through oxidation. Furthermore, the rate of aging in various species correlates [2] inversely with the rate and fidelity of DNA repair [3], and most progeroid syndromes for which the genetic lesion has been identified have impaired DNA replication and/or DNA damage responses. In combination with the fact that cumulative DNA damage accompanies chronological aging [4], these observations suggest that both the indisputable heritable and the environmental components of aging result in large part from changing DNA status during the individual’s lifetime. The next section develops this intellectual framework and relates it to the phenomenon of skin aging, and particularly photoaging, by focusing on mtDNA [5, 1]. The subsequent sections provide detailed information now available with regard to specific aging targets and signaling pathways responsible for photoaging-associated morphological and functional changes in the skin. These include UV-induced alterations of connective tissue components, vascularization patterns, inflammatory cells, and protein oxidation. Finally, a unifying concept is presented that reconciles with the most recent findings in an attempt to provide a novel and comprehensive model to explain photoaging and provide a framework for future investigations.
Mitochondrial DNA Mutations and Photoaging Mitochondria are organelles whose main function is to generate energy for the cell. This is achieved by a multistep process called oxidative phosphorylation or electron transport chain. Located at the inner mitochondrial membrane are five multiprotein complexes that generate an electrochemical proton gradient used in the last step of the process to turn ADP and organophosphate into ATP. This process is not completely error-free and ultimately
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_10, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 10.1 Wavelength-dependent penetration of UV radiation into human skin
leads to the generation of reactive oxygen species (ROS), making the mitochondrion the site of the highest ROS turnover in the cell. In close proximity to this site lies the mitochondrion’s own genomic material, the mtDNA. Human mtDNA is a 16,559-bp long circular doublestranded molecule of which four to ten copies exist per cell. Since mitochondria do not exhibit any repair mechanism to remove bulky DNA lesions, although they do exhibit a base excision repair mechanism and repair mechanisms against oxidative damage, the mutation frequency of mtDNA is approximately 50-fold higher than that of nuclear DNA. Mutations of mtDNA have been found to play a causative role in degenerative diseases such as Alzheimer’s disease, chronic progressive external ophthalmoplegia, and Kearns–Sayre syndrome (KSS) [6]. In addition to degenerative diseases, mutations of mtDNA may play a causative role in the normal aging process with an accumulation of mtDNA mutations accompanied by a decline of mitochondrial functions [7]. Recent evidence indicates that mtDNA mutations are also involved in the process of photoaging [1]. Photoaged skin is characterized by increased mutations of the mitochondrial genome [8, 9, 10]. Intraindividual comparison studies have revealed that the so-called common deletion, a 4,977-bp deletion of mtDNA, is
increased up to tenfold in photoaged skin as compared with sun-protected skin of the same individuals. The amount of the common deletion in human skin does not correlate with chronological aging [11], and it has therefore been proposed that mtDNA mutations such as the common deletion represent molecular markers for photoaging. In support of this concept it has been shown that repetitive, sublethal exposure to UVA radiation at doses that may be acquired during a regular summer holiday induces mutations of mtDNA in cultured primary human dermal fibroblasts in a singlet oxygendependent fashion [3]. Even more importantly, in vivo studies have revealed that repetitive exposure three times daily of previously unirradiated buttock skin for a total of 2 weeks to physiological doses of UVA radiation leads to an approximately 40% increase in the levels of the common deletion in the dermal, but not the epidermal, compartment of irradiated skin [12]. Furthermore, it has been shown that, once induced, these mutations persist for at least 16 months in UV-exposed skin. Interestingly, in a number of individuals, the levels of the common deletion in irradiated skin continued to increase with a magnitude up to 32-fold. It has been postulated for the normal aging process as well as for photoaging that the induction of ROS generates mtDNA mutations, in turn leading to a defective respiratory chain and, in a vicious cycle, inducing even more ROS and subsequently allowing mtDNA mutagenesis independent of the inducing agent [13]. It is the characteristic of vicious cycles that they evolve at everincreasing speeds. Thus, the increase of the common deletion up to levels of 32-fold, independent of UV exposure may represent the first in vivo evidence for the presence of such a vicious cycle in general and in human skin in particular. The mechanisms by which generation of mtDNA mutations by UVA exposure translates into the morphological alterations observed in photoaging human skin are currently being unravelled. In general, a cause-effect relationship between premature aging and mtDNA mutagenesis is strongly suggested by studies employing homozygous knock-in mice that express a proofreading-deficient version of PolgA, the nucleus-encoded subunit of mtDNA polymerase [14]. As expected, these mice develop an mtDNA mutator phenotype with increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations has been found to be associated with a reduced life span and premature onset of aging-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and heart enlargement.
Pathomechanisms of Photoaged Skin
In addition, recent studies have demonstrated that UVA radiation-induced mtDNA mutagenesis is of functional relevance in primary human dermal fibroblasts and apparently has molecular consequences suggestive of a causative role for mtDNA mutations in photoaging of human skin as well [15]. Accordingly, induction of the common deletion in human skin fibroblasts is paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of MMP-1, while tissue-specific inhibitors of MMPs (TIMPs) remain unaltered, an imbalance that is known to be involved in photoaging of human skin (see below). These observations suggest a link not only between mutations of mtDNA and cellular energy metabolism, but also between mtDNA mutagenesis, energy metabolism, and a fibroblast gene expression profile that would functionally correlate with increased matrix degradation and thus premature skin aging. In order to provide further evidence for a role of the energy metabolism in mtDNA mutagenesis and the development of this ‘‘photoaging phenotype,’’ the effect of creatine was studied in these cells. This applied the hypothesis that generation of phosphocreatine, and consequently ATP, is facilitated if creatine is abundant in cells. This would allow easier binding of existing energy-rich phosphates to the energy precursor creatine. Indeed, experimental supplementation of normal human fibroblasts with creatine normalizes mitochondrial mutagenesis as well as the functional parameters, oxygen consumption, and MMP-1, while an inhibitor of creatine uptake abrogates this effect [15] (> Fig. 10.2). A second line of evidence for cause-effect relationship between a disturbance of mtDNA integrity and skin aging is provided by a very recent study, in which a phenocopy of cells bearing large-scale deletions of mtDNA was generated by gradually depleting the mtDNA from unirradiated human skin fibroblasts [16]. Gradual depletion of mtDNA caused a gene expression profile, which was a reminiscent of that observed in photoaged skin. Accordingly, in these cells an increased expression of MMP-1 without a concomitant change in tissue inhibitor metalloproteinase-1 as well as a decreased expression of collagen type 1 alpha-1, that is, a gene involved in collagen de novo synthesis, was observed. This altered gene expression resulted from intracellular, mitochondria-derived oxidative stress. These results support the concept that disruption of mt integrity, for example, by UV-induced mtDNA mutagenesis, is of pathogenetic relevance for photoaging of human skin. Finally, a third line of evidence for a pathogenetic role of mtDNA mutations in photoaging is provided by
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. Figure 10.2 The defective powerhouse model of (photo)aging of the skin
human in vitro studies employing skin fibroblasts from Kearns–Sayre syndrome (KSS) patients, which constitutionally carry large amounts of the UV-inducible common deletion. Accordingly, human dermal skin equivalent models, which were engineered using KSS skin fibroblasts, developed multiple features of photoaged skin including MMP-1 upregulation and increased collagen breakdown even in the absence of any UV exposure ([17] and Krutmann et al., unpublished observation).
Connective-Tissue Alterations in Photoaging: The Role of Matrix Metalloproteinases and Collagen Synthesis Photoaged skin is characterized by alterations to the dermal connective tissue. The extracellular matrix in the dermis mainly consists of type I and type III collagen, elastin, proteoglycans, and fibronectin. In particular,
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collagen fibrils are important for the strength and resilience of skin, and alterations in their number and structure are thought to be responsible for wrinkle formation. In photoaged skin, collagen fibrils are disorganized and abnormal elastin-containing material accumulates [18]. Biochemical studies have revealed that in photoaged skin levels of types I and III collagen precursors and crosslinks are reduced, whereas elastin levels are increased [19, 20]. How does UV radiation cause these alterations? In principle it is reasonable to assume that UV radiation leads to an enhanced and accelerated degradation and/or a decreased synthesis of collagen fibers, and current knowledge indicates that both mechanisms may be involved. A large number of studies unambiguously demonstrate that the induction of matrix metalloproteinases (MMPs) plays a major role in the pathogenesis of photoaging. As indicated by their name, these zinc-dependent endopeptidases show proteolytic activity in their ability to degrade matrix proteins such as collagen and elastin. Each MMP degrades different dermal matrix proteins; for example, MMP-1 cleaves collagen types I, II, and III, whereas MMP-9, which is also called gelatinase, degrades collagen types IV and V and gelatin. Under basal conditions, MMPs are part of a coordinated network and are precisely regulated by their endogenous inhibitors, i.e., TIMPs, which specifically inactivate certain MMPs. An imbalance between activation of MMPs and their respective TIMPs could lead to excessive proteolysis. It is now very well established that UV radiation induces MMPs without affecting the expression or activity of TIMPs [21, 22]. These MMPs can be induced by both UVB and UVA radiations, but the underlying photobiological and molecular mechanisms differ depending on the type of irradiation. In a very simplified scheme, UVA radiation would mostly act indirectly through the generation of ROS, in particular, singlet oxygen, which can subsequently exert a multitude of effects such as lipid peroxidation, activation of transcription factors, and generation of DNA strand breaks [22]. While UVB radiationinduced MMP induction has been shown to involve the generation of ROS as well [23], the main mechanism of action of UVB is by direct interaction with DNA via the induction of DNA damage. Recent studies have indeed provided evidence that enhanced repair of UVB-induced cyclobutane pyrimidine dimers in the DNA of epidermal keratinocytes through topical application of liposomally encapsulated DNA repair enzymes on UVB-irradiated human skin prevents UVB radiation-induced epidermal MMP expression. [24].
The activity of MMPs is tightly regulated by transcriptional regulation and elegant in vivo studies by Fisher et al. have demonstrated that exposure of human skin to UVB radiation leads to the activation of the respective transcription factors [25]. Accordingly, UV exposure of human skin not only leads to the induction of MMPs within hours after irradiation, but already within minutes, transcription factors AP-1 and NF-kB, which are known stimulatory factors of MMP genes, are induced. These effects can be observed at low UVB dose levels, because transcription factor activation and MMP-1 induction can be achieved by exposing human skin to one tenth of the dose necessary for skin reddening (0.1 minimal erythema dose). Subsequent work by the same group clarified the major components of the molecular pathway by which UVB exposure leads to the degradation of matrix proteins in human skin. Low-dose UVB irradiation induces a signaling cascade, which involves upregulation of epidermal growth factor receptors (EGFR), the GTP-binding regulatory protein p21Ras, extracellular signal-regulated kinase (ERK), c-jun aminoterminal kinase (JNK), and p38. Elevated c-jun together with constitutively expressed c-fos increases activation of AP-1. Identification of this UVB-induced signaling pathway not only unravels the complexity of the molecular basis, which underlies UVB radiation-induced gene expression in human skin, but also provides a rationale for the efficacy of tretinoin (alltrans-retinoic acid) in the treatment of photoaged skin. Accordingly, topical pretreatment with tretinoin inhibits the induction and activity of MMPs in UVB-irradiated skin through prevention of AP-1 activation. In addition to destruction of existing collagen through activation of MMPs, failure to replace damaged collagen is thought to contribute to photoaging as well. Accordingly, in chronically photodamaged skin, collagen synthesis is down regulated as compared to sun-protected skin [26]. The mechanism by which UV radiation interferes with collagen synthesis is not yet known, but a recent study has provided evidence that fibroblasts in severely (photo) damaged skin have less interaction with intact collagen and are thus exposed to less mechanical tension, and it has been proposed that this situation might lead to decreased collagen synthesis [27].
UV-Induced Modulation of Vascularization There is increasing evidence that cutaneous blood vessels may play a role in the pathogenesis of photoaging. Photoaged skin shows vascular damage intrinsically aged skin
Pathomechanisms of Photoaged Skin
does not. In mildly photodamaged skin, there is venular wall thickening, while in severely damaged skin the vessel walls are thinned and the supporting perivascular veil cells are reduced in number [28]. The number of vascular cross-sections is reduced [29] and there are local dilations, corresponding to clinical telangiectases. Overall, there is a marked change in the horizontal vascularization pattern with dilated and distorted vessels. Studies in humans and in the hairless skh-1 mouse model for skin aging have demonstrated that acute and chronic UVB irradiation greatly increases skin vascularization [30, 31]. The formation of blood vessels from preexisting vessels is tightly controlled by a number of angiogenic factors and factors that inhibit angiogenesis. These growth factors include basic fibroblast growth factor, interleukin-8, tumor growth factor-beta, platelet-derived growth factor, and vascular endothelial growth factor (VEGF). VEGF appears to be involved in chronic UVB damage because UVB radiation-induced dermal angiogenesis in Skh-1 mice is associated with increased VEGF expression in the hyperplastic epidermis of these animals [31]. Even more importantly, targeted overexpression of the angiogenesis inhibitor thrombospondin-1 not only prevents UVB radiation-induced skin vascularization and endothelial cell proliferation, but also significantly reduces dermal photodamage and wrinkle formation. These studies suggest that UVB radiation-induced angiogenesis plays a direct biological role in photoaging.
Photoaging as a Chronic Inflammatory Process In contrast to intrinsically aged skin, which shows an overall reduction in cell numbers, photoaged skin is characterized by an increase in the number of dermal fibroblasts, which appear hyperplastic, and also by increased numbers of mast cells, histiocytes, and mononuclear cells. The presence of such a dermal infiltrate indicates the possibility that a chronic inflammatory process takes place in photoaged skin, and in order to describe this situation the terms heliodermatitis and dermatoheliosis have been coined [32]. More recent studies have shown that increased numbers of CD4 + T cells are present in the dermis, whereas intraepidermally, infiltrates of indeterminate cells and a concomitant reduction in the number of epidermal Langerhans cells have been described [33]. It is currently not known whether the presence of inflammatory cells represents an epiphenomenon or whether these cells play a causative role in the pathogenesis of photoaging, e.g., through the production of soluble mediators,
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which could affect the production and/or degradation of extracellular matrix proteins.
Protein Oxidation and Photoaging The aging process is accompanied by enhanced oxidative damage. All cellular components including proteins are affected by oxidation [34]. Protein carbonyls may be formed either by oxidative cleavage of proteins or by direct oxidation of lysine, arginine, proline, and threonine residues. In addition, carbonyl groups may be introduced into proteins by reaction with aldehydes produced during lipid peroxidation or with reactive carbonyl derivatives generated as a consequence of the reaction with reducing sugars or their oxidation products with lysine residues of proteins. Within the cell, the proteasome is responsible for the degradation of oxidized proteins. During the aging process, this function of the proteasome is diminished and oxidized proteins accumulate. In addition, lipofuscin, a highly cross-linked and modified protein aggregate, is formed. This aggregate accumulates within cells and is able to inhibit the proteasome. These alterations mainly occur within the cytoplasm and lipofuscin does not accumulate in the nucleus. In biopsies from individuals with histologically confirmed solar elastosis, an accumulation of oxidatively modified proteins was found specifically within the upper dermis [35]. Protein oxidation in photoaged skin is most likely due to UV irradiation because repetitive exposure of human buttock skin over 10 days to increasing UV doses as well as in vitro irradiation of cultured dermal fibroblasts with UVB or UVA have been shown to cause protein oxidation. The functional relevance of increased protein oxidation in UV-irradiated dermal fibroblasts, in particular with regard to the pathogenesis of photoaging, is currently not known. Very recent studies, however, indicate that increased protein oxidation, which may result from a single exposure of cultured human fibroblasts to UVA radiation, inhibits proteasomal functions and thereby affects intracellular signaling pathways, which are involved in MMP-1 expression (Krutmann J et al., unpublished observation).
Conclusion and Towards a Unifying Concept From the above observations, it is evident that major progress has been made recently in identifying molecular
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mechanisms involved in photoaging. In this regard, skin has proven to serve as an excellent model organ to understand basic mechanisms relevant for extrinsic aging. Despite all these progresses, however, a general, unifying concept linking the different mechanisms and molecular targets described in the previous paragraphs is still lacking. In other words, the critical question to answer is: How do mtDNA mutagenesis, neovascularization, protein oxidation, down regulation of collagen synthesis, and increased expression of matrix metalloproteinases together cause photoaging of human skin? Which of these mechanisms are of primary importance and responsible for inducing others? Are some or all of the above-mentioned characteristics of photoaged skin merely epiphenomena and, if so, to what extent are they causally related to premature skin aging? The current state of knowledge does not allow to answer these questions in a definitive manner. Nevertheless, a hypothesis is proposed, which tries to reconcile with most of the research discussed above in one model, for which the term ‘‘the defective powerhouse model of skin aging’’ has been coined [16, 36]. Specifically, the persistence of UV radiation-induced mtDNA mutations and the resulting vicious cycle with further increases in mtDNA mutations lead to a situation resembling a ‘‘defective powerhouse’’ where inadequate energy production leads to chronic oxidative stress. In the dermis, functional consequences of direct DNA damage and aberrant ROS production in human dermal fibroblasts could be (1) an altered gene expression pattern, which would affect neovascularization and collagen metabolism and possibly also the generation of an inflammatory infiltrate, (2) the oxidation of intracellular proteins and inhibition of the proteasome, and (3) possibly also changes in the epidermal compartment such as epidermal atrophy, skin barrier, and dysfunction. As a consequence, measures to prevent, delay, or even reverse photoaging should target the dermal rather than the epidermal compartment of human skin.
Cross-references > Pathomechanisms
of Endogenously Aged Skin
Acknowledgments Part of the work described in this chapter has been supported by the DFG, SFB 728.
References 1. Berneburg M, Plettenberg H, Krutmann J. Photoaging of human skin. Photodermatol Photoimmunol Photomed. 2000;16:239–244. 2. Brash D, Rudolph J, Simon J, Lin A, McKenna G, Baden H, Halperin A, Pontenm JA. Role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Aacad Sci USA. 1998;88:10124–10128. 3. Hart RW, Setlow RB. Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Proc Natl Acad Sci USA. 1974;71:2169–2173. 4. Vijg J. Somatic mutations and aging: a re-evaluation. Mutat Res. 2000;447:117–135. 5. Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J. Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem. 1999;274:15345–15349. 6. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med. 2003;348:2656–2668. 7. Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992;256:628–632. 8. Berneburg M, Gattermann N, Stege H, Grewe M, Vogelsang K, Ruzicka T, Krutmann J. Chronically ultraviolet-exposed human skin shows a higher mutation frequency of mitochondrial DNA as compared to unexposed skin and the hematopoietic system. Photochem Photobiol. 1997;66:271–275. 9. Birch-Machin MA, Tindall M, Turner R, Haldane F, Rees JL. Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J Invest Dermatol. 1998;110:149–152. 10. Yang JH, Lee HC, Wei YH. Photoageing-associated mitochondrial DNA length mutations in human skin. Arch Dermatol Res. 1995;287:641–648. 11. Koch H, Wittern KP, Bergemann J. In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation. J Invest Dermatol. 2001;117:892–897. 12. Berneburg M, Plettenberg H, Medve-Konig K, Pfahlberg A, GersBarlag H, Gefeller O, Krutmann J. Induction of the photoagingassociated mitochondrial common deletion in vivo in normal human skin. J Invest Dermatol. 2004;12:1277–1283. 13. Jacobs HT. The mitochondrial theory of aging: dead or alive? Aging Cell. 2003;2:11–17. 14. Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder E, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, Tornell J. Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429: 417–423. 15. Berneburg M, Gremmel T, Kurten V, Schroeder P, Hertel I, Mikecz AV, Wild S, Chen M, Declercq L, Matsui M, Ruzicka T, Krutmann J. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences. J Invest Dermatol. 2005;125:213–220. 16. Schroeder P, Gremmel T, Berneburg M, Krutmann J. Partial depletion of mitochondrial DNA from human skin fibroblasts induces a gene expression profile reminiscent of photoaged skin. J Invest Dermatol. 2008;128:2297–2303. 17. Majora M, Wittkampf T, Schuermann B, Grether-Beck S, Schroeder P, Krutmann J. Increased expression of lysyl oxidase and production of reactive oxygen species in fibroblasts with deletions of the
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27. Varani J, Schuger L, Dame MK, Leonhard Ch, Fligiel SEG, Kang S, Fisher GJ, Vorhees JJ. Reduced fibroblast interaction with intact collagen as a mechanism for depressed collagen synthesis in photodamaged skin. J Invest Dermatol. 2004;122: 1471–1479. 28. Braverman M, Fonfrko E. Studies in cutaneous aging: II. The microvasculature. J Invest Dermatol. 1982;73:59–66. 29. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–46. 30. Bielenberg DR, Bucana CD, Sanchez R, Donawho CK, Kripke ML, Fidler IJ. Molecular regulation of UVB-induced angiogenesis. J Invest Dermatol. 1998;111:864–872. 31. Yano K, Ouira H, Detmar M. Targeted over expression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photodamage. J Invest Dermatol. 2002;118: 800–805. 32. Lavker RM, Kligman A. Chronic heliodermatitis: a morphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells. J Invest Dermatol. 1988;90:325–330. 33. DeLeo VA, Dawes L, Jackson R. Density of Langerhans cells (LC) in normal versus chronic actinically damaged skin (CADS) of humans. J Invest Dermatol. 1981;76:330–334. 34. Levine RL, Stadtman ER. Oxidative modification of protein during ageing. Exp Gerontol. 2001;36:1495–1502. 35. Sander CS, Chang H, Salzmann S, Muller CS, EkanayakeMudiyanselage S, Elsner P, Thiele JJ. Photoaging is associated with protein oxidation in human skin in vivo. J Invest Dermatol. 2002;118:618–625. 36. Schroeder P, Krutmann J. Role of mitochondria in photoageing of human skin: the defective powerhouse model. J Invest Dermatol Symp P. 2009;in press.
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21 Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin Stacy S. Hawkins . William Shingleton . Jean Adamus . Helen Meldrum
Introduction The outermost layers of the epidermis are critical for providing a barrier against environmental factors, such as damage due to chronic UV irradiation and pollutants. An intact epidermal barrier is critical for preventing moisture loss and maintaining the health, function, and attractive appearance of the skin throughout the aging process [1–3]. Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Other nuclear hormone receptors include steroid hormones, thyroid hormones, and vitamin D hormones, and there is evidence that, similar to other nuclear receptors, PPARs perform a significant role in skin homeostasis [4]. Like other nuclear receptor superfamily members, PPARs contain both a ligand-binding domain and a DNA-binding domain. On ligand activation, PPARs form a heterodimer with retinoic X receptors (RXRs); the resulting complex then binds to PPAR-response elements (PPREs), leading to either an increase or a decrease in the expression of target genes [4, 5]. Although PPAR target genes have been identified across many clinical applications (e.g., cell proliferation, differentiation, inflammation and angiogenesis, atherosclerosis, lipid and glucose metabolism [4]), the emphasis of this summary will be the role of PPARs in the maintenance and improvement of human epidermal skin conditions.
PPARs: Role in Epidermal Structure and Function PPAR-mediated effects on skin homeostasis as well as disease treatment are now well accepted, and it has been shown that activators of PPARs are important regulators
of epidermal differentiation. Since the potential roles of PPARs were first identified by Issemann and Green in 1990 [6], three isotypes have been identified in the human epidermis to date: PPARa, PPAR b/d, and PPARg [7–9]. Over the past 15 years, PPARs have been studied for many clinical applications in dermatology, such as epidermal permeability barrier development and homeostasis [10, 11], stimulation of epidermal keratinocyte differentiation [5, 12–15], and keratinocyte response to inflammation [16, 17], and for treatment of epidermal disorders characterized by inflammation, keratinocyte hyperproliferation, and abnormal differentiation such as psoriasis [18–20]. Several pharmaceutical PPAR ligands have been developed; however, the endogenous ligands are naturally occurring fatty acids. PPAR lipids have also been shown to increase expression of epidermal differentiation proteins in vitro (e.g., transglutaminase [Tgase], involucrin, profilaggrin) and promote cornified cell envelope (CE) maturation in normal human keratinocytes [5]. PPARa agonists have been shown to increase the synthesis of epidermal lipids critical to maintaining a healthy permeability barrier in a living skin equivalent model [21]. Knockout and mutant mouse models have been used to determine the importance of PPARa in epidermal maturation and homeostasis. Analysis has shown that although the barrier function was normal, the stratum granulosum was thinner, with focal parakeratosis, suggesting impaired keratinocyte differentiation, as reflected by a moderate decrease in differentiation markers [22]. A recent study has demonstrated a role for PPAR agonists in improvement in skin barrier in a newborn mouse model, through increased lipid synthesis and acidification [23]. In skin, induction of PPARg results in skin inflammatory diseases such as atopic and allergic contact dermatitis or psoriasis. Topical PPARg agonists troglitazone and ciglitazone increased differentiation markers and
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_21, # Springer-Verlag Berlin Heidelberg 2010
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promoted epidermal barrier recovery in hairless mice [14], possibly through ERK1/2 and p38 signaling [24]. PPAR b/d is the most abundant of the three isotypes found in skin, yet functionally less defined due to its lack of specific ligands [8, 25]. In keratinocytes, it has been shown to be involved in differentiation, lipid accumulation, directional sensing, polarization and migration [15, 16]. PPARb/d may also play a role in the epidermal barrier formation, as evidenced in the LSE model [21] and also in primary keratinocytes [26]. In barrier-compromised skin such as the axillary area, PPAR ligands have been shown to improve epidermal skin condition, as measured by increased involucrin and filaggrin by immunohistochemistry compared to vehicle control, following a repeat patch test [27]. More recently, PPAR lipids have been shown to significantly improve the photoaged appearance after long-term product application [28, 29].
PPARs: Role in Inflammation and Skin Aging Inflammation is a contributing factor in many aging processes in the human body, from intrinsic aging to the pathogenesis of age-related diseases such as atherosclerosis, Alzheimer’s disease, and arthritis. There is a wealth of information in the literature documenting the anti-inflammatory role of PPARs in a variety of animal models, cell types, human tissues and diseases [30–35]. Therefore, this section of the review focuses on the skin-specific influences of PPARs on inflammation. In the field of dermatology, inflammation or irritation is associated with many diseases, and activation of PPARs has been shown to have beneficial effects on a number of skin conditions [17, 34, 36–38]. Marketed pharmaceuticals for the treatment of inflammatory skin disorders include agonists to PPARa and PPARg. The mode of action of these drugs is, in part, through reduction of inflammation. Concomitant with this appears to be a normalization of epidermal turnover [39]. The expression of PPARa and PPARg in psoriatic skin is unchanged or even reduced; however, PPARb/d is upregulated [19]. In mouse skin, PPARb/d is upregulated in direct response to the pro-inflammatory cytokine interferon-g (IFN-g) [16]. Recently, it has been demonstrated that PPARb/d mediates tumor necrosis factor (TNF)-induced survival of activated T-cells within psoriatic lesions [40]. This suggests that PPARb/d may actually contribute to the pathogenesis of psoriasis. However, in a mouse model of wound healing, TNFa via PPARb/d
promotes both epidermal differentiation and moderation of inflammation [16]. This mechanism involves ceramide as a second messenger in the TNF signaling cascade leading to upregulation of PPARb/d. So the role of PPARb/d as a mediator of inflammation and epidermal homeostasis appears to be highly dependent on the environment that the keratinocyte is in (e.g., wounded or inflamed), as well as on the species of animal model or the disease state. It is interesting to note that ceramide can play a role in the upregulation of PPARb/d [16]. It could be speculated that the increased synthesis and secretion of ceramides during epidermal differentiation could concomitantly increase the expression of PPARb/d. This may explain why PPARb/d is the most abundant PPAR found in normal skin [8, 19]. PPARa agonists such as clofibric acid and WY-14643 appear able to reduce inflammation by preventing inflammatory cell infiltrate. In mouse models of irritant and allergen contact dermatitis, these agonists reduced the amount of pro-inflammatory cytokines, TNFa and interleukin-1 (IL-1) [17]. These cytokines are key drivers of the inflammatory cascades that lead to the migration of inflammatory cells into affected tissues. In this model, the authors comment on the fact that the PPARa agonists were of similar potency as the corticosteroid clobetasol, an indication of the potential for PPAR therapies in treating inflammatory skin disease. The levels of inflammatory mediators – such as IL-1b, TNFa, IL-6, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) – increase with age, and aged animals have an increased sensitivity to inflammatory stimuli [35]. A key signaling pathway for both synthesis and activity of pro-inflammatory mediators is the nuclear factor-kB (NK-kB) pathway. Under normal conditions the NF-kB pathway is activated transiently, to enable an appropriate response to the stimulating challenge, such as injury or infection. As resolution of the insult proceeds, the pathway’s activity decreases. However, in aged animals there is evidence for constitutive activation of NF-kB, which may contribute to the acceleration of the aging process [41]. Activation of PPARa has been shown to repress NF-kB signaling and reduce production of IL-6 and IL-12 [42]. A recent article has proposed that the inhibitor of NF-kB kinase-alpha (IKKa) is a master regulator of epidermal differentiation [43]. The role of IKKa within the NF-kB signaling pathway is to phosphorylate inhibitors of NF-kB (IkBs), allowing translocation of NF-kB to the nucleus. Exactly how IKKa is involved in keratinocyte differentiation is not completely clear, but these data suggest that keratinocyte hyperproliferation may be driven, in part, by inflammatory pathways. This
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
appears to demonstrate that epidermal homeostasis is very closely linked to the inflammatory status of the skin, and that PPARs could be intricately involved in regulation of both. Exposure to UV light accelerates skin aging, and UV exposure induces skin inflammation, which is a major player in skin aging. While UVB will only penetrate to the epidermis, UVA can penetrate the skin to the dermis, where it will initiate a cascade of events that leads to the degradation of the dermal extracellular matrix. These changes take the form of increased collagen degradation, increased catabolic activity of dermal fibroblasts, and effects on the vasculature that promote the infiltration of inflammatory cells. This infiltrate adds to the catabolic environment within the dermis. Cytokines and chemokines are key to this process, as they orchestrate the response to the UV insult. As discussed above, many of the pro-inflammatory mediators act via the NF-kB intracellular signaling pathway. As such, PPARs are likely to be involved in mediating the inflammatory reaction to UV exposure. There is evidence that pre-treatment of skin with the PPARa agonist WY-14643 protects against UVB-induced erythema [44]. UVB can only penetrate as far as the epidermis; however, the ultimate effect of UVB irradiation in this model is an increase in erythema, which suggests keratinocyte-induced effects could be driving dermal vasculature changes that lead to the increased blood flow – in other words, an epidermal-induced dermal effect. Potential mediators for this epidermal driven process could be IL-8 and IL-6. IL-8 is a CXC chemokine known to be expressed by keratinocytes [45], and its function is to draw inflammatory cells out of the vasculature to the seat of an infection or injury. IL-6 is a prominent earlyphase cytokine, released in response to insult, which triggers many inflammatory cascades. Expression of IL-6 and IL-8 were both reduced by WY-14643 in UVB-irradiated keratinocyte cultures [44]. In addition to being induced in response to UV irradiation, IL-6 is known to be upregulated with age [46–48], raising the possibility that IL-6 may play a role in skin aging. IL-6 is a member of a family of cytokines known as the gp130 binding cytokines. They are grouped together as their receptor complexes share a common receptor subunit: gp130. The primary signaling pathway utilized by this family is the janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway. It is known that members of the IL-6 family, which includes oncostatin M also present in skin [49, 50], can synergize with IL-1 or TNF to induce matrix-degrading enzymes [51]. Data from this and similar studies [52–54] suggest
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that low, physiologically relevant levels of the cytokines are enough to induce catabolic events that could contribute to the molecular causes of aged skin. The synergies between these cytokines occur at convergence points within their intracellular signaling pathways. There is evidence in the literature that activation of PPARs can repress the activity of the JAK/STAT pathway [55] and the AP-1 pathway [16]. This evidence, and the fact that PPARs may be able to regulate IL-6 expression in UV-induced inflammation and induce repressive pressures on NF-kB signaling, suggests that intervention or prophylactic treatment with PPAR therapies may be able to ameliorate some of the inflammation-driven causes of premature skin aging. Many of the natural ligands for PPARs are lipids; therefore, it is not surprising that some members of the bioactive lipid mediator families, prostaglandins, leukotrienes and thromboxanes, have been identified as PPAR ligands. One such mediator is leukotriene B4 (LTB4). The identification of LTB4 as a PPARa ligand came about during investigations to explore whether induction of PPARa activity would increase the catabolism of LTB4 [56]. The pro-inflammatory actions of LTB4 are controlled via its catabolism by peroxisomal enzymes; thus, increasing peroxisomes via PPARs was investigated. This was indeed the case; the authors demonstrated that PPARa knockout mice took longer to resolved LTB4induced inflammation. Prostagalandin E2 (PGE2) is a pro-inflammatory mediator that is also derived from the membrane-bound lipid arachidoninc acid. Cyclooxygenase enzymes (COX-1 and 2) are responsible for the initial cleavage of arachidonic acid, which, via intermediates, leads to the synthesis of PGE2 and other bioactive lipid mediators. PGE2 is required for UVB-induced inflammation and is the main prostaglandin produced by keratinocytes [57]. While there is no evidence to date that PGE2 can bind directly to PPARs, there is evidence in the literature that activation of PPARg can suppress the transcriptional activation of COX-2 and subsequent synthesis of PGE2 [58]. The PPARg ligands tested in this study were ciglitazone, troglitazone, and 15-deoxy-D12,14 prostaglandin J2 (15dPGJ2). 15d-PGJ2 is another member of the prostaglandin family; however, binding of 15d-PGJ2 to its target receptors tends to elicit anti-inflammatory events. 15d-PGJ2 is a natural ligand for PPARg, and its effects on the synthesis of prostaglandins by suppressing the synthesis of COX2 are PPARg-dependent. 15d-PGJ2 can also mediate antiinflammatory activity independent of PPARg [55]. As discussed above, PPARs can act upon the JAK/STAT pathway to reduce inflammation. It appears that 15d-PGJ2 can reduce the activity of this pathway [59, 60].
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CLA: A Naturally Occurring Lipid with PPAR Activity Dietary literature suggests that Conjugated Linoleic Acid (CLA) provides a number of physiologic health benefits to the immune system from supplements, which may involve regulation of prostaglandin, cytokine, and PPAR pathways. (For reviews, see [60–62] and references therein.) CLA may be extracted from sunflower or safflower oil, and is naturally present in a number of foods, including dairy products (milk, cheese, butter, yoghurt) and meats (beef, lamb). CLA is a collective term for positional and geometric (cis/trans) isomers of linoleic acid (C18:2), in which two double bonds are conjugated (e.g., separated by one carbon-carbon single bond). Principal isomers produced from the conjugation of linoleic acid are cis-9, trans-11 CLA and trans-10, cis-12 CLA (> Fig. 21.1). CLA has been shown to be a natural ligand for all three of the PPAR receptors [61, 63, 64]. While most studies on CLA have been directed at understanding the dietary benefits of CLA supplementation, the potential to use CLA as topically applied cosmetic ingredient to improve the appearance of photoaged skin is being investigated. Previous research has demonstrated that CLA delivers a variety of anti-aging benefits to skin, including a
reduction in the appearance of overall photodamage, mottled hyperpigmentation, lines and wrinkles, and coarse wrinkles [29].
Clinical Studies with CLA: Evidence of Anti-aging Benefit from Topical Application Photodamaged Split-Application Facial Studies Healthy female subjects (ages 40–70 years), with Fitzpatrick skin type I–III and a moderate degree of expert-assessed photodamage, were enrolled in Institutional Review Boardapproved, randomized, split-face application studies. A total of 64 subjects were enrolled to complete two facial photodamage studies. Subjects provided informed consent to participate in each study. Subjects were excluded if they had previous reactions to a-hydroxyl acids, retinoids, fragrances, or soaps. Test products were a cream containing 3% CLA and its vehicle. Subjects applied each test product (approximately 0.35 g for each side at each application) to each side of their face twice daily for 12–16 weeks. Subjects were instructed to use their current cleansing regimen throughout the study, and agreed to discontinue use of
. Figure 21.1 Conjugated Linoleic Acid (CLA). CLA is a collective term for positional and geometric (cis/trans) isomers of linoleic acid (C18:2), in which two double bonds are conjugated (e.g., separated by one carbon-carbon single bond). Principal isomers produced from the conjugation of linoleic acid are cis-9, trans-11 CLA and trans-10, cis-12 CLA
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
their normal moisturizers and other anti-aging products (e.g., masks, eye creams, or gels) throughout the study. Expert visual assessment of photodamage attributes in the periorbital region (rated on a 0–9 scale, where 0 = none, 1–3 = mild, 4–6 = moderate, and 7–9 = severe) was performed at baseline and at monthly time points by an expert clinical evaluator. Fine lines in the crow’s feet area (periorbital), fine lines under the eye, and overall photodamage were assessed on each side of the face. Analysis of between-treatment differences observed by expert visual assessment was calculated using the Wilcoxon signed-rank test. Digital photos were obtained at baseline and Weeks 12 and 16 for each side of the face (approximately 45 views) to record improvement in the appearance of photodamage using the VISIA-CR1 photography system (Canfield Scientific, Inc., Fairfield, NJ, USA). Color calibration cards were used to ensure and monitor quality of the images throughout the studies. 25 subjects completed the first facial study, which was a 16-week double-blind product application study. The CLA cream significantly improved the appearance of photodamage attributes from baseline conditions (> Figs. 21.2–21.4) as assessed by an expert clinical evaluator. Fine lines in the crow’s feet and under-eye areas significantly improved from baseline conditions at Weeks 12 and 16 (p < 0.05). By Week 12, the CLA cream provided significant improvement in expert visual assessment of photodamage attributes compared with its vehicle cream (> Figs. 21.2–21.4), which continued at Week
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16. Sample photos of individual subjects (baseline compared with Week 16) are shown in > Figs. 21.5 and > 21.6. Clinical improvement of fine lines and wrinkles in the periorbital region were observed, as well as improvement in overall appearance. 39 subjects completed the second facial study, which was a 12-week double-blind product application study. Expert visual evaluation of photodamage was performed at baseline and after 12 weeks of product application. Objective measurements with 3D facial scans were obtained using the Cyberware™ 3030/HIREZ facial scanner (Cyberware, Inc., Monterey, CA, USA) at baseline and at Weeks 2, 4, 6, 8 and 12. Virtual 3D images were obtained via facial scanning, across a 15 cm vertical line, 90 section, sampled at a rate of 0.2 . The resulting scans captured images approximately from ear to ear on each subject’s face. The vertical scan data captured images approximately from subjects’ chins upward to mid-forehead, depending on the size of the individual’s face. The nasolabial fold lines, characterized by length and depth parameters were quantified using Echo-TK/Cyscan™ software. At Week 12, fine lines in the crow’s feet area, under the eye, and overall photodamage significantly improved from baseline condition with the CLA cream (> Fig. 21.7). In addition, the CLA-treated side was significantly more improved than its vehicle-treated control side, and the vehicle-treated side was not significantly improved for any visual assessment at Week 12. A subset of the panel (N = 12) were measured using objective 3D facial
. Figure 21.2 Clinical evaluation of periorbital fine lines. Compared with its vehicle, CLA cream significantly improved the appearance of periorbital fine lines (P < 0.05)
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. Figure 21.3 Clinical evaluation of fine lines under the eye. Compared with its vehicle, CLA cream significantly improved the appearance of fine lines under the eye and on the upper cheek (P < 0.05)
. Figure 21.4 Clinical evaluation of overall appearance. Compared with its vehicle, CLA cream significantly improved overall appearance (P < 0.05)
scanning measurements, via measurement of nasolabial fold lines (length in mm). The CLA-treated side showed significant improvement from baseline condition at Weeks 8 and 12; the vehicle-treated side showed no significant improvement at any week (> Fig. 21.8).
Biopsy Study on Photodamaged Forearms Healthy female subjects (ages 40–65 years) with Fitzpatrick Skin Type I–III and mild to moderately photodamaged volar forearms, provided informed consent to
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
21
. Figure 21.5 Courtesy at (a) baseline and (b) Week 16 for individual subject. CLA cream significantly improved the appearance of photodamage attributes such as fine lines, wrinkles, and overall appearance. Application of the CLA cream has reduced the length and depth of this subject’s crow’s feet. The fine lines in this subject’s under-eye area have also decreased in depth and become less apparent
. Figure 21.6 Courtesy at (a) baseline and (b) Week 16 for individual subject. CLA cream significantly improved the appearance of photodamage attributes such as fine lines, wrinkles, and overall appearance. The depth of this subject’s fine lines in the crow’s feet area have greatly decreased with use of the CLA cream. An improvement in photodamage is also seen in the subject’s overall appearance, particularly in the lower cheek area
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Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
. Figure 21.7 Clinical evaluation of CLA cream compared to its vehicle at Week 12, by expert assessment of photodamage. Significant improvement was observed with CLA for all attributes, compared to baseline skin condition. Significant improvement favoring CLA over its vehicle was observed for all attributes
. Figure 21.8 Objective 3D assessment of CLA compared to its vehicle, using Cyberware Facial Scanning
Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin
participate in a 21-day repeat (9 24 h) patch test, followed by a dermatologist-collected punch biopsies test. 12 total subjects were enrolled, and completed all phases of the study. The test methodology and evaluation results with CLA were previously described by Shingleton et al. [65]. Test products included CLA formulated into a simple vehicle, the vehicle, and 0.05% retinoic acid as a positive control. Test products were applied to the skin under occlusive patch for 24 h, with 24-h recovery gaps over the course of 21 days. On the last day of the study the consulting dermatologist took full thickness punch biopsies from each site. Biopsies were formalin-fixed, processed for paraffin wax embedding, and sectioned. Sections were stained for H&E and orcein. Further sections were subjected to immunohistochemical analysis for filaggrin expression. The degree of filaggrin staining was assessed by a trained expert evaluator. The CLA and retinoic acid responses were normalized to degree of staining with the vehicle control, and reported as mean increased percentage of responses. Erythema was assessed by the consulting dermatologist on a 0–4 scale (where 0 = none and 4 = severe). The analysis of biopsies from subjects treated with CLA showed increased keratinocyte proliferation and epidermal thickness (9 out of 12). A significant increase in the expression of filaggrin following CLA cream application was also observed, suggesting a concomitant increase in epidermal differentiation. Detailed histological evaluation of the dermis showed evidence of stimulation of extracellular matrix turnover. In addition, increased removal of elastotic material and a modest increase in damaged collagen degradation was observed in all subjects treated with the CLA cream. After removal of the final patch, there was no significant difference in edema observed between the vehicle and the CLA cream. However, as expected, the retinoic acid control showed significantly increased erythema/edema grades compared to the measured response of the vehicle.
Conclusion Clinically, CLA – a cosmetic PPAR lipid – has recently been shown to improve the appearance of photodamage [29], such as reduced fine lines and wrinkles, overall appearance, and the appearance of coarse rhytides in the nasolabial fold area, in vehicle-controlled photodamaged facial studies as described above. CLA cream increased epidermal proliferation, leading to an increase in the
21
thickness of the epidermis in a photodamaged forearm test, with no significant edema, whereas application of retinoic acid (0.05%) induced significant irritation/ edema. The increase in keratinocyte proliferation was accompanied by an increase in differentiation, as judged by the significant increase in filaggrin expression and a thickening of the stratum granulosum, which is likely to result in improved epidermal quality. The magnitude of the effects described above is significant and similar to the effects described when PPARs are activated in models of epidermal development and disease. There is an apparent increase in epidermal proliferation [10, 11], and a concomitant increase in differentiation [12, 13]. Evidence of dermal effects following CLA treatment in the photodamaged forearm model is well demonstrated; dermal fibroblasts have been shown to respond to activators of PPARa in vitro [66]; however, there is no evidence to suggest that topically applied PPAR agonists can activate fibroblasts directly. It is possible that by inducing beneficial effects on epidermal homeostasis, CLA also triggers an epidermal-induced dermal response that may lead to the observed dermal changes. The role of the PPARs in skin homeostasis is now widely accepted, as is their benefit in treating hyper-proliferative and inflammatory skin disorders. Keratinocytes can respond to the environment they are in via signaling pathways that utilize PPARs; this includes both cell cycle control and inflammatory pathways. Akin to their role in systemic lipid control, PPARs in skin could be regarded as the keratinocyte sensors leading to homeostatic control of inflammation as well as differentiation. As such, they have high potential as targets for cosmetic and therapeutic intervention strategies. The significant improvement in photodamaged attributes with no irritation have been summarized here for CLA, a natural ligand for PPAR, in vehicle controlled, double-blind clinical trials.
Acknowledgment The authors would like to thank Susan Krein, Vickie Foy, Priya Vaidyanathan, and Robert Marriott for their contributions to this research.
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Peroxisome Proliferator-activated Receptors: Role in Skin Health and Appearance of Photoaged Skin 36. Boyd AS. Thiazolidinediones in dermatology. Int J Dermatol. 2007;46:557. 37. Dahten A, Koch C, Ernst D, Schnoller C, Hartmann S, Worm M. Systemic PPAR gamma ligation inhibits allergic immune response in the skin. J Invest Dermatol. 2008;128:2211. 38. Michalik L, Wahli W. Peroxisome proliferator-activated receptors (PPARs) in skin health, repair and disease. Biochim Biophys Acta-Mol Cell Biol Lipids. 2007;1771:991. 39. Demerjian M, Man MQ, Choi EH, Brown BE, Crumrine D, Chang S, Mauro T, Elias PM, Feingold KR. Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptorgamma, normalizes epidermal homeostasis in a murine hyperproliferative disease model. Exp Dermatol. 2006;15:154. 40. Al YN, Romanowska M, Krauss S, Schweiger S, Foerster J. PPAR delta is a type 1 IFN target gene and inhibits apoptosis in T cells. J Invest Dermatol. 2008;128:1940. 41. Spencer NF, Poynter ME, Im SY, Daynes RA. Constitutive activation of NF-kappa B in an animal model of aging. Int Immunol. 1997;9:1581–1588. 42. Poynter ME, Daynes RA. Peroxisome proliferator-activated receptor alpha a´Activation modulates cellular redox status, represses nuclear factor-kappa B signaling, and reduces inflammatory cytokine production in aging. J Biol Chem. 1998;273:32833–32841. 43. Liu B, Zhu F, Xia X, Park E, Hu Y. A tale of terminal differentiation: IKKalpha, the master keratinocyte regulator. [In Process Citation]. Cell Cycle (Georgetown, Tex 8). 2009;8(4):527–531. 44. Kippenberger S, Loitsch SM, Grundmann-Kollmann M, Simon S, Dang TA, Hardt-Weinelt K, Kaufmann R, Bernd A. Activators of peroxisome proliferator-activated receptors protect human skin from ultraviolet-B-light-induced inflammation. J Invest Dermatol. 2001;117:1430–1436. 45. Coquette A, Berna N, Vandenbosch A, Rosdy M, De Wever B, Poumay Y. Analysis of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8) expression and release in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization. Toxicology In Vitro. 2003;17:311–321. 46. Daynes RA, Araneo BA, Ershler WB, Maloney C, Li GZ, Ryu SY. Altered regulation of interleukin-6 production with normal aging. J Immunol. 1993;150:A285. 47. Ershler WB. Interleukin-6 - A cytokine for gerontologists. J Am Geriatr Soc. 1993;41:176–181. 48. Ershler WB, Keller ET. Age-associated increased interleukin-6 gene expression, late-life diseases, and frailty. Ann Rev Med. 2000; 51:245–270. 49. Boniface K, Diveu C, Morel F, Pedretti N, Froger J, Ravon E, Garcia M, Venereau E, Preisser L, Guignouard E, Guillet G, Dagregorio G, Pene J, Moles JP, Yssel H, Chevalier S, Bernard FX, Gascan H, Lecron JC. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. J Immunol. 2007;178:4615. 50. Yu M, Kissling S, Freyschmidt PP, Hoffmann R, Shapiro J, McElwee KJ. Interleukin-6 cytokine family member oncostatin M is a hairfollicle-expressed factor with hair growth inhibitory properties. Exp Dermatol. 2008;17:12. 51. Rowan AD, Koshy PJT, Shingleton WD, Degnan BA, Heath JK, Vernallis AB, Spaull JR, Life PF, Hudson K, Cawston TE. Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown. Arthritis Rheum. 2001;44:1620–1632. 52. Cawston TE, Curry VA, Summers CA, Clark IM, Riley GP, Life PF, Spaull JR, Goldring MB, Koshy PJT, Rowan AD, Shingleton WD. The
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6 Physiological Variations During Aging Ge´rald E. Pie´rard . Philippe Paquet . Emmanuelle Xhauflaire-Uhoda . Pascale Quatresooz
Introduction In affluent societies, an extraordinary shift has taken place over the past decades in the age distribution of the populations. Indeed, older people progressively represent a growing segment of the demographic profile. Nobody can escape from aging. Such evolution is correlated with the individual loss of youthful appearance, which in turn has important social implications. As a result, any newer medical and cosmetic anti-aging modalities are avidly watched by the aging individuals. Moreover, middle-aged and younger subjects may show a craze for cosmetic dermatology beginning at the onset of early signs of wear and tear. Hopefully, breakthroughs in cosmetic procedures and novel treatments fulfill some of the expectations and promises. In addition, technological advances at the forefront of the management of skin aging are supported by a better understanding of the relationships among skin biology, skin physiology, and the ultimate clinical appearance. Both physical growth and senescence are characterized by cumulative progression of interlocking biologic events. They may proceed at some time in life as if they were evolving in tandem. Globally, aging is a physiological process corresponding to a progressive impairment in the homeostatic and adaptive homeodynamic capacities of the body systems, ultimately increasing the susceptibility and vulnerability to environmental threats and internal changes. While aging is associated with a slower rate of cutaneous wound healing, it may be paradoxically associated with an improvement in the quality of scarring. The global aging process is quite complex. It becomes severe in cases where the impaired skin has lost most of its protective biological and mechanical functions. The atrophy becomes considerably disabling when the aspect of the ‘‘transparent skin’’ or ‘‘dermatoporosis’’ is reached [1]. The corresponding clinical manifestations encompass a series of markers of fragility including senile purpura, stellate pseudoscars, and skin thinning. Skin fragility leads to frequent lacerations following minor traumas. A varied combination of delayed wound healing, nonhealing atrophic ulcers, subcutaneous bleeding, and dissecting hematomas is at the origin of large necrotic areas.
From Global to Molecular Aging and Back Again All living organisms are engaged in multifaceted aging processes that show interspecies variations. Two complementary classifications of life histories are of major importance in the biological definition of aging. The first classification distinguishes species that show a clear distinction between germ cells and somatic tissues from those that do not. The second classification distinguishes the semelparous species reproducing only once in their lifetime, from the iteroparous species, which reproduce repeatedly. It is a mistake to regard the postreproductive end of life of semelparous species, which usually occurs in a highly determinate fashion, as being comparable with the more protracted process of senescence in iteroparous species. There is evidence that aging progresses differ among individuals of the same age. In addition, each and every organ of the human body develops and fails at its own rate, which is referred to as its age [2]. Senescence is heterogeneous at other levels including tissues, cells, and subcellular structures [3]. Intracellular and extracellular molecular compounds are involved differently in aging. However, within each organ system, aging manifests as a progressive, almost linear reduction in maximal function and reserve capacity. Although some aspects of aging appear as a predetermined programmed process, many of the age-associated physiological decrements result in part from environmental insults and/or from endogenous failures. The global but heterogeneous aging occurs throughout the entire body from the time of about 30–45 years of age (> Table 6.1). To make the situation more complex, there is regional variability in skin aging over the body. It is indeed quite evident that at any time in adult life the face, scalp, forearms, trunk, and other body sites show different manifestations or stages of aging. In addition, scrutinizing skin aging at the tissue level (epidermis, dermis, hypodermis, hair follicle), and further at the cellular level (keratinocyte, melanocyte, fibroblast, dermal dendrocyte, endothelial cells, etc.) shows a patchwork of aging severity.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_6, # Springer-Verlag Berlin Heidelberg 2010
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Physiological Variations During Aging
. Table 6.1 Core age markers of each of the body system (Adapted from Braverman [2]) Aging type
Decline in
Average onset age
Electropause
Electrical activity of brain waves
45
Biopause
Neurotransmitters
Dopamine 30, Acetylcholine 40, GABA 50, Serotonin 60
Pineal pause
Melatonin
20
Pituitary pause
Hormone feedback loops
30
Sensory pause
Touch, hearing, vision, taste, and smell sensitivity
40
Psychopause
Personality health and mood
30
Thyropause
Calcitonin and thyroid hormone levels
50
Parathyropause
Parathyroid hormone
50
Thymopause
Glandular size and immune system
40
Cardiopause/vasculopause
Ejection fraction and blood flow
50
Pulmonopause
Lung elasticity and function with increase in blood pressure
50
Adrenopause
DHEA
55
Nephropause
Erythropoietin level and creatinine clearance
40
Somatopause
Growth hormone
30
Gastropause
Nutrient absorption
40
Pancropause
Blood sugar level
40
Insulopause
Glucose tolerance
40
Andropause
Testosterone in men
45
Menopause
Estrogen, progesterone, and testosterone in women
40
Osteopause
Bone density
30
Dermopause
Collagen, vitamin D synthesis
35
Onchopause
Nail growth
40
Uropause
Bladder control
45
Genopause
DNA
40
Cellular Senescence in Perspective Several basic cellular mechanisms are involved in the aging process: the telomere shortening, the oxidative stress, the mitochondrial dysfunction, and a series of genetic mechanisms. Granted that death is the ultimate failure of the organisms, the aging process itself is considered to bring about the termination of the replicative ability of cells as the individual becomes progressively older. The age of any tissue appears to be reflected in the behavior of their cultured skin-derived cells [4]. Replicative senescence of human cells is thus related to and perhaps caused by the exhaustion of their proliferative potential. At each
cell division, cells lose some of their telomere repeats corresponding to specific DNA sequences at the end of linear DNA [5]. According to the telomere hypothesis, somatic cells lack a sustained activity of telomerase to maintain the telomere repeats allowing replication. The telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative stress. Since telomere length predicts the replicative capacity of cells, it is thought to provide the best biomarker for cellular aging [6]. Aging is associated to free radical damage by a variety of reactive oxygen species (ROS) including superoxide and hydroxyl radicals, as well as other activated forms of oxygen such as hydrogen peroxide and singlet oxygen [7].
Physiological Variations During Aging
In the intracellular machinery, mitochondria are the primary sites of production of ROS. Enzymes that minimize oxidative injury include superoxide dismutase, catalase, gluthatione peroxidase, glutathione transferases, peroxidases, and thiol-specific antioxidant enzymes. In addition, ROS play a role in normal signaling processes. Their generation is essential to maintain homeostasis and cellular responsiveness [8]. The so-called stress-induced premature senescence (SIPS) occurs following a series of sublethal stresses including those induced by H2O2, other ROS, and a variety of other chemicals [9]. Of note, cells engaged in replicative senescence share common features with cells involved in SIPS [9]. Thus, SIPS could be a mechanism of the in vivo accumulation of senescent-like cells in the skin [10]. Mitochondria are both producers and targets of oxidative stress, thus forming the basis for the mitochondria theory of aging. With advanced age, the activity of the mitochondrial respiratory system declines. The integrity of the mitochondrial DNA is reduced, eventually leading to apoptosis [11–13]. It is acknowledged that the alterations in the mechanisms of apoptosis result from genetically programmed features and oxidative stress as well [14, 15]. Cellular senescence and cancer are closely related by several biological aspects including p53 mutation [16, 17], telomere shortening [5, 18], vitamin A depletion [19], and defects in intercellular communications [20].
6
The age-related mottled faint (subclinical) melanoderma might be a predictive sign for a skin carcinoma-prone condition [21–23].
Clinically Relevant Skin Aging Mechanisms Human aging may be perceived as one single chronologic process of physiological decline with age. However, this process exhibits multiple facets altering differently the organs, tissues, and cells. This is particularly obvious in the skin. For years, the understanding of aging skin has benefited from the distinction between the intrinsic chronologic aging and photoaging [24]. According to this concept, the changes observed in the aging skin appearance reflect two main processes (> Table 6.2). First, the intrinsic changes in the aging skin are caused by the passage of time modulated by hereditary factors, along with modifications occurring inherently in the structure, physiology, and mechanobiology. Second, photodamage is a result of the cumulative exposure of the skin to ultraviolet (UV) light and near-infrared radiations (IRA) [25]. Clinically, these two types of aging may manifest differently, with intrinsic aging being responsible for dry, pale, and smooth-to-finely wrinkled skin. By contrast, photoaging gives rise to coarse, roughened, and deeply wrinkled skin accompanied by pigmentary changes such
. Table 6.2 Comparison of intrinsic aging and photoaging Feature
Intrinsic aging
Photoaging
Clinical appearance
Smooth texture, unblemished surface. Fine Nodular, leathery surface. Sallow complexion wrinkles. Some deepening of skin surface markings yellowish mottled, pigmentation. Coarse wrinkles Some loss of elasticity, redundant skin Severe loss of elasticity
Epidermis
Thin and viable
Marked acanthosis, cellular atypia, skin field carcinogenesis, most skin cancers
Elastic tissue
Increased, but almost normal
Tremendous increase, altered as elastotic amorphous mass
Collagen
Thick, disoriented bundles
Marked decrease of bundles and fibers
Glycosaminoglycans Slightly decreased
Markedly increased
Reticular dermis
Thinner fibroblasts decreased, inactive mast cells decreased, no inflammation
Thickened, elastosis. Fibroblasts increased, hyperactive Mast cells markedly increased, mixed inflammatory infiltrate
Papillary dermis
No Grenz zone
Solar elastosis with Grenz zone
Microvasculature
Moderate loss
Great loss, abnormal, and telangiectatic
Subcutaneous fat
Focal shrinkage. Focal hypertrophy
Unaffected
Hair
Hair thinning Graying hair. Hypertrichosis
Discoloration
47
48
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Physiological Variations During Aging
as solar lentigines and mottled melanosis. Differences between these two types of aging can be seen within one individual when comparing an area of skin commonly exposed to the sun, for example, the face, the neck, and the dorsal forearms, with an area commonly masked from the sun, for example, buttock skin. This concept based on a duality in skin aging has been challenged because it may appear as an oversimplification in clinical practice [26]. In addition, recent evidence indicates that chronologically aged and UV-irradiated skin share important molecular features [7, 8, 27, 28]. Oxidative stress is thought to play a central role in initiating and driving the signaling events that lead to cellular response following UV and IRA exposures. These features suggest that nonionizing radiations accelerate many key aspects of the chronological aging process in human skin [25]. In order to better cope with the diversity of clinical presentations of skin in the elderly, another classification of skin aging in seven distinct types was offered (> Table 6.3). The most important categories included the endocrine and overall metabolic status, the past and present lifestyle, and several environmental threats, including cumulative UV and IRA exposures, and repeated mechanical solicitations by muscles and external forces including earth gravity [26]. In this framework, the past history of the subject is emphasized. Accordingly, the global aging is considered to represent the cumulative or synergistic effects of specific features, each of them being independent from the others. Such a concept allows to individualize or integrate typical processes including among others climacteric aging and smoking effects. Increased awareness of the distinct age-associated physiologic changes in the skin may allow for more effective and specific skin care regimens, preventive measures, and dermatologic treatment strategies in the elderly. As a consequence, the immutability of diverse factors in skin aging was challenged [3, 29]. However, factors of skin
aging share some common mechanisms [30]. For instance, molecular mechanisms imply hyaluronate-CD44 pathways in the control and maintenance of epithelial growth and the viscoelastic properties of the extracellular matrix offer new opportunities for preventive intervention [1].
Environmental Aging and Photoaging Environmental influences produce obvious alterations in the texture and quality of the skin, the major extrinsic insults being chronic exposure to UV and IRA [25]. The action spectrum of photodamages is not fully characterized. The cumulative effects from repeated exposures to suberythemal doses of UVB, UVA, and IRA in human skin are involved in these processes [25, 31]. The role of UVB in elastin promoter activation in photoaging is obvious. In addition, UVA significantly contributes to long-term actinic damage, and the spectral dependence for cumulative damages does not parallel the erythemal spectrum for acute UV injury in human beings. The earliest detectable response of the skin cell to UV irradiation is the activation of multiple cytokine and growth factor cell surface receptors including epidermal growth factor receptor (EGF-R), TNF-a receptor, platelet-activating factor (PAF receptor) insulin receptor, interleukin (IL)-1 receptor, and platelet-derived growth factor (PDGF) receptor [27]. Nonionizing radiations initiate a number of cellular responses, including ROS production within both dermal and epidermal cells. The so-called stress-induced parameter senescence (SIPS) phenomenon is engaged [9]. More specifically, cultures of keratinocytes derived from donors of different ages and from paired sun-exposed and sunprotected sites of older donors demonstrate that both chronological aging and photoaging affect quite distinctly
. Table 6.3 Cutaneous aging types (Adapted from Pie´rard [26]) Aging type
Determinant factor
Genetic
Genetic (premature aging syndromes, phototype-related, ethnic background)
Chronologic
Time
Actinic
Ultraviolet and infrared irradiations
Behavioral
Tobacco, alcoholic abuse, drug addiction, facial expressions
Endocrine
Pregnancy, physiological and hormonal influences (ovaries, testes, thyroid)
Catabolic
Chronic intercurrent debilitating disease (infections, cancers), nutritional deficiencies
Gravitational
Earth gravity
Physiological Variations During Aging
some gene expressions. For instance, chronological aging strikingly increases the baseline expressions of the differentiation-associated gene SPR2 (small proline-rich protein) and of the interleukin (IL)-1 receptor antagonist gene. By contrast, it has relatively little effect on the UV-inducibility of several other genes including the protooncogenes, c-myc and c-fos, the GADD 153, a gene inducible by growth arrest and DNA damage, and the IL-1a and 1L-b genes. Photoaging appears different because it increases the UV-inducibility of c-fos while decreasing the baseline expression of the differentiationassociated genes IL-1ra and SPR2 [32, 33]. The physiological consequences of photodamages occur at variable pace on the different skin structures. For instance, skin loosening and solar elastosis exhibit clinical manifestations independently from the severity in the mottled faint melanoderma [21]. IR are nonionizing, electromagnetic radiations accounting for more than half of the solar energy that reaches the human skin. The IR wavelengths ranges between 760 nm and 1 mm, and are further divided into IRA, IRB, and IRC. While IRB and IRC do not penetrate deeply into the skin, more than 65% of IRA reaches the dermis. Human skin is increasingly exposed to IRA radiation. The most relevant sources are: (a) natural solar radiation consisting of over 30% IRA, (b) artificial IRA sources used for therapeutic or wellness purposes, and (c) artificial UVA sources supplemented with IRA. As part of natural sunlight, IRA significantly contributes to extrinsic photoaging. Photoaging affects both the epidermis and dermis. The epidermis becomes more atrophic than sun-protected areas often with disordered keratinocyte maturation. This may represent an early step in actinic field carcinogenesis. Key histological features of photoaged skin are most apparent in the dermis where the extracellular matrix is altered in its composition [34]. The collagen network is responsible for most skin strength and resiliency, and is intimately involved in the expression of photoaging. The major fibrillar collagen components of the dermis belong to the type I and III collagens. In photoaged human skin, precursors of both proteins are markedly reduced in the papillary dermis, and their reduction correlates with the clinical severity of photoaging [35]. This reduction results from a combination of decreased procollagen biosynthesis contrasting with an increased enzymatic breakdown by matrix metalloproteinases (MMPs) [36]. Furthermore, collagen breakdown products negatively influence procollagen biosynthesis by fibroblasts [37]. Fibrillar collagens are closely associated with the small chondroitin sulfate proteoglycan, and decorin. Its
6
distribution closely mirrors that of type I collagen in the dermis, regardless of level of extrinsic aging [38]. Decorin makes connections between the fibrillar collagens and the microfibril-forming type VI collagen with further interaction with type IV collagen in the basement membrane at the dermo-epidermal junction. Therefore, type VI collagen is likely to play an important physiological role in the organization of the dermal matrix. It is abundant in the papillary dermis and seems little affected by photoaging [39]. Type VII collagen is the major constituent of anchoring fibrils below the basement membrane providing cohesiveness between the epidermis and the dermis. In photoaged skin, the number of anchoring fibrils along the basement membrane is reduced, thus increasing the potential for fragility and blistering in photoaged skin [40]. It was reported to be involved in the mechanism of wrinkle formation. The elastic fiber network is responsible for recoil and elasticity of the skin. The process of elastic fiber formation is under tight developmental control, involving tropoelastin deposition on a preformed framework made of fibrillin-rich microfibrils. Mature elastic fibers are encased in fibrillin and form a continuous network throughout the dermis. The elastic fiber network comprises thick elastin-rich fibers within the reticular dermis, finer fibers with reduced elastin content in the lower papillary dermis, and a meshwork of discrete fibrillin-rich microfibrillar bundles, with only discrete elastin, in the upper papillary dermis merging with the dermo-epidermal junction. Fibrillin is a product of both fibroblasts and keratinocytes. The elastic fiber network is considerably disrupted and clumped in chronically photoaged skin. First, photoaged skin contains abundant amounts of dystrophic elastotic material in the reticular dermis [41], which is immunopositive for tropoelastin, fibrillin, lysozyme, and immunoglobulins [42] Versican, a large chondroitin sulfate proteoglycan, appears to be regulated along with dystrophic elastin resulting in a relative increase in photoaging. The fibrillin-rich microfibrils are markedly altered since the early stages of photoaging [43]. All structural changes found in the dermis particularly affect the biomechanical properties of the skin. A cutaneous extrinsic aging score was derived from the difference between comparative photoexposed and photoprotected areas [44].
Phototype and Ethnicity-Related Aging The most obvious ethnic skin difference relates to skin color, which is dominated by the presence of melanin [45, 46]
49
50
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Physiological Variations During Aging
affording relative photoprotection. The rate of skin aging varies between different racial groups, however, although none of the groups escape the photoaging process. Generally, Caucasians present an earlier onset and a deeper skin wrinkling and sagging than other skin phototypes. In general, increased pigmentary problems develop in skin of color, although East Asians living in Europe and North America exhibit less pigment spots with age. Induction of a melanotic response to sun exposure is thought to occur through signaling by the protease-activated receptor (PAR)-2, which together with its activating protease is increased in the epidermis of subjects with skin of color [47]. Changes in skin biophysical properties with age demonstrate that subjects of dark complexion keep younger skin properties compared with the more lightly pigmented individuals.
. Table 6.4 Neuroendocrine receptors active in the skin (Adapted from Quatresooz [50]) 1. Adrenergic receptors 2. Androgen and estrogen receptors 3. Calcitonin gene-related peptide receptor (CGRP-R) 4. Cholinergic receptors 5. Corticotropin-releasing hormone and urocortin receptors (CRH-R) 6. Glucocorticoid and mineralocorticoid receptors 7. Glutamate receptors 8. Growth hormone receptor (GH-R) 9. Histamine receptors 10. Melanocortin receptors (MC-R) 11. Miscellaneous neuropeptide receptors 12. Miscellaneous receptors
Endocrine Aging
13. Neurokinin receptors (NK-R) 14. Neutrophin receptors (NT-R)
Skin is recognized as a hormone-dependent organ [48–51]. Irrespective of age, most of the skin components are under the physiological control of endocrine and neuroendocrine factors (> Table 6.4). The whole endocrine system is affected by the global aging process. Like any other system in the body, aging of the hormonal functions basically results in deteriorations expressed by deficiencies, which in turn influence the aging machinery operative in the skin [50]. Quite distinct are the skin manifestations of some endocrinopathies, which mimic or interfere with skin aging [48–51]. They are mostly related to the declined activity of the pituitary gland, adrenal glands, ovaries, and testes. Some hormones and neurotransmitters are synthesized by nerves, as well as by epithelial and dermal cells in the skin (> Table 6.5). A number of environmental and intrinsic factors regulate the level of the cutaneous neuroendocrine system activity. Solar radiation, temperature, environmental moisture, and diverse chemical and biological xenobiotics represent important environmental factors. Some internal mechanisms affecting the neuroendocrine system of the skin may be generated in reaction to some environmental signals or result from local biological rhythms, or from local or general disease processes [49]. The paradigm of deleterious hormonal effects is represented by the induction of skin atrophy by corticosteroids. Cushing syndrome and iatrogenic effects of topical and systemic corticotherapy are equally involved. Corticosteroids are known to regulate the expression of genes
15. Opioid receptors 16. Parathormone (PTH) and PTH-related protein (PTHrP) receptors 17. PRL and LH-CG receptors (LH/CG-R) 18. Serotonin receptors 19. Thyroid hormone receptors 20. Vasoactive intestinal peptide receptor (VIP-R) 21. Vitamin D receptor (VDR)
. Table 6.5 Hormones and neurotransmitters produced by the skin (Adapted from Quatresooz [50]) 1. Hypothalamic and pituitary hormones 2. Neuropeptides and neurotrophins 3. Neurotransmitters/neurohormones 4. Other steroid hormones 5. Parathormone-related protein 6. Sex steroid hormones 7. Thyroid hormones
encoding collagens I, III, IV, V, decorin, elastin, MMPs 1, 2, 3, tenascin, and tissue inhibitors of MMPs 1 and 2 [52]. However, the precise molecular mechanisms of skin atrophy induced by corticosteroids are not yet known. The corticosteroid-induced atrophy can be one
Physiological Variations During Aging
of the most severe form of skin aging corresponding to dermatoporosis [1]. The most important endocrine compound produced by the skin is vitamin D, which is a regulator of the calcium metabolism and exhibits other systemic effects as well. For example, epidemiological evidence suggests that sunlight deprivation with associated reduction in the circulating level of vitamin D3 may result in increased incidence of carcinomas of the breast, colon, and prostate [53]. Vitamin D3 and its analogs also modulate the biology of keratinocytes and melanocytes of the skin in vivo [54]. Growth hormone (GH) is secreted by the pituitary gland under the control of several hypothalamic and peripheral modulators that exert either positive or negative influences [40]. The final balance among the modulating factors determines the pulsatile and circadian secretion of GH. Moreover, physiological changes occurring during aging affect the GH secretion. The peripheral effects of GH are mainly exerted by insulin-like growth factor (IGF), produced by the liver upon GH stimulation. The circulating IGF-1 is bioavailable and functionally active depending upon its binding with the IGF-binding proteins (IGF-Bps). Skin is a target of the GH-IGF system that exerts a significant influence on the dermal and epidermal physiology [55]. GH, IGF-1, IGF-2, and IGF-Bps are present in the skin and are involved in its physiological homeostasis, including the dermo-epidermal cross talking. Thus, systemic paracrine and/or autocrine cutaneous activity of the GH-IGF system contributes to skin homeostasis [55, 56]. While GH system is abated during aging, GH supplementation induces skin changes, a part of which may correspond to some corrective effects on aging skin [57, 58]. The progressive decline in DHEA serum concentration with age, and conversely its supplementation have not demonstrated prominent effects on the skin except on sebum production. Sex hormones manifest a variety of biological and immunological effects in the skin [59]. In responsive women, estrogen, alone or together with progesterone, has been reported to prevent or reverse skin atrophy, dryness, and wrinkles associated with chronological aging or photoaging. In responsive women, estrogen and progesterone stimulate proliferation of keratinocytes while estrogen suppresses apoptosis and thus prevents epidermal atrophy. Estrogen also enhances collagen synthesis, and estrogen and progesterone suppress collagenolysis by reducing MMP activity in fibroblasts, thereby maintaining skin thickness. Estrogen maintains skin moisture by
6
increasing hyaluronic acid levels in the dermis. Progesterone increases sebum excretion. Both the climacteric period around menopause and the andropause decade may negatively affect the skin [59–61]. Hormone replacement therapy (HRT) during the climacteric period helps limiting these changes [62–65]. However, there is a limitation because it seems that good and poor responders exist [66]. Smoking habit may also interfere with the treatment result [67].
Catabolic Aging The elderly are often accompanied by a substandard diet deficiency in many of the nutrients thought to be essential for maintaining health. Protein-containing foods such as meat and fish tend to be too expensive or troublesome to prepare. Dietary faddism, confusional states, and forgetfulness are also responsible for an inadequate diet. These situations predispose to skin changes that often amplify the alterations induced by age-related deficiencies. Any insufficient intake of fresh fruits and/or vegetables leads to vitamin C deficiency and to scurvy. It results in a defect in coagulation and purpura, particularly in a typical punctate perifollicular pattern on the legs. In the elderly, iron deficiency is common and may result in anemia, generalized pruritus, and diffuse hair loss. Essential fatty acid and vitamin A deficiencies due to dietary faddism or deprivation in the elderly are responsible for xerosis [68]. Many of the elderly are also deficient in zinc, and this may impair wound healing. Zinc supplementation, however, does not improve healing. Chronic hemodialysis is another model of catabolic aging affecting the mechanical properties of skin [69, 70].
Gravitational Aging Skin of any part of the body is subjected to intrinsic and extrinsic mechanical forces. Among them, earth gravitation influences skin folding and sagging during aging. Any force generated by the skin or applied to it, transduces information to cells that may in turn respond to it [71, 72]. The effects of mechanobiology are particularly evidenced in the fibroblasts, dermal dendrocytes, keratinocytes, and melanocytes [73–75]. Physical forces of gravity involve mechanotransduction in the skin [76] and affect cell tensegrity and the cell mechanosensitive ion channels. As a result, the structure of the dermal extracellular matrix is affected.
51
52
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Physiological Variations During Aging
Diversity of Wrinkles There is evidence that wrinkles are not related to the genuine microrelief [77, 78]. In addition, the microanatomical support of wrinkles is varied [77–80]. It depends on subtle changes in the structure of the superficial dermis elastotic deposits in the upper reticular dermis, loosening of the hypodermal connective tissue strands, or, inversely, on focal hypertrophic binding of the dermis to the underlying facial muscles [78, 80]. The wrinkle severity rating [81] is influenced by the nature of the altered connective tissue. Similarly, the skin mechanical properties are under these influences [82, 83]. Photoaged facial skin does not always present clinically with characteristic wrinkling. In some individuals, usually of light phototype, smooth unwrinkled skin and telangiectasia predominate. These people appear to be more at risk of developing basal cell carcinomas on sun-exposed facial skin [84, 85]. There is an apparent inverse relationship between the degree of facial wrinkling and the occurrence of facial basal cell carcinomas [85]. Mechanistically, little is known regarding how these two clinical outcomes occur in response to the same sun-exposure stimulus. Smoking effects on skin aging are probably mediated by the increased production of collagenase and elastase, which is an additional cause of wrinkling [86–88]. Degradation of elastic fibers by ROS and repeated mechanical solicitations by some muscle contractions play a putative role in the formation of the smoker’s wrinkles.
2. 3. 4. 5. 6.
7. 8. 9.
10.
11. 12. 13.
14.
15. 16.
Conclusion Aging is apparent at all levels of the physiology and anatomy of the body. Organs, tissues, cells, and molecules have their own aging processes, which differ in their clinical relevance. The individual may perceive a global appearance of skin aging. By contrast, prevention and correction of skin aging may benefit from targeting some of the specific underlying biological processes.
17. 18.
19. 20.
21.
Cross-references > The
22.
Stratum Corneum and Aging 23.
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67. Castelo-Branco C, Figueras F, Martinez de O saba MJ, Vanrell JA. Facial wrinkling in postmenopausal women effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 68. Uhoda E, Petit L, Pie´rard-Franchimont C, Pie´rard GE. Ultraviolet light-enhanced visualization of cutaneous signs of carotene and vitamin A dietary deficiency. Acta Clin Belg. 2004;59:97–101. 69. Deleixhe-Mauhin F, Pie´rard-Franchimont C, Rorive G, Pie´rard GE. Influence of chronic haemodialysis on the mechanical properties of skin. Clin Exp Dermatol. 1994;19:130–133. 70. Uhoda I, Petit L, Krzesinski JM, Pie´rard-Franchimont C, Pie´rard GE. Effect of haemodialysis on acoustic shear wave propagation in the skin. Dermatology. 2004;209:95–100. 71. Wang N, Butler JP, Ingber DE. Mechano-transduction across the cell surface and through the cytoskeleton. Science. 1993;260: 1124–1127. 72. Silver FH, Siperko M, Seehra GP. Mechanobiology of force transduction in dermal tissue. Skin Res Technol. 2003;9:3–23. 73. Ingber DE. Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol. 1997;59:575–599. 74. Hermanns-Leˆ T, Uhoda I, Pie´rard-Franchimont C, Pie´rard GE. Factor XIIIa-positive dermal dendrocytes and shear wave propagation in human skin. Eur J Clin Invest. 2002;32:847–851. 75. Quatresooz P, Hermanns JF, Paquet P, Pie´rard GE. Mechanobiology and force transduction in scars developed in darker skin types. Skin Res Technol. 2006;12:279–282. 76. Nizet JL, Pie´rard-Franchimont C, Pie´rard GE. Influence of the body posture and gravitational forces on shear wave propagation in the skin. Dermatology. 2001;2002:177–180. 77. Pie´rard GE, Uhoda I, Pie´rard-Franchimont C. From skin microrelief to wrinkles. An area ripe for investigation. J Cosmet Dermatol. 2003;2:21–28.
78. Quatresooz P, Thirion L, Pie´rard-Franchimont C, Pie´rard GE. The riddle of genuine skin microrelief and wrinkles. Int J Cosmet Sci. 2006;28:389–395. 79. Bosset S, Barre´ P, Chalon A, Kurfurst R, Bonte´ F, Andre´ P, et al. Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol. 2002;12:247–252. 80. Pie´rard GE, Lapie`re ChM. The microanatomical basis of facial frown lines. Arch Dermatol. 1989;125:1090–1092. 81. Day DJ, Littler CM, Swift RW, Gottlieb S. The wrinkle severity rating scale: a validation study. Am J Clin Dermatol. 2004;5:49–52. 82. Hermanns-Leˆ T, Jonlet F, Scheen A, Pie´rard GE. Age-and-body mass index-related changes in cutaneous shear wave velocity. Exp Gerontol. 2001;36:363–372. 83. Hermanns-Leˆ T, Uhoda I, Smitz S, Pie´rard GE. Skin tensile properties revisited during aging. Where now, where next? J Cosmet Dermatol. 2004;3:35–40. 84. Kricker A, Armstrong BK, English DR. Sun exposure and nonmelanocytic skin cancer. Cancer Causes Control. 1994;5:367–392. 85. Brooke RCC, Newbold SA, Telfer NR, Griffiths CE. Discordance between facial wrinkling and the presence of basal cell carcinoma. Arch Dermatol. 2001;137:751–754. 86. Ernster VL, Grady D, Miike R, Back D, Selby J, Kerlikowske K. Facial wrinkling in men and women by smoking status. Am J Public Health. 1995;85:78–82. 87. Koh JS. Cigarette smoking associated with premature facial wrinkling: image analysis of facial skin replicas. Int J Dermatol. 2002;41:21–27. 88. Raitio A, Tuomas H, Kokkonen N, Salo T, Sorsa T, Hanemaaijer R, et al. Levels of matrix metalloproteinase-2, -9 and -8 in the skin, serum and saliva of smokers and non-smokers. Arch Dermatol Res. 2005;297:242–248.
52 Pigmentation in Ethnic Groups Rupa Pugashetti . Howard I. Maibach
Introduction
Mechanism of Pigmentation
In order to help understand the pigmentary changes which occur throughout the aging process and in the management of aging skin, it is necessary to examine how pigmentation varies among different ethnicities. Skin color among different ethnicities has important social and political connotations, both historically and at the present time. While it is clear that human skin is an important external feature which can distinguish people of different races, less clear is how variations in human skin pigmentation may contribute to differences in skin structure, function, and pathophysiology. As the demographics of the United States change, the US Census Bureau estimates that by 2050, approximately half of the US resident population will comprise individuals of color [1]. The question of associating race and pigmentation is repeatedly raised, and a recent study demonstrated the need for caution when using pigmentation as a proxy for race or genetic ancestry [2]. Nonetheless, as dermatologists treat more patients of varying ethnic backgrounds and with diverse skin types, an understanding of differences in pigmentation and skin structure and function becomes more important.
Human skin color is determined primarily by melanin synthesis. Melanin can act as an effective sunscreen, protecting darker-pigmented individuals from burning and development of skin cancer [3]. It is now well established that heavy pigmentation in areas of high solar radiation helps protect the skin from carcinogenic effects of ultraviolet light [4]. This might have played a role in selection of darker skin colors evolutionarily in African populations, but how lighter skin was produced as early humans migrated out of Africa and moved into Europe still remains controversial. Additionally, there is a correlation between latitude and skin color; skin is lighter in regions towards the north. This correlates with ultraviolet radiation incidence, which is higher near the equator and lower at higher latitudes. In human skin pigmentation, constitutive skin color refers to genetically determined levels of melanin pigmentation without exposure to ultraviolet radiation or other environmental influences, while facultative skin color refers to increases in melanin pigmentation above the constitutive level induced by exposure to ultraviolet light [5]. Human skin color is dominated by both hemoglobin, which provides red color via the network of capillaries in the skin, and melanin, which provides various gradations of brown coloring to the skin surface. There does not appear to be any significant difference in hemoglobin content among various ethnicities, and there is a constant vascular supply under conditions of constant activity and temperature [6]. Furthermore, the effect of hemoglobin in blood flowing in the dermal papillary layer is easily masked by pigments present in the overlying epidermal layers. Thus, it is primarily differences in the amount of melanin which are responsible for normal variation in human skin pigmentation. Melanin synthesis takes place inside melanosomes, organelles present in the melanocyte. Melanin pigment granules are subsequently transferred to keratinocytes [7]. Specifically, melanosomes are trafficked along dendrites; they are moved along microtubules before being captured
Methods The EMBASE database was searched using the terms ‘‘race,’’ ‘‘race difference,’’ ‘‘minority group,’’ ‘‘ethnicity,’’ ‘‘ethnic difference,’’ ‘‘ethnic or racial aspects,’’ and ‘‘skin pigmentation’’ in different combinations. Altogether, 165 articles were retrieved, and abstracts were subsequently reviewed for relevance to the topic. The PubMed database was searched with the specific terms ‘‘minority groups,’’ ‘‘population groups,’’ and ‘‘skin pigmentation’’ in different combinations. Altogether, 295 results were retrieved, and abstracts were reviewed for relevance to the topic. Editorial articles, population-based studies, basic science, and clinical studies were included provided the subject matter was relevant to the topic discussed.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_52, # Springer-Verlag Berlin Heidelberg 2010
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at dendrite tips through a molecular complex which includes myosin [8]. Melanosomes are pinched off or released from the melanocyte dendrite and subsequently phagocytosed by adjacent keratinocytes. Suprabasal keratinocytes move upwards through epidermal keratinocyte layers where melanocyte dendrites continuously transfer melanosomes to maturing keratinocytes. Eventually, melanin is shed from top layers of stratum corneum with the cornified keratinocytes. The proteinase activated receptor-2 (PAR-2) is a receptor expressed in keratinocytes, and is thought to play a fundamental role in this process of melanosome transfer and skin pigmentation [9]. The overall regulation of melanin formation is complex, and numerous factors affect mammalian melanocytes to regulate melanin synthesis; these include melanotropin, estrogen and progesterone, and agouti signal protein [10]. Further studies must elucidate the complex regulation of melanin formation, the regulation of phagocytosis of melanosomes by keratinocytes, and how signaling intermediates stimulate melanosome transfer. Additionally, it is not entirely clear whether melanosomes are transferred from melanocytes to keratinocytes in membrane-bound clusters or as individual melanosomes, or both.
Variations in Pigmentation and Ultrastructural Differences in Skin Montagna and Carlisle examined the morphology of black and white facial skin among adult women 22–50 years of age [11]. When comparing the epidermis of black skin to white skin, the epidermis of black skin demonstrated more and larger singly distributed melanosomes in corneocytes and keratinocytes. Additionally, the epidermal stratum lucidum was not altered by sunlight exposure in black skin whereas the stratum lucidum was usually distorted on exposure to the sun in white skin. White skin demonstrated frequent areas of atrophy, while only 1 of 19 black women in the study demonstrated atrophic spots in the epidermis. One of the key indices of dermal photodamage is the presence of elastotic material. Black skin demonstrated minimal elastosis while white skin showed variable amounts of moderate to extensive elastosis. The authors speculated that the greater numbers of melanosomes and distribution in black skin perhaps protect the epidermis from photodamage. Additionally, the dermis of black skin demonstrated more fiber fragments made of collagen fibrils and glycoproteins, as well as numerous and larger fibroblasts. Finally, black skin showed more mixed apocrine-eccrine sweat glands, as well as more blood and lymphatic vessels when compared to white skin.
Overall, the most striking difference demonstrated between black and white skin appears to be the size of melanosomes and distribution pattern of melanosomes. In darkly pigmented skin, large melanosomes are surrounded by the membrane whereas smaller melanosomes are grouped or clustered together in a single membrane in lighter skin [12]. Additionally, melanosomal packaging is closer to the basal layer in more darkly pigmented skin as compared to Caucasian skin [13]. Regarding variations in human skin color, the density of pigment producing melanocytes in the skin (approximately 1,000/mm2) has not been known to vary with ethnicity [14]. Conversely, the ratio between eumelanin and pheomelanin synthesis was demonstrated to be higher in black compared to white skin [15]. Thong et al. examined the patterns of melanosome distribution in keratinocytes of Asian skin and compared this to light Caucasian skin and dark African-American skin [16]. In this study, the distribution pattern of melanosomes transferred to keratinocytes in the photoprotected skin (volar forearm) from normal Asian individuals was examined. Results demonstrated that melanosomes in keratinocytes of Asian skin are distributed as both individual and clustered melanosomes, with 62.6% individual and 37.4% clustered. In dark skin keratinocytes, melanosomes are predominantly individual (88.9%) and in light Caucasian skin keratinocytes melanosomes are predominantly clustered (84.5%). Thus, the melanosome distribution in Asian keratinocytes seems to be intermediate between light Caucasian and dark keratinocytes. When examining the size of melanosomes, there appeared to be a variation in size with ethnicity; melanosomes in dark skin were the largest, followed by melanosomes of Asian and then Caucasian skin. Furthermore, melanosomes which are distributed individually tend to be larger as compared to clustered melanosomes. Minwalla et al. examined how keratinocytes play a role in regulating the distribution patterns of recipient melanosomes in vitro [17]. Cocultures using melanocytes and keratinocytes from different racial backgrounds were studied with electron microscopy. When keratinocytes from dark skin were cocultured with melanocytes from either dark or light skin, recipient melanosomes were predominantly individual as opposed to clustered. However, when keratinocytes from light skin were cocultured with melanocytes from dark or light skin, recipient melanosomes were predominantly clustered as opposed to individual. Thus, recipient melanosomes overall are predominantly distributed in membrane-bound clusters from light skin keratinocytes, and distributed individually by dark skin keratinocytes. Furthermore, melanosome
Pigmentation in Ethnic Groups
size was not related to how melanosomes were distributed. Authors suggested that keratinocyte regulatory factors may determine how exactly recipient melanosomes are distributed. Melanin content in photoexposed and photoprotected skin has been examined among varying ethnicities including African, Indian, Mexican, Chinese, and European skin [18]. The lightly pigmented skin types had approximately half as much epidermal melanin as compared to more darkly pigmented skin types. Furthermore, the melanin composition among the lighter skin types (Mexican, Chinese, and European skin) was more enriched with lightly colored, alkali-soluble melanin pigments such as pheomelanin and eumelanin. Epidermal melanin content is greater in chronically photoexposed skin as compared to photoprotected skin, regardless of ethnic background. This analysis, like previous studies, also demonstrated that melanosome size varies with ethnicity: African skin had the largest melanosomes, followed by Indian, Mexican, Chinese, and finally European skin. Thus, the amount of melanin, composition of melanin, and differences in melanosome size may all play roles in determining skin pigmentation. Halprin et al. reported that glutathione may play a role in the genetically determined differences in skin color among different races [19]. This sulfydryl-containing epidermal compound plays a role in melanin formation. Halprin described that the tripeptide glutathione (g-glutamyl-cysteinyl-glycine) is present in the human epidermis in sufficient concentrations to be the inhibitor of melanin formation from tyrosine by tyrosinase. Overall, reduced glutathione and the enzyme glutathione reductase, which is needed to maintain glutathione in the reduced state, are found in lower concentrations in African epidermal skin as compared to Caucasian epidermis.
The Role of Tyrosinase Iwata et al. examined the relationship between tyrosinase activity and skin color in human foreskins [20]. Darker skin types appear to have a higher level of melanin production due to the constitutively higher level of activity of tyrosinase, the rate limiting enzyme in melanin synthesis. Tyrosinase activity was measured with two separate assays, a tyrosinase hydroxylase assay and a [14C] melanin assay, measuring both the hydroxylation of tyrosine to dopa and the conversion of [14C] tyrosine to [14C] melanin. In black foreskin homogenates, tyrosinase activity was measured at nearly three times the activity compared to white skin samples. Tyrosinase activity was generally
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correlated with melanin content in the skin. Variations in tyrosinase activity may be due to different amounts of enzyme, but also potentially due to differences in catalytic activity of the enzyme in melanocytes. Tyrosinase is the rate-limiting enzyme in melanin synthesis, and its overall activity is higher in melanocytes of black skin as compared to Caucasian skin [21]. Fuller et al. examined the regulation of tyrosinase in black and Caucasian human melanocyte cell cultures. Their studies showed that variation in enzyme activity is due to differences in the catalytic activity of preexisting tyrosinase rather than differences in tyrosinase abundance or gene activity. In the melanosomes of black melanocytes, tyrosinase has high catalytic activity while in Caucasian melanocytes, the melanosome-bound enzyme is largely inactive. Furthermore, staining of Caucasian melanocytes with a weak base demonstrated that Caucasian melanosomes are acidic organelles as compared to more neutral in darker skin. Thus, differences in melanosome pH may contribute to variations in pigmentation. Moreover, tyrosinase is inactive in an acidic environment, so it is largely inactive in Caucasian melanosomes as compared to higher activity in melanosomes of black skin. Maeda et al. compared melanogenesis in human black and light brown melanocytes [22]. Melanin pigment in both human black and light brown melanocytes contains eumelanin and pheomelanin, with black melanocytes containing a larger amount. Tyrosinase activity was higher in the black melanocytes as compared to light brown melanocytes. The differences in pigmentation of the two human melanocyte cell lines (black and light brown) seemed to be derived from differences in activity of tyrosinase, as well as other specific proteins affecting the constitution of melanin polymers. These other proteins include tyrosinase-related protein-1 and dopachrome tautomerase, which modulate distal steps in melanogenesis.
Genetics of Skin Pigmentation Only recently have investigators begun to develop a better understanding of the complex genetic basis for normal variation in human skin pigmentation. Much of the research investigating the genes playing a role in melanin synthesis have been carried out in animal models. With the help of mouse coat color mutations, many of the biochemical pathways involved in melanin synthesis have been studied and elucidated; over 100 genes have been identified which can affect mouse coat color [23]. Many of these genes have corresponding human phenotypes. One of the genes affecting normal variation in skin
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pigmentation is the melanocortin 1 receptor (MC1R) gene. Mutations in this gene affect pigmentation in humans, mice, cattle, horses, sheep, pigs, and chickens among other animals. Specifically, this gene product is in the melanocyte cell membrane, and is the receptor for a-melanocyte stimulating hormone. While polymorphisms in MC1R may play a vital role in shaping human pigmentation, this process is complex and multiple genes likely determine normal variation in skin pigmentation [24]. In addition, some variation in human skin color is associated with variation in TYR and OCA2, two of the known pigmentation genes [25]. More recent studies have focused on SLC24A5, a putative cation exchanger, which was originally studied in zebrafish and is suggested to play a key role in human skin pigmentation [26]. The SLC24A5 exchanger localizes to an intracellular membrane, likely the melanosome or its precursor; it is thought that variations in this gene may help explain some of the difference in pigmentation between European-Americans and AfricanAmericans. Studies have shown that the genes MATP, TYR, and SLC24A5 may play a predominant role in the evolution of lighter skin in Europeans but not East Asians, suggesting that there is a recent convergent evolution of lighter skin pigmentation in East Asians and Europeans [27]. Interestingly, such data also suggest that European skin turned lighter approximately 6,000–12,000 years ago, contradicting a previous hypothesis that European skin grew more pale approximately 40,000 years ago [28].
Response to Ultraviolet Radiation Tadokoro et al. examined the mechanism of skin tanning in different ethnic groups [29]. The effect of ultraviolet radiation after one minimal erythemal dose exposure was studied. Overall, the density of melanocytes present at the epidermal–dermal junction did not change significantly 1 week following ultraviolet light exposure, and this density was similar among different ethnic skin types. However, the distribution of melanin from the lower layers to middle layers of the skin epidermis was more dramatic in darker skin as compared to lighter skin following ultraviolet exposure. Erythema responses have also been examined in patients of different complexions [30]. The minimal erythema dose (MED), defined as the smallest quantity of radiation needed to produce a barely perceptible erythema, was determined in Caucasians and in differing complexions of African-American skin. Among light, medium, and dark-complexioned African-Americans, no minimal
erythema response was typical. Instead, a spectrum of responses was found which was directly proportional to the degree of pigmentation. Additionally, the average MED of the darker-complexioned African-Americans was 33 times greater than that of Caucasians. Caucasian skin had the smallest amount of pigment and smallest melanosomes, which were mostly contained within melanosome complexes. Further findings included that melanosome size is directly proportional to the intensity of skin pigmentation, and darkly pigmented subjects have larger, wider and denser melanosomes. Generally, with increasing pigmentation, the size of melanosomes, the proportion of singly dispersed melanosomes, and the MED were all shown to increase. Authors suggested that the increased resistance of darker skin to the damaging effects of ultraviolet radiation may be due to larger, more light-absorbing, individually dispersed melanosomes. Furthermore, melanosomes in darkly pigmented skin are degraded less by lysosomes, resulting in more light-absorbing bodies in the stratum corneum [31].
Skin Structure and Function The skin barrier, made primarily of terminally differentiated keratinocytes in a lipid matrix composed of fatty acids, ceramides, and cholesterols, helps to determine the skin’s integrity [32]. When examining the skin barrier and its components, darkly pigmented skin does appear to have some inherent structural and functional differences as compared to lighter skin [33]. One study compared transepidermal water loss and water content among black, white, Latino, and Asian populations [34]. The transepidermal water loss measurements were highest in black, followed by white, Latino, and Asian populations in decreasing order. Furthermore, stratum corneum lipids were significantly lower in black epidermal layers as compared to other races. The skin barrier of more darkly pigmented skin is thought to be more resistant to injury and recover more quickly from injury [35]. Marshall et al. demonstrated that black skin may have decreased susceptibility to cutaneous irritants, suggesting that the black skin barrier may be stronger [36]. Another study by Weigand et al. demonstrated that there may be greater numbers of stratum corneum cell layers in black skin and possibly greater cell cohesiveness as well [37]. However, it has also been suggested that darkly pigmented persons are more susceptible to cold injury as compared to those with lighter pigmentation [38]. One concern about varying levels of pigmentation among human skin of different ethnic backgrounds is
Pigmentation in Ethnic Groups
the possible effect on cutaneous synthesis of vitamin D. Matsuoka et al. examined this further by testing serum vitamin D levels and levels of active serum metabolites among white, East Asian, South Asian, and black subjects following a fixed dose of ultraviolet B radiation [39]. Because the amount of epidermal melanin determines the number of photons, which reach the lower epidermal layers where vitamin D3 synthesis takes place, vitamin D formation could be affected by individual characteristics of melanization. However, when assessing vitamin D nutritional status by measuring serum 25-hydroxyvitamin D, ethnic background had only a marginal effect with higher levels in whites compared to blacks. Regarding its active serum metabolite 1, 25-dihydroxyvitamin D, levels were similar across all groups. Authors concluded that varying levels of pigmentation do not prevent the generation of normal levels of active vitamin D metabolites, while increasing pigmentation in the epidermis still exerts a strong photoprotective effect.
Objective Measurements of Skin Color Advances have been made in pigmentation measurement devices, allowing for easy measurement of epidermal melanin using tristimulus reflectometry, narrow-band spectroscopy, and diffuse reflectance spectroscopy. The tristimulus chromameter utilizes the L*a*b* color system to determine skin color: L* represents skin reflectance or lightness, a* measures color saturation from red to green, and b* measures color saturation from yellow to blue. The aforementioned measurement devices have been utilized in objectively measuring human skin pigmentation and in examining the impact of melanin on human skin color. Lee et al. evaluated the Minolta CR-400 chromameter (Tokyo, Japan) as an objective measurement of periocular and facial pigmentation in subjects form different ethnic backgrounds [40]. African-American, Caucasian, and Hispanic subjects had facial and periocular skin color measurements performed. Using the L*a*b* color system, significant differences in L* were observed among all ethnic groups, while a* and b* were less sensitive to pigmentation differences. Additionally, the value L* (i.e. skin reflectance or lightness) demonstrated significant differences between different Fitzpatrick skin types III–VI, the more heavily pigmented groups. The Minolta CR-400 chromameter reliably measures facial pigmentation, and can be utilized when evaluating changes in skin pigmentation in studies; the chromameter showed good inter- and intra-instrument reliability as well.
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Shriver and Parra compared two methods to measure pigmentation in skin and hair in a group of subjects including European-Americans, African-Americans, South Asians, and East Asians [41]. The tristimulus colorimeter Photovolt ColorWalk (Indianapolis, IN) which uses the L*a*b* color system was compared to the DermaSpectrometer narrow-band reflectometer (Hadsund, Denmark) which measures pigment in terms of erythema and melanin indices. Both types of instruments did provide accurate estimates of pigment level in the skin. However, measurements performed by the narrow-band reflectometer were less affected by the greater redness of specific body sites due to increased vascularization. Alaluf et al. examined the impact of epidermal melanin on objective measurements of human skin color [42]. The tristimulus chromameter was used to obtain measurements of human skin color in different ethnic skin types. Tristimulus L*a*b* measurements were made in European, Mexican, Chinese, Indian, and African subjects. Overall, darker skin types tend to have lower L* values, higher a* values and higher b* values when compared to constitutively lighter skin types. Results demonstrated that total epidermal melanin is the primary determinant of L* values (i.e., skin reflectance or lightness). Melanosome size also has a significant influence on L* values and larger melanosomes are associated with a darker skin color, as discussed previously. Based on the strength of correlations observed in this study, epidermal melanin content still seems to play a greater role than melanosome size in determining skin color.
Conclusion Many advances have been made in understanding the genetic, molecular, and cellular differences underlying normal variation in human skin pigmentation. However, further studies must be carried out in order to investigate the complex genetic pathway underlying melanin synthesis, the role of genetic variation in epidermal pigmentation, and to elucidate differences in skin pathophysiology among humans from different ethnic backgrounds. As knowledge develops about the intricate process of pigmentation and distinctions between ethnic groups, it can be further understood how pigmentary changes occur with aging, and consequently develop more effective management.
Cross-references > Hyperpigmentation > The
in Aging Skin New Face of Pigmentation and Aging
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References 1. US Census Bureau. Interim projections by age, sex, race, and Hispanic origin, 2004. Available at: http://www.census.gov/ipc/www/ userinterimproj/. Accessed September 22, 2004. 2. Parra EJ, Kittles RA, Shriver MD. Implications of correlations between skin color and genetic ancestry for biomedical research. Nat Genet. 2004;36:S54–60. 3. Jackson IJ. Identifying the genes causing human diversity. Eur J Hum Gen. 2006;14:978–980. 4. Harrison GA. Differences in human pigmentation: measurement, geographic variation, and causes. J Invest Dermatol. 1973;60: 418–426. 5. Quevedo WC Jr., Fitzpatrick TB, Pathak MA, Jimbow K. Role of light in human skin color variation. Am J Phys Anthrop. 1975;43: 393–408. 6. Kalla AK. Human skin pigmentation, its genetics and variation. Humangenetik. 1974;21:289–300. 7. Westerhof W. A few more grains of melanin. Int J of Dermatol. 1997;36:573–574. 8. Scott GA. Melanosome trafficking and transfer. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Oetting WS, Ortonne JP (eds) The Pigmentary System, Physiology and Pathophysiology, 2nd ed. Massachusetts: Blackwell Publishing Ltd, 2006, pp. 171–180. 9. Seiberg M. Keratinocyte-melanocyte interactions during melanosome transfer. Pigment Cell Res. 2001;14:236–242. 10. Hearing VJ. The regulation of melanin formation. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, Oetting WS, Ortonne JP (eds) The Pigmentary System, Physiology and Pathophysiology, 2nd ed. Massachusetts: Blackwell Publishing Ltd, 2006, pp. 191–212. 11. Montagna W, Carlisle K. The architecture of black and white facial skin. J Am Acad Dermatol. 1991;24:929–937. 12. Szabo G, Gerald AB, Pathak MA. Racial differences in human pigmentation on the ultrastructural level. J Cell Biol. 1968;39: 132a–133a. 13. Toda K, Pathak MA, Parrish JA, Fitzpatrick TB, Quevedo WC Jr. Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light. Nat New Biol. 1972;236:143–145. 14. Szabo G. The number of melanocytes in human epidermis. Br Med J. 1954;1:1016–1017. 15. Thody AJ, Burchill SA, Ito S. Epidermal eumelanin and phaeomelanin concentrations in different skin types and in response to PUVA. Br J Dermatol. 1990;123:842–845. 16. Thong HY, Jee SH, Sun CC, Boissy RE. The patterns of melanosome distribution in keratinocytes of human skin as one determining factor of skin colour. Br J Dermatol. 2003;149:498–505. 17. Minwalla L, Zhao Y, Le Poole IC, Wicket RR, Boissy RE. J Invest Dermatol. 2001;117:341–347. 18. Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A. Ethnic variation in melanin content and composition in photoexposed and photoprotected human skin. Pigment Cell Res. 2002;15:112–118. 19. Halprin KM, Ohkawara A. Glutathione and human pigmentation. Arch Derm. 1966;94:355–357. 20. Iwata M, Corn T, Iwata S, Everett MA, Fuller BB. The relationship between tyrosinase activity and skin colour in human foreskins. J Invest Dermatol. 1990;95:9–15. 21. Fuller BB, Spaulding DT, Smith DR. Regulation of the catalytic activity of preexisting tyrosinase in black and caucasian human melanocyte cell cultures. Exp Cell Res. 2001;262:197–208.
22. Maeda K, Yokokawa Y, Hatao M, Naganuma M, Tomita Y. Comparison of the melanogenesis in human black and light brown melanocytes. J Dermatol Sci. 1997;14:19–206. 23. Westerhof W. Evolutionary, biologic, and social aspects of skin color. Dermatol Clin. 2007;25:293–302. 24. Makova K, Norton H. Worldwide polymorphism at the MC1R locus and normal pigmentation variation in humans. Peptides. 2005;26:1901–1908. 25. Shriver MD, Parra EJ, Dios S, Bonilla C, Norton H, Jovel C, et al. Skin pigmentation, biogeographical ancestry and admixture mapping. Hum Genet. 2003;112:387–399. 26. Lamason RL, Mohideen MPK, Mest JR, Wong AC, Norton HL, Aros MC, et al. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science. 2005;310:1782–1786. 27. Norton HL, Kittles RA, Parra E, McKeigue P, Mao X, Cheng K, et al. Genetic evidence for the convergent evolution of light skin in Europeans and East Asians. Mol Biol Evol. 2007;24:710–722. 28. Gibbons A. American Association of physical anthropologists meeting. European skin turned pale only recently, gene suggests. Science. 2007;316:364. 29. Tadokoro T, Yamaguchi Y, Batzer J, Coelho SG, Zmudzka BZ, Miller SA, et al. Mechanisms of skin tanning in different racial/ ethnic groups in response to ultraviolet radiation. J Invest Dermatol. 2005;124:1326–1332. 30. Olson RL, Gaylor J, Everett MA. Skin color, melanin, and erythema. Arch Dermatol. 1973;108:541–544. 31. Olson RL, Nordquist J, Everett MA. The role of lysosomes in melanin physiology. Br J Dermatol. 1970;83:189–199. 32. Baumann L, Rodriguez D, Taylor SC, Wu J. Natural considerations for skin of color. Cutis. 2006;78(6):2–20. 33. Taylor S, Woolery-Lloyd H. Pigmentation disorders in skin of color: the role of natural substances. Semin Cutan Med Surg. 2008;27:14–15. 34. Sugino K, Imokawa G, Maibach H. Ethnic difference of stratum corneum lipid in relation to stratum corneum function [abstract]. J Invest Dermatol. 1993;100:597. 35. Reed JT, Ghadially R, Elias PM. Effect of race, gender, and skin type of epidermal permeability barrier function. J Invest Dermatol. 1994;102:537. Abstract. 36. Marshall E, Lynch V, Smith H. Variation in the susceptibility of the skin to dichloroethylsulfide. J Pharmacol Exp Ther. 1919;12: 291–301. 37. Weigand D, Haygood C, Gaylor J. Cell layers and density of negro and caucasian stratum corneum. J Invest Dermatol. 1974;62: 563–568. 38. Post PW, Farrington D Jr., Binford RT Jr. Cold injury and the evolution of ‘‘white’’ skin. Human Biology. 1975;47:65–80. 39. Matsuoka LY, Wortsman J, Haddad JG, Kolm P, Hollis BW. Racial pigmentation and the cutaneous synthesis of vitamin D. Arch Dermatol. 1991;127:536–538. 40. Lee JA, Osmanovic S, Viana MAG, Kapur R, Meghpara B, Edward DP. Objective measurement of periocular pigmentation. Photodermatol Photoimmunol Photomed. 2008;24:285–290. 41. Shriver MD, Parra EJ. Comparison of narrow-band reflectance spectroscopy and tristimulus colorimetry for measurements of skin and hair color in persons of different biological ancestry. Am J Phys Anthropol. 2000;112:17–27. 42. Alaluf S, Atkins D, Barrett K, Blount M, Carter N, Heath A. The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell Res. 2002;15:119–126.
12 Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases in Skin Aging Process Satoshi Amano
Introduction Skin aging can be classified into two types: intrinsic aging and photoaging [1]. Intrinsic aging is the basic biological process common to all living things and is characterized as an age-dependent deterioration of skin functions and structures, such as epidermal atrophy and epidermal– dermal junctional flattening [2]. Photoaging is well known to be a consequence of chronic exposure of the skin to sunlight. Sun-exposed skin, such as face or neck skin, clearly appears to be ‘‘prematurely aged’’ in comparison with the relatively sun-protected skin of the trunk or thigh, and is characterized by various clinical features, including wrinkles, sagging, roughness, sallowness, pigmentary changes, telangiectasis, and neoplasia [3, 4]. The histological features of sun-exposed skin include cellular atypia, loss of polarity, flattening of the dermal–epidermal junctions (DEJ), a decrease in collagen, and dermal elastosis [2, 5]. The basement membrane (BM) at the DEJ has many functions, of which the most obvious is to tightly link the epidermis to the dermis [6]. It also determines the polarity of the epidermis and provides a barrier against epidermal migration. Once the BM has been assembled, the epidermal cells recognize the surface adjacent to the BM as the basal surface. Stratification of the epidermis proceeds with the proliferating cells remaining attached to the BM and the daughter cells migrating into the upper layers [7–9]. It is thought that the BM influences epidermal differentiation and maintains the proliferative state of the basal layer. Under normal circumstances, the BM prevents direct contact of epidermal cells with the dermis. Another important function of the BM derives from the positioning of the structure between the epidermal and the dermal cells. The epidermis and the dermis do not function independently [10]. Instead, normal skin
homeostasis requires the constant passage of signals back and forth between the two cell types. In general, these signals are small molecules, synthesized in one compartment, that diffuse to the other compartment. In other words, these signals must cross the BM. Components of the BM can selectively facilitate or prevent the passage of these signals. In some cases, the signaling molecules are stored by the BM and released only if the BM is damaged or destroyed. Thus, epidermal–dermal communication through the BM is extremely important. The BM may be divided into three layers on the basis of morphological studies: the lamina lucida, the lamina densa, and the lamina fibroreticularis [11], as shown in > Fig. 12.1. Lamina densa is a sheet-like structure, which is mainly composed of type IV collagen. Lamina lucida is a region between the lamina densa and the epithelia, forming electron-dense plaques, hemidesmosomes, which mainly consist of a6b4integrin and the bullous pemphigoid antigen 2 (180 kDa). The BM contains unique structures that maintain the attachment of the epidermis. The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis. One of the key components of the anchoring complex is laminin 5 (332). Past work has shown that laminin 332 is essential to epidermal attachment, as mutations in the genes encoding the laminin 332 chains underlie the severe blistering phenotype of Herlitz’ junctional epidermolysis bullosa [12]. Laminin 332 is processed extracellularly to a mature form at the a3 and g2 chains by BMP-1 and other enzymes [13–15]. It is clear that laminin 332 constitutes the anchoring filaments and binds the transmembrane hemidesmosomal integrin a6b4, which is known to be the receptor of laminin 332. With regard to binding of laminin 332 with other components of the BM or of the papillary dermis, it
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_12, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 12.1 Basement membrane structure at DEJ and damaged BM structures. In the top row, the DEJ is visualized at stepwise magnification in transmission electron microscope scale. Hemidesmosome (HD), anchoring filament (af), lamina densa, and anchoring fibrils (Af) are observed and form the special anchoring complex for the attachment of the epidermis to the dermis. The lower pictures are transmission electron microscopic images of the DEJ of human skin. Disruption and reduplication of the lamina densa can be seen at the DEJ in the sun-exposed cheek skin of a 30-year-old female, while neither duplication nor disruption can be observed in the sun-protected abdomen skin of a 34-year-old female
has recently been elucidated that (1) laminin 332 directly binds type VII collagen, which forms the anchoring fibrils that insert into the papillary dermis, and (2) laminin 332 forms a covalent complex with laminin 311 or 321, and this laminin 332–311/321 complex interacts with type IV collagen in the BM through nidogen. The matrix metalloproteinases (MMPs) are zincdependent endopeptidases, and they are involved in remodeling of the extracellular matrix and also play important roles in morphogenesis, angiogenesis, arthritis, skin ulcer, tumor invasion, and metastasis [16]. Five families of MMPs have been recognized: collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs. These
enzymes are composed of several domains, including propeptide, catalytic, and hemopexin (except for matrilysin) domains. They are involved in the degradation of collagens, proteoglycans, and various glycoproteins [16]. Among them, gelatinase A (MMP-2 or 72 kDa type IV collagenase) and gelatinase B (MMP-9 or 92 kDa type IV collagenase) digest type IV and VII collagens, while stromelysins (MMP-3, MMP-10) degrade laminins of the BM [17]. MMPs are secreted as inactive zymogens (proMMPs) and activation of proMMPs (to active MMPs) is a prerequisite for function. Stimulation or repression of proMMP synthesis is mostly regulated at the transcriptional level by growth factors or cytokines [17]. Furthermore,
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases
posttranscriptional regulation of MMPs activity is controlled by tissue inhibitors of metalloproteinases (TIMPs), of which TIMP-1, TIMP-2, TIMP-3, and TIMP-4 have been characterized [18]. MMP-2 binds specifically to TIMP-2, whereas MMP-9 binds to TIMP-1 [19]. MMP-2 is constitutively expressed in many cells, including dermal fibroblasts, and has a ubiquitous tissue distribution. ProMMP-2 is activated at the cell surface by a membrane-type MMP known as MT1-MMP [20]. Plasminogen activators (PAs)/plasmin represent one of the most potent and widely expressed systems for extracellular proteolysis [21]. PAs can be produced by many cell types, including human epidermal keratinocytes [22], and they convert the widely distributed zymogen, plasminogen, to plasmin, which degrades most extracellular proteins either directly or by activating other proteases [22, 23]. Tissue-type PA (tPA) and urokinase-type PA (uPA) are the products of distinct but related genes with different patterns of expression and regulation. Physiologically, tPA is predominantly responsible for fibrinolysis, while uPA appears to be involved in pericellular proteolysis by binding to cell surfaces through a specific, high-affinity, glycosylphosphatidylinositol-anchored plasma membrane receptor [24]. Binding increases the catalytic efficiency and targets generation of plasmin to the immediate pericellular space. In skin, urokinase-type plasminogen activator activity was found to be present in the stratum corneum, as well as the basal layer after barrier disruption [25, 26]. The plasminogen/plasmin system in the epidermis is thought to be the major protease activity involved in the delay of barrier recovery [26]. The first skin-equivalent model was developed in 1981 by Bell et al. [27], who plated human keratinocytes on top of contracted collagen gel containing dermal fibroblasts. It is useful for grafting on wounds [27] or burned skin [27], to explore the dynamics of the BM [28, 29], and for studies of epidermal differentiation, dermal–epidermal interaction, tumor cell invasion, and so on [28, 30, 31].
Ultrastructural Alteration of Epidermal BM in Sun-Exposed Skin At the DEJ of sun-exposed skin, duplication of lamina densa was reported in both aged adults [2] and mouse [32], and these changes may result in a more fragile epidermal–dermal interface and weaker resistance of the epidermis to shearing forces in aged skin. It was observed that,
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even in cheek (sun-exposed) skin of a 30-year-old female, severe disruption and reduplication of lamina densa were frequently observed beneath keratinocytes and anchoring fibrils were also associated with detached lamina densa, mainly on the dermis side (> Fig. 12.1). On the other hand, in young, sun-protected skin, such as abdominal skin of a 34-year-old female, scarcely any alteration of the epidermal BM structure was apparent at the DEJ (> Fig. 12.1). In old sun-exposed skin from cheek of a 60-year-old female, the layer number of reduplicated lamina densa was increased and laminae densae branched in various directions. In contrast, skin from upper thigh of an 83-year-old female showed very little disruption or reduplication at the DEJ. However, following injury that penetrates or disrupts the BM, the epidermal cells lose contact with the BM, and come in contact with naked dermis. Under these conditions, the epidermal cells modify their behavior to cover and close the wound. Such behavior changes include the upregulation of proteolytic enzymes and other changes that accompany conversion to a migratory phenotype [33, 34].
Involvement of Matrix Metalloproteinases in Damage to Epidermal BM in Sun-Exposed Skin MMPs are considered to be involved in photoaging, since MMPs-1, 2, 3, and 9 were found to be increased by ultraviolet irradiation in experiments using human fibroblasts [28, 35–38] and human skin [39, 40]. In particular, Fisher et al. demonstrated an increase of MMPs in human skin following exposure even to an extremely low level of UVB [40], and suggested that MMPs are UV-induced aging factors. In fact, gelatinase activities have been detected in the epidermis of forehead skin by in situ gelatin zymography [41]. For further study, the skin equivalent was selected as a model for BM damage, which partially mimics the photoaging process because of missing BM structure at the DEJ and the presence of large amounts of MMPs, including gelatinases (MMP-2 and MMP-9), in the culture medium as shown in > Fig. 12.2 [28, 35]. An MMP inhibitor, CGS27023A, enhances the assembly of BM at the DEJ in the skin equivalent (> Fig. 12.3) [28, 35], suggesting that MMPs play an important role in the degradation of BM components and induce BM structural damage, such as detachment of BM from basal keratinocytes and disruption of lamina densa, which are observed in sun-exposed skin (> Fig. 12.1).
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. Figure 12.2 Skin-equivalent model. Human skin-equivalent models are prepared by using human fibroblasts and keratinocytes. On the top of contracted collagen gel containing fibroblasts, keratinocytes are plated and the next day, the keratinocyte layer is exposed to air. Multilayered epidermis with cornified layer, like normal human skin, is formed within a week on the top. However, no lamina densa structure is observed at the DEJ. The activities of MMP-2 and MMP-9 are detected in the conditioned medium by gelatin zymography
. Figure 12.3 Protective effects of MMP inhibitors against BM damage at the DEJ. Skin equivalents were cultured from day 7 through day 14 with or without a synthetic MMP inhibitor, CGS27023A. Each sample was processed for electron microscopy on day 14. Lamina densa (arrows) was observed along the DEJ in the presence of CGS27023A
Possible Involvement of Basement Membrane Damage by Matrix Metalloproteinases and Serine Proteinases
Involvement of Plasminogen Activator/ Plasmin in Damage to Epidermal BM, Degradation of Laminin 332, and Reduced Activities of Laminin 332 in Keratinocyte Adhesion and Type VII Collagen Binding UVB exposure increases the synthesis of urokinase-type plasminogen activator (uPA) [23, 42, 43], as well as matrix metalloproteinases [40, 44]. Both uPA activity and uPA are present in the conditioned medium of skin equivalents, and the addition of plasminogen enhances the degradation of BM components and impairs the assembly of BM structure at the DEJ even in the presence of MMP inhibitor (> Fig. 12.4) [42]. Aprotinin, a plasmin inhibitor, restores the assembly of BM structure at the DEJ damaged by the addition of plasminogen (> Fig. 12.4). Since lamina densa structure is detached at the anchoring filament from basal keratinocytes in sun-exposed skin (> Fig. 12.1), laminin 332, a major component of anchoring filament, is a candidate target of plasmin. As expected, plasmin degrades the a3 and b3 chains of laminin 332, although g2 is unaffected (> Fig. 12.5). Plasmin cleaves both the amino and carboxy terminals of a3 chain, and these cleavages reduce the keratinocyte adhesion activity of laminin 332 (> Fig. 12.6). Similarly, the removal of an amino terminal fragment (domain VI) of the b3 chain by plasmin (> Fig. 12.5) results in a reduction of its affinity for type VII collagen (> Fig. 12.6). Therefore,
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degradation of laminin 332 by plasmin may induce BM damage such as the detachment of lamina densa from basal keratinocytes.
Enhanced BM Assembly in the Presence of Laminin 332 and in Response to Increased Synthesis of BM Components in a Skin-Equivalent Model Damaged BM must be repaired, since BM at the DEJ plays important roles in maintaining a healthy epidermis and dermis. In order to find substances that stimulate the repair of damaged BM, a skin-equivalent model was selected for screening purposes, which is suitable for investigating the assembly of BM through cooperation between keratinocytes and fibroblasts. Purified laminin 332, a glycoprotein (MW: 410 kDa) composed of 165 kDa (a3), 140 kDa (b3), and 105 kDa (g2) chains, enhances the formation of hemidesmosome-like structures and BM at the DEJ in the skin equivalent [29]. Keratinocytes synthesize BM components, except nidogen [45]. Fibroblasts also produce BM components, other than laminin 332. Recently, it was found that increasing the production of BM components such as laminin 332, collagen IV, and collagen VII in keratinocytes and/or fibroblasts with/without inhibitors of gelatinases and/or serine proteinases is also effective to enhance the repair or assembly of BM at the DEJ.
. Figure 12.4 Ultrastructural analyses of BM structures at the DEJ of SE. Fourteen days after plating keratinocytes, the SEs were processed for the analysis of BM structures at the DEJ using electron microscopy. CGS27023A (10 mM) enhances the formation of a linear and continuous lamina densa-like structure (indicated by arrows) at the DEJ just beneath the basal keratinocytes. The addition of plasminogen (0.06 mM) impaired the assembly of the BM structure at the DEJ. Aprotinin (1.5 mM), a serine proteinase inhibitor, restored the linear and continuous lamina densa-like structures (indicated by arrows) at the DEJ
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. Figure 12.5 Degradation of laminin 332 by plasmin and analyses of the cleavage sites of laminin 332. The processed form of laminin 332 was purified from a conditioned medium of human keratinocytes and incubated with plasmin for 24 h at 37 C. SDS-PAGE on 5% acrylamide gel under reducing conditions and western blotting using polyclonal anti-laminin 332 antibodies were carried out. Lane 1, processed form of laminin 332; lane 2, plasmin-treated laminin 332. From the N-terminal amino acid sequences of the separated 145, 140, 110, and 105 kDa bands, cleavage sites in a3, b3, and g2 are indicated by arrows on schematic domain structures of laminin 332. Predicted cleavage sites of a3 chain are shown in G-3. Since the N-terminal sequence of a3 chain is DSSPA, which is the same as that of the 150 kDa fragment of the a3 chain, plasmin cleaved a 5 or 10 kDa fragment from the LG3 domain of the 150 kDa a3 chain with the putative cell adhesion region, LRD, is present
. Figure 12.6 Dysfunction of plasmin-treated laminin 332. Laminin 332 degraded by plasmin showed lower keratinocyte adhesive activity than control laminin 332. Purified plasmin-treated laminin 332 (open circle) and control laminin 332 (closed circle) were coated on 96-well plates and keratinocytes were added. The attached cells were quantitatively measured with the AlamarBlue assay. Laminin 332 degraded by plasmin (open circle) showed reduced binding to type VII collagen as compared with control laminin 332 (closed circle). Purified type VII collagen was coated on 96-well plates and purified plasmin-treated laminin 332 (open circle) or control laminin 332 (closed circle) was added. After washing, bound laminin 332 was detected using polyclonal anti-laminin 332 antibodies
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. Figure 12.7 Schematic representation of the involvement of BM damage in the process of photoaging. The disruption and reduplication of BM at the DEJ in sun-exposed skin may be induced by increased levels of BM-damaging enzymes, such as plasmin and MMPs, through the degradation of BM components: laminin 332, and type IV and VII collagens. The impairment of the BM structure may be associated with functional changes of the epidermal and dermal cells and consequently, facilitating aging processes by damaging dermal extracellular matrices and inducing keratinocyte abnormality
Conclusion The disruption and reduplication of BM at the DEJ in sun-exposed skin may be associated with increases of BM-damaging enzymes, such as plasmin and MMPs, which degrade BM components (laminin 332, and type IV and VII collagens). The impairment of BM structure may be associated with functional changes of epidermal cells and dermal cells and consequently may facilitate aging processes by damaging dermal extracellular matrices and inducing keratinocyte abnormality, as summarized in > Fig. 12.7. Laminin 332 is able to enhance BM assembly [29]. Some cosmetic ingredients also promote BM repair by increasing synthesis of BM components such as laminin 332, and type IV and VII collagens in the epidermis and/or the dermis. Therefore, the BM represents a good target for skin-care cosmetics; components that enhance BM repair may improve epidermal– dermal communication and skin homeostasis, thereby strengthening defenses against ‘‘skin aging.’’
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29. Tsunenaga M, Adachi E, Amano S, et al. Laminin 5 can promote assembly of the lamina densa in the skin equivalent model. Matrix Biol. 1998;17:603–613. 30. Tsunenaga M, Kohno Y, Horii I, et al. Growth and differentiation properties of normal and transformed human keratinocytes in organotypic culture. Jpn J Cancer Res. 1994;85:238–244. 31. Nishiyama T, Amano S, Tsunenaga M, et al. The importance of laminin 5 in the dermal-epidermal basement membrane. J Dermatol Sci. 2000;24:S51–59. 32. Feldman D, Bryce GF, Shapiro SS. Mitochondrial inclusions in keratinocytes of hairless mouse skin exposed to UVB radiation. J Cutan Pathol. 1990;17:96–100. 33. Sarret Y, Woodley DT, Goldberg GS, et al. Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices. J Invest Dermatol. 1992;99:836–841. 34. Sudbeck BD, Parks WC, Welgus HG, et al. Collagen-stimulated induction of keratinocyte collagenase is mediated via tyrosine kinase and protein kinase C activities. J Biol Chem. 1994;269: 30022–30029. 35. Amano S, Ogura Y, Akutsu N, et al. Protective effect of matrix metalloproteinase inhibitors against epidermal basement membrane damage: skin equivalents partially mimic photoageing process. Br J Dermatol. 2005;153(2):37–46. 36. Herrmann G, Wlaschek M, Lange TS, et al. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMPs) in cultured human fibroblasts. Exp Dermatol. 1993;2:92–97. 37. Kawaguchi Y, Tanaka H, Okada T, et al. The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts. Arch Dermatol Res. 1996;288:39–44. 38. Brenneisen P, Wenk J, Klotz LO, et al. Central role of Ferrous/Ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts. J Biol Chem. 1998; 273:5279–5287. 39. Koivukangas V, Kallioinen M, Autio-Harmainen H, et al. UV irradiation induces the expression of gelatinases in human skin in vivo. Acta Dermatol Venereol. 1994;74:279–282. 40. Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature. 1996;379:335–339. 41. Inomata S, Matsunaga Y, Amano S, et al. Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse. J Invest Dermatol. 2003;120:128–134. 42. Ogura Y, Matsunaga Y, Nishiyama T, et al. Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction. Br J Dermatol. 2008;159:49–60. 43. Miralles F, Parra M, Caelles C, et al. UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element. Mol Cell Biol. 1998;18:4537–4547. 44. Scharffetter K, Wlaschek M, Hogg A, et al. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res. 1991;283:506–511. 45. Fleischmajer R, Schechter A, Bruns M, et al. Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro. J Invest Dermatol. 1995;105:597–601.
11 Proteoglycans in Skin Aging Franc¸ois-Xavier Maquart . Ste´phane Bre´zillon . Yanusz Wegrowski
Introduction Proteoglycans are ubiquitous macromolecules of extracellular matrices, cell surfaces, and some intracellular granules [1]. They are composed of a glycoprotein core to which one or several sulfated glycosaminoglycan (GAG) chains are attached by covalent linkage. Four different classes of sulfated glycosaminoglycans (GAGs) exist in vertebrates: chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and heparan sulfate/heparin (HS). Hyaluronic acid is not esterified with sulfate and not linked to a protein core. The properties, structure, and functions of glycosaminoglycan are discussed in another chapter. One protein core may bear one type or several types of glycosaminoglycans of the same or different classes. For example, syndecan-1 may contain heparan sulfate only or heparan sulfate and chondroitin sulfate glycosaminoglycan chains. Depending on cell context, the same proteoglycan may be substituted with different GAG chains. For example, serglycin of mast cells is substituted with heparin and serglycin of macrophages is substituted with chondroitin sulfate. A third feature of these macromolecules is the possibility of absence of the glycosaminoglycan chain in the same proteoglycan, depending on the organ. Lumican in cornea is a keratan sulfate proteoglycan, whereas in skin it is a glycoprotein without any glycosaminoglycan chain. Finally, the presence of GAG may be time dependent in the so-called part-time proteoglycans. Often, it is done by the synthesis of splicing variants lacking the GAG attachment sequence, as in the versican V3 variant. The GAG chains of galactosaminoglycans (CS/DS) and HS are attached to a serine residue of the core protein via a short common tetrasaccharide, by a beta 1–4 bond in the case of CS/DS and by alpha 1–4 bond in the case of HS. This region is composed of a tetrasaccharide GlcA (b1-3)Gal(b1-3)Gal(b1-4)Xylb1-O-Ser, where GlcA is glucuronic acid, Gal is galactose, and Xyl is xylose. Keratan sulfate chains of aggrecan (KS type II) are also attached to a serine residue via O-glycosidic linkage, but the composition of this linkage is similar to those of the O-substituted oligosaccharides of glycoproteins. Keratan sulfate chains of lumican or keratocan in cornea (KS I)
possess a mannose-rich region attached by N-glycosidic linkage to an asparagine, similar to the N-substituted oligosaccharides of glycoproteins. Although the presence of keratan sulfate in skin has been reported [2], no detailed structure of the linkage region of these molecules is known. Several glyco and sulfotransferases are involved in the assembly of the GAGs on the protein core. The xylosyltransferases are the enzymes, which start the GAG synthesis. Although there is no consensus sequence for a given exported protein to be substituted with GAG chain, the xylosyl-transferases recognize Ser-Gly sequences in the vicinity of acid-amino acids [3]. It seems that the presence of hydrophobic clusters and the conformation of the protein also contribute to the activity of N-acetyl-glucosamine transferase I and N-acetyl-galactosamine transferase I, two enzymes which initiate the synthesis of HS or CS/DS, respectively. At present, over 50 proteoglycan genes are known. The mRNAs of most of them are expressed in skin cells (> Table 11.1) [4]. However, only some of the protein products have been confirmed to reside in the skin. The extracellular proteoglycans are regrouped in different groups or classes. First and most abundant class of extracellular matrix proteoglycans of the dermis belong to the small, leucine-rich proteoglycan (SLRP) family [1]. Although there is no comparative quantification of different proteoglycans of human dermis, decorin seems to be the most aboundant representative in the skin, associated with collagen fibers. The same localization concerns lumican, which, in the skin, carries no GAG chain. Biglycan is present mostly in the skin pericellular matrix [5]. Fibromodulin was recently reported to be present in the epidermis [6]. Second group contains large, aggregating proteoglycans that are able to bind hyaluronic acid, usually called hyalectans (or lecticans). These proteoglycans are able to form very high molecular mass complexes, composed of numerous proteoglycan molecules settled down on a hyaluronan molecule [1]. From this group, only versican was documented in human dermis and epidermis [7]. Basement membrane of the dermo–epidermal junction contains a major heparan sulfate proteoglycan: perlecan
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_11, # Springer-Verlag Berlin Heidelberg 2010
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. Table 11.1 The proteoglycans mRNA expressed by keratinocytes (K) or skin fibroblasts (F) screened by cDNA array obtained from GEO profiles of NCBI [4] (A) Extracellular proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
SLRP class Decorin
DCN
NM_001920a
K/F
DS/CS
Biglycan
BGN
NM_001711
K/F
DS/CS
Asporin
ASPN
NM_017680
K/F
CS
Extracellular matrix protein 2
ECM2
NM_001393
K
Fibromodulin
FMOD
NM_002023
K
Lumican
LUM
NM_002345
F
KS
Pro/Arg-rich end LRR protein
PRELP
NM_002725a
K
CS
Keratocan
KERA
NM_007035
K/F
KS
Osteomodulin
OMD
NM_005014
K
CS
Epiphycan
EPYC
NM_004950
K/F
CS
a
KS
Osteoglycin
OGN
NM_033014
F
CS
Chondroadherin
CHAD
NM_001267
K
CS
Nyctalopin
NYX
NM_022567
K/F
VCAN
NM_004385a
F
a
Hyalectans Versican
CS/(DS)
Aggrecan
ACAN
NM_013227
K
CS + KS
Neurocan
NCAN
NM_004386
K
CS
Brevican
BCAN
NM_021948a
K/F
CS
Perlecan (heparan sulfate proteoglycan 2)
HSPG2
NM_005529
K/F
HS/(CS)
Agrin
AGRN
NM_198576
K/F
HS
Bamacan (structural maintenance of chromosome 3)
SMC3
NM_005445
K/F
CS
Leprecan (PRP, prolyl-3-hydroxylase)b
LEPRE1
NM_022356a
K/F
CS
Type IX
COL9A2
NM_001852
K
CS
Type XII
COL12A1
NM_004370
F
CS
Basement membrane PGs
b
Collagens
Type XIV
COL14A1
NM_021110
F
CS
Type XVIIIb
COL18A1
NM_030582a
K/F
HS
Testican 1
SPOCK1
NM_004598
K/F
CS
Testican 2
SPOCK2
NM_014767a
K
CS
Adlican
MXRA5
NM_015419
K/F
?
Colony simulating factor-1 (CSF-1, M-CSF)
CSF-1
NM_000757a
K/F
CS
Testicans
Miscellaneous
(B) Cell membrane proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
Syndecans Syndecan-1
SDC1a
NM_001006946
K/F
HS/(CS)
Syndecan-2
SDC2
NM_002998
F
HS
Proteoglycans in Skin Aging
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. Table 11.1 (Continued) (B) Cell membrane proteoglycans
Gene
NCBI Ref.Seq
mRNA expression
GAG chain
Syndecan-3
SDC3
NM_014654
K/F
HS
Syndecan-4
SDC4
NM_002999
K/F
HS
Glypican-1
GPC1
NM_002081
K/F
HS
Glypican-3
GPC3
NM_004484
K
HS
Glypican-4
GPC4
NM_001448
K
HS
Glypican-5
GPC5
NM_004466
K
HS
Glypican-6
GPC6
NM_005708
F
HS
CD44
NM_000610a
K
CS/HS
Neuroglycan C chondroitin sulfate proteoglycan 5
CSPG5
NM_006574
K
CS
Phosphacan
PTPRZ1
NM_002851
K
CS
ENG,
NM_000118a
K/F
CS
Glypicans
Hyalectans CD44 (lymphocyte homing receptor) Neural PGs
Endothelial PGs Endoglin (CD105)
a
Endothelial cell-specific molecule 1 (endocan)
ESM1
NM_007036
K
DS
Thrombomodulin (CD 141)
THBD
NM_000361
K
HS
NG2 (melanoma-associated antigen, MAA), chondroitin CSPG4 sulfate proteoglycan 4
NM_001897
K/F
CS
Betaglycan
NM_003243
K
CS
Miscellaneous
(C) Granules proteoglycan Serglycin
TGFBR3 Gene SRGN
NCBI Ref.Seq
mRNA expression
GAG chain
NM_002727
K
Heparin/ CS
a
Different splicing variants or mRNAs Basement membrane-associated proteoglycans
b
(see below). Some collagens (types IX, XII, XIV, and XVIII) also contain a covalently linked GAG chain. Apart from type IX collagen, they are all expressed in collagen-rich dermis [8]. Testicans form another family of extracellular proteoglycans. Four testican genes have been cloned, but only testican-1 and testican-2 mRNAs are expressed in the skin, by keratinocytes (> Table 11.1). Among miscellaneous proteoglycans, the mRNA of adlican was reported to be expressed in the fibroblasts of very old subjects, but nothing is known about the protein. Another proteoglycan, CSF-1, is expressed by resident skin cells of the immune system, e.g., Langerhans cells [9]. Cell membrane proteoglycans, which are characterized by a very rapid turnover, include two main families of heparan sulfate macromolecules. The family of syndecans (four genes cloned) comprises integral type I membrane proteins, with a transmembrane domain and a
short cytoplasmic tail (> Table 11.1). Syndecan-1 is expressed in epidermis including hair follicles [10]. The family of glypicans (six genes cloned) has glycosylphosphatidylinositol-anchored molecules with a glycanic chain attached at the vicinity of plasma membrane [11]. Only the expression of neural glypican-2 was not detected in the skin (> Table 11.1). Syndecans and glypicans are easily shedded by the action of metalloproteinases and phospholipases, respectively, and are often found in pericellular space. CD44 and one of its splicing epidermal variants, epican, is an integral plasma membrane proteoglycan, which binds hyaluronic acid [12]. Keratinocytes also express the mRNAs of two brain proteoglycans, neuroglycan-C and phosphacan (> Table 11.1). One of the splicing variants of phosphacan possesses protein tyrosine phosphatase activity. Some proteoglycans characteristic for endothelial cells are also
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expressed by keratinocytes. The proangiogenic transforming growth factor-beta (TGF-b) co-receptor, endoglin, is expressed in epidermis [13], as well as the thrombin receptor, thrombomodulin [14]. Another TGF-b co-receptor, betaglycan, also known as TGF-beta receptor type III, is also expressed in epidermis at the mRNA level, but the presence of the protein is not documented. Characteristic for embryonic development, the melanoma-associated antigen/NG2 is an integral membrane, chondroitin sulfate containing high molecular mass proteoglycan overexpressed in aggressive melanoma lesions [15]. NG2 was proposed to be a marker of melanoma invasion in the lymphatic glands. This proteoglycan is also a characteristic for stem cell population of hair follicles root sheet [16]. Skin is a niche of mast cells, which contain heparin in their secretory granules. Heparin is linked to the proteoglycan serglycin core protein. This is also expressed by macrophages and Langerhans cells but, in this case, it is substituted with chondroitin sulfate chains [1]. The knowledge about the roles of proteoglycans in physiologic and pathologic conditions started when the first demonstration of a dermatan sulfate–protein complex in the skin was done [17]. The cloning era, followed by the creation of knockout mice, permitted to this knowledge to expand rapidly. Numerous excellent reviews, either general or devoted to particular class/families of proteoglycans give up-to-date state of the art in this area (cited in [1]). In general, skin proteoglycans retain all the properties and functions of particular glycosaminoglycans, contributing to tissue hydratation, resistance and resilience, molecular filtration, cell behavior, and cell– cell or cell–matrix interaction. Additionally, by their polyanionic properties, they contribute to cation exchange and to the retention of all positively charged proteins. As a consequence, they constitute the biological reservoir of many cytokines and growth factors. Such a diversity of functions supposes that any changes of their expression or structure during aging may perturb the tissue homeostasis in important manner. The clinically relevant morphological changes of the skin during aging can be summarized by the term ‘‘senile atrophy.’’ Main changes are a diminished thickness of epidermis with a reduced mitosis rate of epidermal basal cells and a decreased number of fibroblasts and capillaries in the dermis. The mesenchymal changes in the dermis have been morphologically described by the term ‘‘senile elastosis’’ or ‘‘elastoid collagen degeneration,’’ corresponding to a progressive collagen denaturation with aging. Analysis of the glycosaminoglycan content of the senile skin showed a minimal increase of the total content of hexosamines and
uronic acids with a significant increase of DS and KS, a decrease of hyaluronic acid and, also partly, a decrease of chondroitin-4-sulfate and chondroitin-6-sulfate. The neosynthesis of sulfated glycosaminoglycans is only slightly increased in aged skin, whereas the activities of the enzymes specific for the glycosaminoglycan catabolism (beta-glucuronidase, beta-N-acetyl-glucosaminidase) are significantly decreased [18].
Proteoglycans of the Dermis Extracellular Matrix Small Leucine-Rich Proteoglycans Small leucine-rich proteoglycans (SLRPs) are a family of proteoglycans present in a large number of tissues. They are characterized by their relatively low molecular weight and by the presence of similar structural motifs, the leucine-rich repeats (LRR) [19]. They are presently classified into five distinct families [20], according to their structural features and chromosomal organization (> Table 11.2). Dermis contains several SLRPs, particularly decorin and biglycan [5], lumican [21], and keratocan [22]. SLRPs . Table 11.2 Classification of human Small Leucine-rich proteoglycans (SLRPs) Schaefer L, Iozzo RV. [20] SLRP class I
Name Biglycan Decorin Asporin ECM2
II
Fibromodulin Lumican PRELP Keratocan Osteoadherin
III
Epiphycan Opticin Osteoglycin
IV
Chondroadherin Nyctalopsin Tsukushi
V
Podocan Podocan-like protein 1
Proteoglycans in Skin Aging
play important roles in the regulation of cell activity and in the organization and functional properties of skin connective tissue [23]. Several lines of mice deficient in SLRPs have been generated, particularly for the most prominent and widely expressed SLRPs: decorin, biglycan, fibromodulin, and lumican. All these SLRP deficiencies result in the formation of abnormal collagen fibrils and induce a wide array of diseases in the deficient mices (for review, see [24]). The implication of SLRPs modifications in the alterations of skin function, which may occur in skin aging has been suggested by several studies devoted to decorin and, more recently lumican.
Decorin Decorin was the most studied among the SLRP family. It is composed of a 40 kDa protein core, bearing a unique DS/CS chain. It is the most abundant SLRP in adult human dermis, where it is observed in association with collagen fibers. It is synthesized and secreted by dermal fibroblasts and might represent 30–40% of total proteoglycans of the skin [25]. Previous data from Bernstein et al. [26] reported that decorin was greatly decreased within photoaged skin, as appreciated by immunohistochemical staining and confocal laser scanning microscopy. In addition, Northern blot analysis showed that decorin steady-state mRNA levels measured in fibroblasts derived from photoaged skin was decreased by 46% in cultures derived from photo-damaged sites. Since collagen degradation was strongly increased in such areas, and collagen amounts strongly decreased, it is possible, however, that the amount of decorin necessary to bind available collagen fibers may decrease in photoaged skin as collagen degradation takes place. The decorin alterations in skin chronological aging seem very different from that observed in photoaging. As soon as 1997, Passi et al. [27] studied the modifications of proteoglycans secreted into their growth medium by young and senescent fibroblasts. In this study, senescence was induced in human skin fibroblasts by serial passages in culture, and their proteoglycan synthesis was studied by incorporation of radiolabeled precursors. The authors observed that, whereas the biosynthesis of total proteoglycan fraction was not altered with replicative senescence, the relative proportions of the different proteoglycan populations secreted into the growth medium changed. Particularly, the relative content of small CS/DS-proteoglycans that consisted mainly of decorin, as shown by immunological identification, was increased by 50% in senescent (28–31 passages) versus young (4–5 passages)
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fibroblasts. Interestingly, the authors suggested that the increased secretion of decorin into the growth medium by the late passage fibroblasts might contribute to their decline of proliferative capacity, a characteristic common to every aging cells. In a study of human skin samples from fetal skin, mature skin, and senescent skin, Carrino et al. [28] showed an increase in the proportion of decorin with a concomitant decrease of the large chondroitin sulfate proteoglycan, versican, in senescent skin. In addition, the authors reported that decorin from postnatal skin is smaller in size than the decorin of mature skin, probably due to shorter glycosaminoglycan chains. More importantly, the authors reported the presence in aged skin of a shortened (Mr about 45 kDa) form of decorin, with a core protein of about 27 kDa only. Analysis of this molecule, named ‘‘decorunt’’ by the authors, indicated that it was a catabolic fragment of decorin, representing the amino-terminal 43% of the mature decorin molecule [29]. Decorunt contains the first four leucine-rich repeats and three amino acids of the fifth leucine-rich repeat. Since decorunt lacks almost half of the collagen-binding domain of decorin, it was expected to have impaired capacity to stabilize collagen fibers. Moreover, since the sixth leucine-rich repeat, the most important for the interaction between decorin and the epidermal growth factor receptor, is lacking, decorunt should be unable to bind to this receptor, which may induce a defect of cell stimulation in aged skin. In addition, the absence of the transforming growth factor-b binding site in decorunt might impair the sequestering of this growth factor in the extracellular matrix. In an immunohistochemical study of rat dermis, Ito et al. [30] reported that decorin was only faintly visible in young (22 days old) rats, whereas it was abundant on the collagenous network of the dermis of old (24–30 months old) rats. Since decorin was shown to have a growth inhibitory effect, the authors suggested that decorin excess might be one of the cell growth inhibitory factors implicated in skin senescence. Analysis of rat skin performed by Nomura et al. [31] confirmed that the amount of decorin in rat skin increased with rat’s age (postpartus 0.5 days until 90 days of age). Interestingly, the authors noted that the molecular size of decorin decreased during aging, from about 111 kDa in 18.5 days embryo to about 70 kDa at postpartus 90 days. This decrease of size was not due to a decreased size of the core protein but to a decreased length of the glycosaminoglycan chain. The mean length of the chain was 78.58 13.94 nm in the skin of the 18.5 days embryos versus 54.05 4.79 nm in the 90 days postpartus
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rat skin. Reducing the length of the decorin glycosaminoglycans was supposed to reduce the distance between the collagen fibers [32]. In a clinical study performed on full thickness punch biopsies obtained from five young (25–35 years) and five older (61–68 years) volunteers, Lockner et al. [33] reported that decorin mRNA in skin biopsies from older volunteers was approximately 100% higher that in younger volunteers. In contrast, type I and type III collagens mRNAs were decreased. The authors suggested that the resulting decreased collagen to decorin ratio in older skin might be responsible for decreased collagen bundle diameters in aged human skin, which might affect their tensile strength [34, 35].
Lumican Lumican was first identified as a major proteoglycan of the cornea [36]. It is present in this tissue under the form of a keratan-sulfate (KS)-containing proteoglycan, with a 38 kDa core protein. In adult human skin, however, it is present under the form of a glycoprotein, devoid of glycosaminoglycan chain [37]. As decorin, lumican seems to play an important role in the preservation of skin functional properties. For instance, lumican-null mice showed abnormal collagen fibril assembly, with large and abnormally shaped collagen fibrils and an extremely loose and fragile skin [38]. In a recent study, Vuillermoz et al. [39] studied the expression of SLRPs by early passage cultured fibroblasts, obtained from 36 normal donors of 1 month to 83 years old. By Northern blot analysis, the authors showed a significant negative correlation of lumican mRNA expression with donor’s age. By contrast, no correlation with age was found for either decorin or biglycan mRNAs. Immunohistochemistry associated with image analysis quantitation showed that lumican core protein was preferentially located in the superficial, papillary layer of the dermis and that it was strongly decreased ( 81% and 85% respectively, p < 0.01) in aged skin (donors over 50) compared to young (0–15 years old) and adult (16–50 years old) skin. The strong decrease of lumican expression in aged skin, associated with the increase of decorin, induced important modification of the lumican to decorin ratio. Due to the particular importance of these two SLRPs in dermal organization and properties, the authors suggested that these alterations might be involved in the functional defects, which characterize aged skin.
Versican and Other Proteoglycans of the Dermis Extracellular Matrix Although skin fibroblasts express mRNAs for different proteoglycans (> Table 11.1) only type XII and XIV collagens and versican were studied in the dermis. Versican or chondroitin sulfate-glycoprotein 2 (CS-PG2), or PG-M, has a tridomain structure. The amino-terminal end, designated as G1, binds to hyaluronan. The carboxy-terminal domain, G3, possesses a lectin domain adjacent to two epidermal growth factor domains, and a complement regulatory domain. The central domain (G2) is encoded by two exons that specify CS attachment regions. RNA splicing of these two exons results in four different forms of versican, called V0–V3 (molecular weight 370, 262, 180, and 72 kDa, respectively) with different numbers of GAG attachment domains [40–42]. The distribution of the V0–V3 forms in adult varies with tissues and organs. Versican is a widespread ECM components of rodent and human skin and has also been characterized in the kidney in the lamina propria of blood vessels [43]. Versican expression was reported to decrease in aging skin [26, 28]. However, in photoaged skin, an accumulation of versican in elastotic material was reported [44]. After acute UV irradiation, there was a rapid upregulation of versican mRNA, which persisted up to 72 h after exposure. The accumulation of versican was accompanied by the formation of a truncated molecule, which lacked the hyaluronan-binding region of this proteoglycan [45]. In chronic sun exposure, this accumulation of versican led to the decrease of its synthesis, accelerating the loss of the molecule. As versican plays an important role in cell–hyaluronan interaction, the loss of this proteoglycan may substantially contribute to the loss of skin elasticity and resilience, especially in photoaged skin.
Cell Surface Proteoglycans in Skin Aging Cell surface proteoglycans may be divided into two main families: the syndecans and the glypicans. The main structural difference between both families is that syndecans have a transmembrane core protein, whereas, for glypicans, the core protein is linked to the cell surface by a glycosyl-phosphatidyl-inositol (GPI) anchor. Both families belong to the HS proteoglycans group even if syndecans may carry CS glycosaminoglycan chains in addition to HS.
Proteoglycans in Skin Aging
Syndecans In vertebrates, the syndecan family is composed of four members: syndecan-1, -2, -3, and -4 (for recent review, see [46]). Each core protein is composed of an extracellular domain bearing three to five chains of HS and CS, a single-span transmembrane domain and a short cytoplasmic domain. The molecular weight of the four syndecan core proteins are respectively 33, 23, 43, and 22 kDa for syndecans 1, 2, 3, and 4. The extracellular domains of syndecans are able to interact with a number of extracellular proteins such as fibronectin and growth factors, mainly fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-b), and platelet-derived growth factor (PDGF). These interactions essentially occur through the heparan-sulfate chains of syndecans. On the other hand, the intracellular domain is able to bind to other syndecan molecules, which permits their multimerization, and to a number of intracellular molecules, involved either in cytoskeleton formation or in intracellular signaling. Recent data indicates that syndecans are involved in complex signaling and cytoplasmic interactions, and could be present in membrane microdomains specialized in signal transduction [47]. Syndecan-1 is the most abundant syndecan on epithelial keratinocytes. It is strongly expressed in the suprabasal, differentiating epidermal cells, whereas it is expressed at low levels in the basal layer [48, 49]. Its expression is, however, markedly upregulated in all cell layers of the epidermis during wound healing [50]. Recent data demonstrated that overexpression of syndecan-1 in transgenic mouse epidermis induces epidermal proliferation, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers. The expression of terminal differentiation markers, keratin 10 and involucrin, was not disrupted in the epidermis of transgenic animals, showing that epidermal differentiation was not altered [51]. On the other hand, the loss of syndecan-1 in syndecan-1-null mice was shown to induce a decreased migration rate, linked to an increased adhesion of syndecan-1-defective keratinocytes [52]. Taken together, these results suggest that syndecan-1 is an important modulator of epidermal cell proliferation, migration, and adhesion and may control the behavior of epithelial cells. It may be suggested that the decreased proliferation potential of keratinocytes in aging epidermis might be linked to a defect in syndecan-1 expression.
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Syndecan-4 is another important component of the skin. In normal skin, it is detectable in the epidermis but not in the dermis. It is, however, strongly expressed by fibroblasts and endothelial cells in wounded skin [53]. Mice lacking syndecan-4 are characterized by delayed wound repair and impaired angiogenesis [54], which demonstrate that this proteoglycan is essential for skin repair. This finding may be important for understanding the physiopathology of skin aging. Few works have been devoted to the variations of syndecan expression in aging skin. Preliminary data from the authors’ laboratory indicated, however, that syndecan expression may be altered during aging. Reverse transcription polymerase chain reaction (RT-PCR) analysis of syndecan-1 mRNA indicated that keratinocytes may express syndecans 1, 2, and 4. Syndecan-1 expression was decreased in keratinocytes from donors over 50 years old [55]. That decrease was confirmed by immunocytochemical analysis of syndecan-1 in the epidermis of donors over 50 compared to donors between 16 and 50 years old [56]. Such a decrease in syndecan-1 might alter signalization processes in aged skin.
Glypicans Glypicans were identified about 15 years ago as phosphatidyl-inositol-anchored membrane heparansulfate proteoglycans [57]. In mammals, the glypican family is presently composed of six members, glypican-1 to glypican-6, with a core protein of 60–70 kDa [11]. The insertion sites for the heparan sulfate chains seem to be restricted to the last 50 amino acids in the C-terminus, placing the chains close to the cell membrane [58]. Glypicans are predominantly expressed during development [57]. Their main function is to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins. Depending on the context, they may have either a stimulatory or inhibitory activity on signaling [11]. Part of glypicans may be shedded from the cell membrane into the extracellular matrix. This shedding is dependent on an extracellular lipase, Notum [59]. Little data is available about the presence of glypicans in the skin and their function. Preliminary data from the authors’ laboratory, however, indicated that human keratinocytes may express glypicans 1, 3, 4, 5, and 6 mRNAs [55]. However, no variation was observed on glypican expression during skin aging.
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Previous data from Litwack et al. [60] detected glypican-1 mRNA and protein expression in rat skin. Significant glypican-1 protein staining was detected in all layers of epidermis, except stratum corneum, and slight expression was found in the dermis. No study of glypican variation during aging was, however, performed by the authors.
Role of the Heparan-Sulfate Chains Since both syndecan and glypican are heparan-sulfate proteoglycans, these molecules may bind and modulate the activity of several matrix components, growth factors, proteinase inhibitors, cell–cell, and cell–matrix adhesion molecules [61]. Such an ability likely plays an important role in the regulation of skin cell functions, especially in the aging process. Further investigations have, however, to be performed to better precise the exact place of the molecules in skin physiology.
Proteoglycans of the Dermo–Epidermal Junction Components of the dermo–epidermal junction include collagen IV, collagen VII, laminin, entactin/nidogen, fibronectin, as well as subepidermal dermal markers (collagen I and fibrillin 1), and proteoglycans (mainly HS proteoglycans). Collagen type IV, HS proteoglycans, laminin, entactin/nidogen, and fibronectin are products of the epidermal cells of the skin [62].
Heparan-Sulfate Proteoglycans (HS-PGs) HS-PGs are common constituents of cell surfaces and of the ECM, including the basement membrane [63, 64]. The HS-PGs found in the ECM are perlecan, agrin, and collagen XVIII [65, 66]. HS-PGs are implicated in regulating the integrity of basement membranes, morphogenesis, angiogenesis, tumor metastasis, and tissue repair. These activities are attributed to the ability of HS-PGs to bind mitogenic and angiogenic growth factors [67], and to modulate their biological activities. Severe structural changes, including deterioration of the mechanical properties of the dermis, occur during skin aging. Skin HS-PGs, which regulate cell proliferation and proteolysis, as well as matrix adhesion and assembly decrease during aging and thus, may be implicated in the functional alterations linked to the aging process. They
may represent important targets in dermo-cosmetology for fighting skin aging. For instance, Pineau et al. [68] demonstrated the potential interest of a new C-xylopyranoside derivative (C-beta-D-xylopyranoside2-hydroxy-propane, simplified as C-xyloside) to improve HS-PG production in human skin. In an organotypic model of corticosteroid atrophied human skin, characterized by a decrease of PGs expression, treatment with C-xyloside improved expression of HS-PGs. Improvement of the dermo–epidermal junction in human reconstructed skin treated by C-xyloside was also reported [69]. Basement membranes contain several proteoglycans, and those bearing heparan-sulfate chains such as perlecan and agrin, usually predominate.
Perlecan Perlecan is a large proteoglycan, with a core protein of 396 kDa, divided into five domains. It is predominantly substituted with HS chains, but on some occasions it may be substituted with CS, DS, hybrid HS/CS, or CS/DS. It may be also secreted as a glycosaminoglycan-free glycoprotein [70, 71]. Perlecan is present in virtually all basement membranes [72, 73]. It interacts with basement membrane components such as laminin-1 and collagen IV, but also with cell adhesion molecules such as b1-integrin [74]. Inactivation of the perlecan gene in mice results in embryonic lethality and prenatal death. Similarly, the functional null mutation in the perlecan gene is characterized by perinatal death in humans [75]. Perlecan was shown to regulate both the survival and terminal steps of differentiation of keratinocytes: Sher et al. [76] demonstrated that perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors (particularly keratinocyte growth factor or FGF-7), involved in epidermal morphogenesis. Consequently, it may be suggested that perlecan is important for the preservation of skin epithelium. Pineau et al. [68] reported a decreased expression of perlecan during skin aging. This decrease might be involved in the alterations of basement membrane observed in aged skin.
Agrin Agrin is a ubiquitously expressed proteoglycan, with a 200 kDa core protein [77]. It contains not only HS chains,
Proteoglycans in Skin Aging
but also CS chains. The synaptic basal lamina, a component of ECM in the synaptic cleft at the neuromuscular junction, is rich in agrin. However, many other structures, which have basal laminae, including skin, also contain agrin. Available information indicates, however, that no data on agrin expression in skin aging has been published.
Collagen XVIII Type XVIII collagen is an ubiquitous basement membrane zone component, occurring prominently at vascular and epithelial basement membranes including dermo– epidermal junction [78]. While it contains collagen domains, it is also a member of the proteoglycan family since it carries a heparan-sulfate chain. Endostatin, a proteolytic fragment of type XVIII collagen, has been shown to inhibit angiogenesis, tumor growth, and endothelial cell proliferation and migration. Overexpression of endostatin in the skin [79] induced a widening of the epidermal basement membrane zone, as observed by electron microscopy. Immunoelectron microscopy of the type XVIII collagen in mouse skin showed a polarized orientation of this molecule in the basement membrane, with the C-terminal endostatin region localized in the lamina densa. In transgenic mice overexpressing endostatin, type XVIII collagen was dispersed in the skin, suggesting that the transgene-derived endostatin might displace the full-length collagen XVIII. This may impair the anchoring of the lamina densa to the dermis and thereby lead to loosening of the basement membrane zone, resembling the previously observed situation in collagen XVIII-null mice. It was reported that desorganization of the basement membrane zone was one of the features of aged skin [80].
Chondroitin-Sulfate Proteoglycans Although less abundant than HS-PG, several CS-PGs have been described in the basement membrane.
Versican General characteristics of versican were presented in Section ‘‘Versican and Other Proteoglycans of the Dermis Extracellular Matrix.’’ While mainly present in the dermis, traces of versican have been found in some basement
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membranes. No data is available, however, about alterations of versican in the dermo–epidermal junction of aging skin.
Leprecan Leprecan has been described as a CS-PG with a 100-kDa core protein [81]. Immunostaining with polyclonal antibodies showed the localization of its protein core into the basement membrane of the vasculature. Rat-homolog of Gros1, a human growth suppressor gene, was identified to be the leucine–proline-enriched basement membraneassociated proteoglycan leprecan [82]. Leprecan was predicted to be a protein hydroxylase that might be involved in the generation of substrates for protein glycosylation [83]. It was suggested that regulated expression of leprecan coupled with its reported prolyl hydroxylase activity might play a role during basement membrane assembly in the kidney [84]. Available information indicates, however, that no data about alterations of leprecan expression during skin aging has been published.
Bamacan Bamacan is a CS-PG that abounds in basement membranes. Bamacan can occur in certain cell types as either a secreted proteoglycan involved in basement membrane assembly or as an intracellular protein contributing to the structure of chromosome 3 (SMC3) [85]. The entire bamacan core protein is characterized by an Mr of 138 kDa. A stabilizing role for bamacan in the basement membrane has been proposed [86]. Available information indicates, however, that no data on bamacan expression during skin aging has been published.
Conclusion It is clear from the data reported in this chapter that proteoglycans may play a major role in skin homeostasis, and in the functional and architectural properties of this tissue. While many data are available concerning some particular proteoglycans of the dermal or epidermal compartments, for instance, decorin in the dermis or syndecans in the epidermis, large areas remain, however, unknown at the present time. It is particularly the case of the alterations of proteoglycan expression, distribution,
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or structure, which may occur during the aging process and, more importantly, on their potential consequences on skin physiology. Since these alterations may affect not only the glycosaminoglycan chains, but also the protein core of the proteoglycans itself, it will be important to design new experimental models, especially in vivo, which will permit to better study their consequences on skin physiology and their implication in the defects linked to skin aging.
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59. Traister A, Shi W, Filmus J. Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem J. 2008;410:503–511. 60. Litwack ED, Ivins JK, Kumbesar A. Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent. Dev Dyn. 1998;211:72–87. 61. Jackson RL, Busch SJ, Cardin AD. Glycosaminoglycans: molecular properties, protein interactions and role in physiological processes. Physiol Rev. 1991;71:481–539. 62. Tamiolakis D, Papadopoulos N, Anastasiadis P, et al. Expression of laminin, type IV collagen and fibronectin molecules is related to embryonal skin and epidermal appendage morphogenesis. Clin Exp Obstet Gynecol. 2001;28:179–182. 63. David G. Integral membrane heparan sulfate proteoglycans. FASEB J. 1993;7:1023–1030. 64. Erickson AC, Couchman JR. Still more complexity in mammalian basement membranes. J Histochem Cytochem. 2000;48:1291–1306. 65. Blackhall FH, Merry CL, Davies EJ, Jayson GC. Heparan sulfate proteoglycans and cancer. Br J Cancer. 2001;85:1094–1098. 66. Jiang X, Multhaupt H, Chan E, et al. Essential contribution of tumor-derived perlecan to epidermal tumor growth and angiogenesis. J Histochem Cytochem. 2004;52:1575–1590. 67. Iozzo RV. Heparan sulfate proteoglycans: intricate molecules with intriguing functions. J Clin Invest. 2001;108:165–177. 68. Pineau N, Bernerd F, Cavezza A, et al. A new C-xylopyranoside derivative induces skin expression of glycosaminoglycans and heparan sulphate proteoglycans. Eur J Dermatol. 2008;18:36–40. 69. Sok J, Pineau N, Dalko-Csiba M, et al. Improvement of the dermal epidermal junction in human reconstructed skin by a new c-xylopyranoside derivative. Eur J Dermatol. 2008;18:297–302. 70. Isemura M, Sato N, Yamaguchi Y, et al. Isolation and characterization of fibronectin-binding proteoglycan carrying both heparan sulfate and dermatan sulfate chains from human placenta. J Biol Chem. 1987;262:8926–8933. 71. Iozzo RV, Hassell JR. Identification of the precursor protein for the heparan sulfate proteoglycan of human colon carcinoma cells and its post-translational modifications. Arch Biochem Biophys. 1989;269: 239–249. 72. Iozzo RV, Cohen IR, Gra¨ssel S, Murdoch AD. The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J. 1994;302:625–639. 73. Murdoch AD, Liu B, Schwarting R, et al. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem. 1994;42:239–249. 74. Brown JC, Sasaki T, Go¨hring W, et al. The C-terminal domain V of perlecan promotes beta1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem. 1997;250:39–46. 75. Arikawa-Hirasawa E, Yamada Y. Roles of perlecan in development and disease: studies in knockout mice and human disorders. Seikagaku 2001;73:1257–1261. 76. Sher I, Zisman-Rozen S, Eliahu L, et al. Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation. J Biol Chem. 2006;281:5178–5187. 77. Godfrey EW, Dietz ME, Morstad AL, et al. Acetylcholine receptoraggregating proteins are associated with the extracellular matrix of many tissues in Torpedo. J Cell Biol. 1988;106:1263–1272. 78. Saarela J, Rehn M, Oikarinen A, et al. The short and long forms of type XVIII collagen show clear tissue specificities in their expression
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83. Aravind L, Koonin EV. The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2001;2:0007.1–0007.8. 84. Lauer M, Scruggs B, Chen S, et al. Leprecan distribution in the developing and adult kidney. Kidney Int. 2007;72:82–91. 85. Ghiselli G, Siracusa LD, Iozzo RV. Complete cDNA cloning, genomic organization, chromosomal assignment, functional characterization of the promoter, and expression of the murine Bamacan gene. J Biol Chem. 1999;274:17384–17393. 86. Wu RR, Couchman JR. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs. J Cell Biol. 1997;136:433–444.
Endocrinology
33 Sebum Production Claudine Pie´rard-Franchimont . Pascale Quatresooz . Ge´rald E. Pie´rard
Introduction Sebum is produced exclusively by the sebaceous glands. It serves as a vehicle for odors involved in sexual and social attraction. By a similar mechanism, the newborn child commonly recognizes his/her mother’s body odor. The reciprocal recognition is likely during the first weeks of life when the sebaceous glands are active in the newborn. It is noteworthy that the individual sebum-driven scents of each human being are commonly detected by dogs on skin and clothes. Other volatile compounds corresponding to pheromones are produced by mammalian skin in a mixture of apocrine sweat and sebum. In addition, sebum brings vitamin E, the melanocyte-stimulating hormone isotype a (a-MSH), and other various compounds to the stratum corneum (SC). Sebum interferes with the skin microbial biocenosis. It is fungistatic to dermatophyte species. Tinea capitis caused by Microsporum or Trichophyton spp typically occurs only before puberty when sebum production is minimal to absent. In addition, sebum exhibits some bacteriostatic properties. It promotes the growth of specific anaerobic and lipophilic microorganisms and parasites including Propionibacterium spp, Staphylococcus epidermidis, Malassezia spp, and Demodex mites. Other fields of biology are influenced by sebum. The high squalene content in sebum supposedly constitutes, after resorption, a substrate for cholesterol and vitamin D synthesis by the epidermis. Plasticity and cohesion between corneocytes are somewhat related to the sebum amount. Sebum protects the skin and the hair shaft from damage induced by mild acid solutions and friction. Indeed, combing and hairdressing generate friction effects on the cuticlar cells. Skin and hair surfaces are primarily hydrophobic. They paradoxically become more wettable following deposit of sebum components including free fatty acids [1]. In fact, surface wettability is involved in various protective functions of the SC including biocenosis preservation, smoothness, resiliency, and barrier effect to various xenobiotics. Sebum production shows large interindividual differences. Nonetheless, there are global influences of age and gender. In adults, the critical period in life regarding sebum production begins after menopause and andropause.
In older subjects, the sebum flow at the skin surface usually runs dry. From global perspective, aging of the sebaceous glands appears quite complex.
Seborrhea in a Cosmetic Perspective Sebum production shows large interindividual variations as well as intraindividual fluctuations with age. Any excess in the sebum output defines seborrhea that leads to unpleasant cosmetic aspects, and possibly fuels-specific disorders. Most seborrheic subjects exhibit both greasy hair and greasy skin on the forehead and nose. However, some relative differences may be found between these locations. After wiping or washing the skin, the sebum coating is often quickly restored. Hence, seborrhea commonly represents a matter of concern to the affected individual. Observing the skin under ultraviolet light discloses specific subclinical or faint patterns of epidermal melanization [2, 3]. Typically, a speckled perifollicular melanotic pattern (SPMP) is disclosed on the face and scalp of seborrheic individuals, in particular, those with androgenic alopecia [2]. SPMP is evidenced well before the development of other distinct patterns of photodamage-related melanosis. In addition, SPMP is absent in children when sebaceous glands are quiescent. It is also absent in the nonseborrheic parts of the body, including sun-exposed areas. It has been postulated that SPMP on seborrheic skin result from the melanocyte activation by a-MSH produced by the follicular infundibulum and present in sebum [2]. Greasy hair loses its natural luster, and it looks dull, darker, and moist. Hair shafts are weighted down with leaking sebum, which makes them adherent and flattens all hairstyles in thick masses on the scalp. They are difficult to comb. This condition is accompanied by adverse effects on hold. Sebum appears to have the same effect as a humid environment on hair hold. When feeling the oily tresses of sticky hairs, their limpness is perceptible and it is difficult to separate the individual hairs. Sebum is left on the fingers and clothes. The possibility to render a piece of paper translucent helps to distinguish sweaty and greasy hair. On heating, an aqueous impregnation disappears
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rapidly by evaporation, while lipids remain. It should be noted that sustained sweating may be associated with increased seborrhea. A scaly dermatitis is commonly present on seborrheic scalp. This condition is sometimes associated with pruritus or slight discomfort during the days preceding a shampoo. While these symptoms disappear with a regular shampoo, dandruff becomes more visible, since scales are no longer stuck to the scalp by sebum, and they are more easily shed on to the clothes and pillow.
Structure of the Sebaceous Gland The mature sebaceous glands are holocrine lobulated structures distributed all over the skin except on the palms and soles (> Fig. 33.1). Apart from specialized sites such as the eyelids and prepuce, sebaceous glands open indirectly at the skin surface via the hair follicle. The density of sebaceous glands differs from site to site on the body, being highest on the face and scalp followed by the back, chest, abdomen, arms, and legs. The face contains 300–1,500 glands/cm2, the scalp about 300–500 glands/cm2, and other sites present 100 glands/cm2 or less. Three distinct types of pilosebaceous follicles are identified according to the volume of the sebaceous glands and the size of the associated hairs. They are termed terminal hair follicle, vellus hair follicle, and sebaceous follicle, respectively. Terminal hair follicles are present on the scalp and beard region in men. The corresponding hair shaft is thick and the sebaceous gland is of medium to large size reaching about 1 mm3. Vellus hair is found over
. Figure 33.1 Sebaceous glands highlighted by an anti-EMA (epithelial membrane antigen) antibody
the entire body surface except in the palms, soles, and areas with terminal hair follicles. In vellus hair follicle, the hair shaft is short and thin, and the sebaceous gland is tiny when present. The sebaceous follicle is only found in humans. The gland is quite large, whereas the hair shaft is miniature and does not reach the skin surface. The holocrine process of sebum production begins with the proliferation of basaloid undifferentiated cells located at the periphery of the acini as well as in transglandular partitioning epithelial strands. During sebocyte maturation and lipid synthesis, cells enlarge up to 150fold in volume and they express the epithelial membrane antigen (EMA). They move toward the ostia of the glands. Finally, the cell wall ruptures, the lipid content and the cellular remnants form the sebum that is discharged into the sebaceous duct and further to the pilosebaceous infundibulum. The pilosebaceous infundibulum forms a reservoir, which may contain a sizable amount of sebum. The overall transit time of sebocytes takes about 2–3 weeks within the gland and a further week or so through the follicular reservoir before reaching the SC surface. The SC acts as a sponge trapping part of the sebum and eventually resorbing it [4, 5]. In these respects, the bulk of lipids present at the skin surface depends on so many variables that it does not directly reflect the metabolic events taking place in the glands themselves.
Lipid Composition of the Sebum Skin surface lipids originate from two distinct sources: the keratinizing epithelium and sebum. The composition of lipids from these two origins greatly differs. Native sebum is made of triglycerides, wax esters, squalene, and cholesterol esters (> Table 33.1). In mature sebocytes, vacuoles almost filling the cytoplasm contain two components, clearly distinctive on electron microscopy. One is opaque, cloudy, and osmium-positive, probably enriched in squalene. The other is translucent and osmium-negative reflecting the presence of saturated lipids. Synthesis of the various components of sebum involves two different pathways including (a) squalene synthesis following the classical mevalonate and farnesyl pyrophosphate route and (b) fatty acids and wax esters synthesis. Cholesterol is only present in trace amounts in native sebum because it is part of the structural compounds of the cell rather than to the sebum itself. Indeed sebocytes do not process the necessary enzymatic equipment to synthesize cholesterol from squalene. Wax esters or squalene are
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. Table 33.1 Average lipid composition of sebum (%) Component
Native sebum
Skin surface
Triglycerides
57
30–40
Wax esters
25
22–25
Squalene
15
12
Cholesterol esters
3
7
Diglycerides
0
2
Free fatty acids
0
16–25
Ceramides
0
2
generally a marker of sebum excretion helping to differentiate the sebum contribution from epidermal lipids. The fatty acids of the triglycerides are varied, corresponding to either saturated or unsaturated compounds branched or not. They show straight hydrocarbon chains with even or odd numbers of carbon atoms. Wax esters contain the longest chains, the C16 and C18 fatty acids being the most abundant. During its transit up to the skin surface, the sebum composition is altered by oxidative processes and biodegradation partly induced by specific microorganisms [6]. Indeed, triglyceride hydrolysis by bacterial lipases gives rise to free fatty acids, and mono- and diglycerides. At the skin surface, epidermal lipids are admixed with the sebum forming a spotty or continuous lipid film. In adults, the relative contribution of epidermal lipids over the SC is minimal in areas rich in sebaceous glands, although it affects the surface lipid composition on the limbs and trunk away from the midline. On the scalp and forehead, for example, epidermal lipids amount at 5–10 mg/cm2 of skin, whereas sebum is present at 100–700 mg/cm2. Quantitative variations in sebum production following drug intake (e.g., retinoids, antibiotics, etc.) are likely associated with subtle changes in its molecular composition, which in turn may alter the cornification of the infundibulum and be a key factor for comedogenesis [7, 8].
Sebum Excretion and Spreading Sebaceous gland dynamics involves four distinct and successive features: sebum production (a secretion rate function), storage (a volume function), skin surface output (a delivery rate function), and SC permeation (an influx rate function). The oily appearance of skin and hair results
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from an excess of sebum excretion, spreading and interacting with sweat, SC, and hair. On the scalp, sebum appears partly as discrete droplets emerging from follicular outlets, and partly as a surface coating. The droplets are unevenly spread on the hair. In seborrheic subjects, the whole hairshaft appears to be fully coated with sebum. On the skin surface, sebum is usually accumulated in the follicular funnels from where it flows out. Sebum permeates the superficial layers of SC, but a homogenous film is only found in seborrheic subjects. Sebum migration occurs between contiguous hairs or in a swatch, due to capillary forces. After degreasing hairs, the initial refatting rate reaches about 2–3.5 mm/min.
Methods of Sebum Excretion Measurement Subjective methods for evaluating skin and hair greasiness are available based on tactile and visual scales. Their correlation with an overall rating into five classes (very dry, dry, medium, greasy, and very greasy) is convenient. These assessments are useful for the appraisal of sebumcontrolling products. Objective measurements of the sebum excretion provide a better evidence of greasy skin and hair. Over the years, a wide variety of ingenious methods have been developed for measuring the amount of sebum excreted at skin surface and present on hair [1]. Indeed, scalp and hair sebum amounts must be distinguished because they differ significantly. Estimating the amount of scalp sebum needs a miniature sampling method, and hair must be shaved 24–30 h before measurement. The sebum amount present at the skin surface is conveniently measured in vivo using photometric assessment, and lipid-sensitive tapes. A reproducibility of about 10% and a sensitivity threshold in the 5 mg range of lipid amounts are usually considered to be satisfactory [1]. Sebum production and secretion are not measurable in the gland itself. In contrast, sebum excretion at the skin surface after its transit within the storage and delivery units corresponding to the infundibulum reservoir is routinely measured. Several major methods contributed over the years in the evaluation of certain parameters quantifying the sebum bulk and rheology. It should be kept in mind that components of the excreted sebum are partly trapped as any xenobiotic by the SC [6]. Hence, part of sebum is free inside the infundibulum and at the skin surface, while another part permeates onto the SC before eventually being metabolized and further resorbed.
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Photometric Method The basic principle of the photometric method relies on the fact that opalescent glass, sapphire plate, or lipid-sensitive tapes of a given opacity to light becomes translucent when their surfaces are coated with lipids. The photometric procedure is time-saving, highly reproducible, and does not require specially trained scientific staff. The Sebumeter1 SM810 (C + K Electronic, Cologne) is a commercially available device, which has gained popularity [1]. Sebum is absorbed into a piece of matted plastic strip of 0.1 mm thickness placed on a roller, which is manually rewound before each measurement. The probe is pressed under constant pressure against the skin surface. Pitfalls may arise from skin microrelief and roughness, which impair the close contact between the probe and the SC. After the probe has been in contact with skin surface for 30 s, measured by an internal timer, it is placed back into the main unit of the Sebumeter1. Transparency of the plastic film is measured by a photocell linked to a microprocessor after the emitted light has passed back and forth through the strip. It is acknowledged that the measures are in good agreement with the actual amount of lipids present on the strip. In fact, it is estimated that an average of about 40% of total skin surface lipids is absorbed with one sampling. The digital readout displayed as micrograms per square centimeters gives the estimated total amount of lipids on the skin. In order to get valid data, it is necessary to take several samples within a given area so as to avoid problems associated with the heterogeneity of sebaceous gland activity. However, the calculated amounts may be inaccurate when seborrhea is intense because there is a saturation effect of the plastic strip. The photometric method yields a single global estimate of the casual bulk of lipids present on a given surface of the skin at one point in time. The test area is large compared to the size of the ostia of sebaceous follicles. Thus, any difference between the activity of individual sebaceous follicles remains impossible to evaluate by this method. A few overactive sebaceous follicles releasing a large amount of sebum contribute to a disproportionate large effect on measurements.
Sebum-Sensitive Tape Techniques The method using standardized hydrophobic lipid-absorbent tapes relies on opaque, open-celled, microporous polymeric films [1, 8, 9]. It is considered as complementary to and as useful as the photometric method [1, 10, 11]. The tape material has to be affixed to the skin by gentle
pressure ensuring the elimination of any air bubbles. When the sebum-sensitive tape is placed on a skin area possibly moved by muscles, the investigator should periodically check that the uniform contact between tape and SC is maintained throughout the test. Two proprietary tapes are currently available. One type is the regular Sebutape1 (Cuderm Corporation, Dallas) characterized by the presence of an adhesive coat on one side of the tape designed to adhere tightly to the SC. Such adhesive coat likely impairs the swift penetration of sebum into the tape. The other type of lipid-sensitive tape is designed to be applied for only a very short time to the skin surface without interfacing any adhesive coating. These commercially available tapes are the Instant Sebutape1 (Cuderm Corporation, Dallas) and the Sebufix1 (C + K Electronic, Cologne). Depending on the study design, the skin may be prepared prior to the timed collection by removing sebum from the skin surface. The collection time should be determined according to the type of tape. With the regular Sebutape1, the adhesive interposed between the lipid-sensitive film and the skin is a limiting factor to the transfer of lipids. This may be of importance when the rate of sebum excretion is low and/or when the duration of the test is short. On the other hand, a saturation effect occurs on regular Sebutape1 when evaluating intense seborrhea during a test period beyond 1 h. The amount of sebum collected over uninterrupted successive hours is in fact lower than the addition of hourly sebum collections during a similar cumulative period of time. This is associated with confluence of lipid droplets and inaccuracy in identifying each spot as a single sebaceous follicle. These features are the main reasons why sebum samplings longer than 1 h should be avoided when using regular Sebutape1. When using one of the uncoated tapes, a contact time of 30 s or less is appropriate. During the procedure, each follicular outlet enriched in sebum pours out lipids, which fill pores of the tape rendering it focally transparent. The size of the clear spots is proportional to the amount of sebum delivered. The number of spots reflects the number of sebum-rich follicular ostia. These parameters are conveniently evaluated by visual inspection alone. Looking at samples against a black background in reflection mode results in a black and white pattern that can be assessed using an ordinal scale. The method allows to obtain a rough, but reasonable estimate of skin greasiness without requiring sophisticated equipment. Better quantitative assessments are achieved using computerized image analysis [9–11], which represents the most sensitive and accurate method (> Fig. 33.2)
Sebum Production
when offering the possibility of recording the number and size of individual spots and calculating the mean and total area of spots (TAS). The free sebum content of follicular reservoir is conveniently assessed using Sebufix1 tapes affixed onto a recording video camera working under ultraviolet light illumination (Visioscan VC 981, C + K Electronic, Cologne). Computer-assisted image analysis provides proper readings [12]. A built-in microprocessor providing such information is present in the apparatus. A photometric evaluation obtained by measuring the intensity of light transmitted through the samples is an alternative rapid approach [13] and is roughly equivalent to the TAS value obtained by image analysis. A variant is represented by reflectance colorimetric assessment of the samples placed against a colored background [14]. However, these overall quantitative approaches lose one of the main benefits of the lipid-absorbent tape method, namely the evaluation of sebum excretion at each individual sebaceous follicle. A similar quantitative method was designed aiming at collecting data at any time during the tape application onto the skin. The basic principle relies on the measurement of the modifications in color of the tape that occur when it becomes transparent. It shifts the natural ‘‘white’’ color of the tape to a color closer to that of the skin itself. Reflectance colorimetry is conveniently expressed as DE*ab. The benefit of such an approach is the ability to obtain multiple measurements without removing the tape, and therefore to explore a continuous reading of the kinetics of sebum output. . Figure 33.2 Aspect of a computerized image from a lipid-sensitive tape. The black dots represent the sebum output at the follicular orifices
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There are some limitations to the proper interpretation of the data. Some are specifically related to the material itself. Sebum spots are subject to changes in their size and transparency depending on storage time and temperature. At 20 C or so, they should be evaluated at a defined time after removal, preferably within 24 h. When immediate evaluation is not possible, storage in a freezer at 30 C is advisable.
Combined Methods When using lipid-sensitive tapes alone, the interpretation of the number and size of lipid droplets with regard to the sebaceous glands may be uncertain. In fact, it is not valid to ascribe a single follicular outlet to each spot, particularly when the latter is large. In order to solve such an uncertainty, a two-step method was designed [9]. Before removing the sebum-sensitive tape from the skin, its outlines are delineated on the SC with an ink mark. In a second step, a follicular biopsy using a cyanoacrylatecoated polyester film (Melinex O, ICI Plastic division) is collected from that site. The follicular biopsy [15], which is an extension of the cyanoacrylate skin surface stripping conveniently, exhibits follicular casts and microcomedones [1, 3, 16]. The ink marks of the outlines of sebumsensitive film are harvested on this material. The skin surface stripping and the corresponding lipid-sensitive tape are then exactly superposed using the ink imprint as an adjusting mark. This dual material is examined under the microscope and processed in a computerized image analyzer (> Fig. 33.3). This method allows simultaneous . Figure 33.3 Cyanoacrylate skin surface stripping superposed to the corresponding lipid-sensitive tape. This method highlights the active sebaceous follicles contrasting with the inactive follicles
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assessment of the size of lipid droplets and that of the corresponding follicular ostia and microcomedones [10]. A surrogate method relies on examination using a video camera working under ultraviolet light illumination (Visioscan VC981, C + K Electronic, Cologne). A frame designed to precisely attach and locate the camera is first affixed onto the test site. The aspect of skin surface and follicular outlets is recorded. In a second step, a Sebufix1 is interposed between the SC and the camera. The picture of lipid droplets is recorded after a 45-s collection. The comparison of both pictures identifies sebum-poor and sebum-rich follicles [12].
Quantitative Parameters of Sebum Excretion
2.5 mg/cm2/min on the forehead. On the scalp, it varies from 0.1 to 0.8 mg/cm2/min. TAS values yielded by the lipid-sensitive tape method represent a surrogate of SER evaluations. As a rule, SER is roughly correlated with CL. A linear relationship is commonly yielded between four successive 1-h SER and TAS measurements at least in the medium range of severity degree of seborrhea. The correlation is lost when the sebum output is either very low or quite high. This finding indicates that these parameters are related to the delivery of the pool of sebum already secreted by the gland and stored in the outer portion of pilosebaceous duct. Thus, it is clear that an initial 3–4-h collection is a measurement of sebum excretion rather than sebum secretion.
Sebum Replacement Time
The various sampling procedures of sebum provide information about a series of specific parameters. As a rule, the values obtained for these parameters may differ from subject to subject by a tenfold coefficient, but each value is a representative of a given subject in a precise environment.
Sebum replacement time appears to be a cumbersome parameter difficult to manage. It refers to the time needed to recover CL after removing sebum from the skin surface. It has been reported to take about 4 h in subjects with sebum excretion in the medium range.
Sebum Casual Level
Density in Sebum-Enriched Reservoirs
Sebum casual level (CL) is defined as the amount of lipid present at equilibrium when the skin surface remains untouched for several hours. It is a global estimate of skin greasiness. For practical reasons, most researchers record CL after a 4-h lag time following uncontrolled removal of the sebum film from the skin surface. It is expected but uncertain that the measurement reflects a plateau value, and hence CL is not recommended as a single parameter for in-depth studies of the sebaceous system. Therefore, it is believed to be a constant value for each normal adult. In contrast, interindividual variations are large as shown by CL ranging from 100 to 700 mg/cm2 on the forehead of healthy subjects. Similar wide variations are found also on the scalp.
The number of spots over a lipid-sensitive tape is a rough indicator of the density of follicular reservoirs enriched in sebum. Such a figure is usually lower than the number of sebaceous glands present on that area of skin This information can be confirmed by staining cyanoacrylate follicular biopsies for lipids. The sebum delivery at a given follicular ostium may represent a clue for the presence of an actively secreting sebaceous gland. It may also represent the site of a follicular reservoir passively filled by the sebum coming from the skin surface. Using the combination of lipid-sensitive tape and follicular biopsy it was clear that many follicular ostia neither store sebum nor are a route for sebum outflow. It is also possible that one single droplet corresponds to merging of several smaller ones. The size of the follicular outlet at skin surface is not correlated with the presence or absence of sebum [10].
Sebum Excretion Rate Sebum excretion rate (SER) refers to the amount of sebum excreted on a given skin area during a defined period of time. The duration of the definitive collection period is important because SER progressively decreases over the first hours after degreasing the skin. SER of the first hour sampling usually ranges from 0.5 to
Instant Sebum Delivery SER and TAS decrease almost linearly during the first hours of collection time. Calculating the regression line for the cumulative data at 1 h intervals allows to extrapolate a theoretical value for instant sebum delivery (ISD) at
Sebum Production
T0. This parameter supposedly reflects the spontaneous leakage of free sebum from follicular reservoir. ISD is not always correlated with SER. Similar information to ISD is provided by the Sebufix1 tape applied to the skin for a few seconds.
Follicular Excretion Rate The slope of the above-mentioned regression line between cumulative TAS over time has been coined follicular excretion rate (FER). It is a measure of the delivery rate of sebum from the follicular reservoirs. FER is not infrequently related to the first hour TAS value although physiological influences and some topical compounds may interfere with such a relationship.
Factors Affecting Sebum Excretion Physicochemical Regulation SER and FER are influenced by the physicochemical characteristics of sebum. Environmental and skin temperatures, and the balance between the sebum molecular components affect the viscosity and rheology of lipids at the skin surface [17]. This could explain in part some chronobiological variations in SER and FER including seasonal fluctuations [18, 19] as well as the influence of the ovarian cycle [20, 21] and perhaps other chronobiological rhythms of unknown periodicity. A circadian rhythm has been described with sebum output being optimal in the mid morning, and minimal during late evening and early morning hours [21]. The width of follicular ostium may greatly influence sebum rheology. There is an inverse relationship between the fluid flux and the fourth power of the radius of the tube in which the fluid passes through. Hence, variations in corneocyte accumulation and swelling at the lips of follicular outlets, as may occur during the ovarian cycle or after occlusion may influence sebum rheology [20, 21]. Skin surface energy phenomena result from molecular interactions. They are involved in sebum and sweat dispersion. Sweating is indeed an important confounding factor in rating sebum excretion. Even in dry skinned subjects, intensive or continuous sweating increases the CL. The sebum of seborrheic subjects appears as an oily and homogeneous fluid barely emulsified with water at ordinary temperatures. However, in some instances, it can be emulsified with sweat though it may take several hours.
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The theory of a continuous sebum excretion is opposed to the concept of a discontinuous excretion with a feedback control by the CL. SER appears to decline progressively as CL regains its initial value. This observation supported the hypothesis of a feedback mechanism controlling sebaceous excretion by the lipid film developed on skin surface. This concept was further strengthened by the fact that the amounts of sebum collected at constant time intervals seem to increase with the number of degreasing procedures. Sebum excretion stops spontaneously even in highly seborrheic subjects if the area, although isolated, remains uncovered. However, the plateau effect in excretion kinetics is only apparent and due to the spreading of sebum over a larger surface, or to its permeation into the upper layers of the SC. It is concluded that the initial phase of sebaceous secretion is likely continuous or fluctuating as a result of the combination of chronobiological rhythms. Any feedback mechanism from CL could only affect the sebum excretion thus modifying sebum storage rather than sebum production. Shampoos dedicated to greasy hair are commonly used as sebum-controlling products. Some chitosanderived molecules hinder sebum coating of the hairshaft. Particles of dry shampoos adhere to hair, retain lipids, and exert electrostatic forces repulsing the hairshafts. Frequent regular shampoos do not increase the sebum output on the scalp and do not influence the sebum coating of hair. In contrast, cationic polymers and silicone oils used in some shampoos facilitate sebum spreading. Regreasing studies on scalp and hair show general differences according to the level of greasiness. Hair sebum amount is typically lower than scalp sebum. Scalp CL recovers completely after 1–4 days following hair washing, at least for greasy hair types [22] and the aspect remains fairly constant in the following days. Scalp SER has been reported to progressively increase until 24 h after shampooing when it reaches its maximum value.
Hormonal Regulation Human skin, in particular the sebaceous glands, receive, produce, and coordinate hormone activation and inactivation through various molecular signals. The involved physiological mechanisms belong to the endocrine, paracrine, juxtacrine, autocrine, and intracrine hormonal repertoire. Free testosterone and 3a-dihydrotestosterone (DHT) are considered to exert a major boosting and dose-related effect on sebocyte proliferation and sebum secretion in
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man [23]. The levels of type 1 isoform of 5a-reductase are significantly higher in sebaceous glands than in other skin structures [23, 24]. Cells of the infundibulum are also reported to be sensitive to the same hormones. In women, the most important androgen is D4-androstenedione, which is produced by adrenal gland and ovaries. It may be converted into testosterone, but it possesses some intrinsic androgenic activity. 5a-androstane-3b, 17b-diol is a potent androgen and is the main metabolite of testosterone in back and scalp skin. Other androgen precursors of purely adrenal origin such as dehydroepiandrosterone (DHEA) explain sebaceous development in the fetus, after birth, and in the prepubertal years. DHEA can be converted into androstenedione and testosterone inside the sebocytes. By contrast, conversion of DHEA sulfate to DHEA only occurs with the assistance of monocytes exhibiting steroid sulfatase activity. The amount of circulating androgens is important to consider. However, the local production of sexual steroids provides autonomous control adjusting sexual steroid metabolism according to the body area. Facial and scalp sebocytes are particularly involved in this mechanism. The glucocorticoid receptor is another nuclear steroid receptor present in sebocytes. Glucocorticoids have been reported to stimulate sebocyte proliferation [23]. The second group of nuclear receptors, namely the thyroid receptor family, encompasses different soluble receptors in sebocytes. They correspond to the thyroid hormone receptor isotype b1, the estrogen receptor b, the retinoic acid receptors (RAR) isotypes a and g, retinoid X receptors (RXR) isotypes a, b, and g, and the peroxisome proliferator-related receptors (PPAR) isotypes a, d, and g. PPAR ligands augment the androgen stimulation of sebocyte differentiation [25]. Estrogens exert the opposite effect of androgens but with a much weaker potency. However, any decrease in size and excretion of sebaceous glands is only achieved with high doses of estrogens that are nonphysiological in women and feminizing in men. It is thus unlikely that normal estrogen levels play a role in inhibiting sebaceous gland activity. Following estrogen suppression, administration of testosterone restores sebum secretion. Estrogens at sebum-suppressive doses have been shown to reduce plasma and urine levels of testosterone. They also inhibit gonadotropin-releasing hormone synthesis and 5 a˜ reductase activity. There is no relationship between the growth rate of vellus hairs and sebum excretion. In contrast, the intensity of seborrhea and severity of evolving androgenic alopecia are often related. In this condition, GH and IGF-1 might play a role.
Neuropeptide Regulation A neurovegetative nerve plexus surrounds the sebaceous gland, but acetylcholine and adrenaline do not seem to influence sebum secretion. Sympathectomy has no effect on the level of surface lipids. A localized neurological lesion may conversely induce seborrhea in the involved site. Several types of neuropeptide receptors are present in sebocytes. They include the m-opiate receptor which bind b-endorphin, the vasoactive intestinal polypeptide (VIP) receptor, the neuropeptide Y receptor, and the calcitonin gene-related peptide (CGRP) receptor. Psychotropic drugs, often dopamine inhibitors, increase sebaceous secretion considerably. The same observation is found in Parkinson disease [26] perhaps due to high a-MSH serum levels. During treatment with levodopa, seborrhea decreases, but the drug is inactive on sebaceous excretion in normal subjects.
Ethnic, Gender, and Age Effects Some specific attributes related to sebum excretion have been ascribed to ethnic groups [27]. Such observation awaits for confirmation. As a consequence of the diversity of hormonal signals, sebum excretion varies according to age, gender, and climacteric period [28–30]. At any given age in men and women, both sebum excretion and secretion rates differ between individuals over a wide range. In addition, there is a huge overlap between data gained in both genders. Hence, it is not the amount of circulating androgens but rather the receptivity of the target tissues that accounts for interindividual differences in sebum excretion. It is clear that additional factors are likely to be involved. SER and FER values remain high in men until the eighth decade. In women, the rates remain unchanged until menopause. During the climacteric period, seborrhea may either increase or steadily decrease with age. Using the lipid-sensitive tapes, it is possible to evidence distinct patterns according to age and physiopathological conditions [27]. The size of follicular reservoirs and pores shows no tendency to shrink with age. A series of endocrine imbalances and a few drug treatments aiming or not at direct or indirect hormonal effects affect the activity of sebaceous apparatus. The most potent inhibitor of sebum excretion is the synthetic retinoid 13-cis-retinoic acid or isotretinoin, which at oral doses of 0.1–1 mg/kg/day inhibits sebum production by up to 80% within 6–8 weeks. Isotretinoin reduces cell renewal and lipid formation in the sebaceous gland.
Sebum Production
Isotretinoin reduces not only SER, but also the follicular reservoir, and both remain significantly suppressed for up to 1 year after therapy. Topical products are far less potent although of cosmetic interest [31, 32].
Conclusion The current knowledge of sebaceous gland physiology and sebum rheology on skin, scalp, and hair shaft has made some progress, leading to the introduction of new antiseborrheic agents. This breakthrough was made possible through the development of qualitative and quantitative methods for measuring the amounts of excreted sebum, and evaluating its lipid composition. Seborrhea on the scalp or forehead may represent a single phenomenon, or be part of a more complex system of multiple disturbances. As an interesting index of neuroendocrine physiology, it plays an increasing role in the understanding of biology, mainly due to the reliability of its measurement. The sebum flow is altered with aging. In any study in this field ethnicity, age range, gender, adequate skin profile, and test area on the body must be appropriate for a relevant design. The environmental conditions including seasons, relative humidity, and temperature should be controlled. In addition, skin temperature affects sebum rheology. A number of other physiological parameters modulate sebum excretion and some of them are responsible for chronobiological variations. Despite clever experimental designs, it should be stressed that panelists, perception, clinical gradings, and biometrological measurements are not always matched. Several objective methods have been devised for measuring the greasiness of skin, most of which involve the collection of sebum once it runs off the sebaceous apparatus. Preconditioning the skin by prior removal of sebum from the skin surface is a common procedure. Part of the sebum present inside the follicular reservoir can also be ignored by measurements. Uncontrolled depletion of the sebum pool impedes collection of reliable data. Conceptually, aging of the sebum production in women begins in the climacteric period after menopause. It is manifest in men well after entering the andropause period. In both genders, the early changes are often erratic and they progressively evolve to a reduction of the sebum flow.
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References 1. Pie´rard GE, Pie´rard-Franchimont C, Marks R, Paye M, Rogiers V. EEMCO guidance for the in vivo assessment of skin greasiness. Skin Pharmacol Appl Physiol. 2000;13:372–389. 2. Petit L, Pie´rard-Franchimont C, Saint Le´ger D, Loussouarn G, Pie´rard GE. Subclinical speckled perifollicular melanosis of the scalp. Eur J Dermatol. 2002;12:565–568. 3. Uhoda E, Pie´rard-Franchimont C, Petit L, Pie´rard GE. The conundrum of skin pores in dermocosmetology. Dermatology. 2005;210:3–7. 4. Blanc D, Saint Le´ger D, Brandt J, Constans S, Agache P. An original procedure for quantification of cutaneous resorption of sebum. Arch Dermatol Res. 1989;281:346–350. 5. Pie´rard-Franchimont C, Martalo O, Richard A, Rougier A, Pie´rard GE. Sebum rheology evaluated by two methods in vivo. Split-face study of the effect of a cosmetic formulation. Eur J Dermatol. 1999;9:455–457. 6. Saint-Le´ger D, Bague A, Lefebvre E, Cohen E, Chivot M. A possible role for squalene oxides in skin surface and intra-comedonal lipids of acne patients. Br J Dermatol. 1986;114:543–552. 7. Pie´rard-Franchimont C, Henry F, Fraiture AL, Fumal I, Pie´rard GE. Split face clinical and bio-instrumental comparison of 0.1% adapalene and 0.05% tretinoin in facial acne. Dermatology. 1999;198:218–222. 8. Nordstro¨m KM, Schmus HG, McGinley KJ, Leyden JJ. Measurement of sebum output using a lipid absorbent tape. J Invest Dermatol. 1986;87:260–263. 9. Pie´rard GE. Follicle to follicle heterogeneity of sebum excretion. Dermatologica. 1986;173:61–65. 10. Pie´rard GE. Rate and topography of follicular sebum excretion. Dermatologica. 1987;175:280–283. 11. Pagnoni A, Kligman AM, El Gammal S, Popp C, Stoudemayer T, et al. An improved procedure for quantitative analysis of sebum production using Sebutape. J Soc Cosmet Chem. 1994;45:221–225. 12. Pie´rard-Franchimont C, Pie´rard GE. Post-menopausal aging of the sebaceous follicle. A comparison between women receiving or not hormone replacement therapy. Dermatology. 2002;204:17–22. 13. Pie´rard GE, Pie´rard-Franchimont C. Effect of topical erythromycinzinc formulation on sebum delivery. Evaluation by a combined photometric multistep samplings with Sebutape. Clin Exp Dermatol. 1993;18:410–413. 14. Pie´rard GE, Pie´rard-Franchimont C, Kligman A. Kinetics of sebum excretion evaluated by the Sebutape-Chromameter technique. Skin Pharmacol. 1993;6:38–40. 15. Mills OH, Kligman AM. The follicular biopsy. Dermatologica. 1983;167:57–63. 16. Pie´rard GE, Pie´rard-Franchimont C, Goffin V. Digital image analysis of microcomedones. Dermatology. 1995;190:99–103. 17. El Khyat A, Mavon A, Leduc M, Agache P, Humbert P. Skin critical surface tension. A way to access the skin wettability quantitatively. Skin Res Technol. 1996;2:91–96. 18. Pie´rard-Franchimont C, Pie´rard GE, Kligman A. Seasonal modulation of the sebum excretion. Dermatologica. 1990;181:21–22. 19. Youn SW, Na JI, Choi SY, Huh CH, Park KC. Regional and seasonal variations in facial sebum secretions: a proposal for the definition of combination skin tpe. Skin Res Technol. 2005;11:189–195.
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20. Pie´rard-Franchimont C, Pie´rard GE, Kligman A. Rhythm of sebum excretion during the menstrual cycle. Dermatologica. 1991;182:211–213. 21. Verschoore M, Poncet M, Krebs B, Ortonne JP. Circadian variations in the number of actively secreting sebaceous follicles and androgen circadian rhythms. Chronobiol Int. 1993;10:349–359. 22. Black D, Lagarde JM, Auzoux C, Gall Y. An improved method for the measurement of scalp sebum. Curr Probl Dermatol. 1998;26:61–68. 23. Thiboutot D, Harris G, Iles V, Cimis G, Gilliland K, Hagari S. Activity of the type 1-5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin. J Invest Dermatol. 1995;105:209–214. 24. Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S, et al. The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology. 1998;196:21–31. 25. Rosenfield RL, Deplewski D, Kentsis A, Ciletti N. Mechanisms of androgen induction of sebocyte differentiation. Dermatology. 1998;196:43–46. 26. Martignoni E, Godi L, Pacchetti C, Berardesca E, Vignoli GP, Albani G, et al. Is seborrhea a sign of autonomic impairment in Parkinson’s disease? J Neural Transm. 1997;104:1295–1304.
27. Nouveau-Richard S, Zhu W, Li YH, Zhang YZ, Yang FZ, Yang ZL, et al. Oily skin: specific features in Chinese women. Skin Res Technol. 2007;13:43–48. 28. Pie´rard GE, Pie´rard-Franchimont C, Leˆ T, Lapie`re C. Patterns of follicular sebum excretion rate during lifetime. Arch Dermatol Res. 1987;279:S104–S107. 29. Callens A, Vaillant L, Lecomte P, Berson M, Gall Y, Lorette G. Does hormonal skin aging exist? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non invasive measurement techniques. Dermatology. 1996;193:289–294. 30. Caisey L, Gubanova E, Camus C, Lapatina N, Smetnik V, Le´veˆque JL. Influence of age and hormone replacement therapy on the functional properties of the lips. Skin Res Technol. 2008;14:220–225. 31. Pie´rard GE, Cauwenbergh G. Modulation of sebum excretion from the follicular reservoir by a dichlorophenyl-imidazoldioxolan. Int J Cosmet Sci. 1996;18:219–228. 32. Pie´rard GE, Ries G, Cauwenbergh G. New insight in the topical management of excessive sebum flow at the skin surface. Dermatology. 1998;196:126–129.
5 Skin Aging: A Brief Summary of Characteristic Changes Christina Raschke . Peter Elsner
Introduction In the last few decades, life expectancy in many industrialized countries has consistently increased and is predicted to be a continuing process [1]. Appropriate care of elderly skin gains increasing medical importance. Since agedependent effects are so manifest in skin appearance, structure, mechanics and barrier function, much effort has been placed in research to better understand them. Aging is a consequence of genetic program and cumulative environmental damage, which contributes to a progressive loss of structural integrity and physiological tasks of the skin [2, 3]. The intrinsic aging (physiological-, chronological-, UV-protected aging) of the skin is determined by genetic influences and internal factors such as hormones or metabolic substances [4]. Intrinsic aging is mainly controlled by progressive telomere shortening [5]. Telomeres, situated at the ends of eukaryotic chromosomes, are repeated DNA sequences that undergo a base-pair loss during DNA replication. Telomere shortening acts as a mitotic clock, leading to replicative senescence. The clinical aspect of intrinsically aged skin is visible on the consistently cloth-covered skin areas of elderly persons. Extrinsic aging is a similar process that superimposes on the process of intrinsic aging. It is caused by exogenous factors such as ultraviolet (UV) radiation, environmental toxins and infectious agents that induce DNA alterations and damage the skin [6]. The most important extrinsic factor inducing preterm skin aging is ultraviolet irradiation, which causes photoaging of the skin. UV irradiation accelerates telomere shortening in sun-exposed skin [5]. UV-induced extrinsic aging is visible on chronically UV-exposed skin areas in persons frequently engaged in outdoor activities. Acute and chronic sun exposure induces short-term and long-term effects on the skin, ranging from sunburn (erythema) and suntan up to the development of skin aging and skin cancer. Besides natural sunlight there is an increasing influence of artificial UV radiation (solarium). The long-wave UVA radiation (320–400 nm) enters the deep dermis, while the more energetic UVB light (290–320 nm) is absorbed mainly in
the epidermis, especially in the keratinocytes and melanocytes [7]. The direct interaction of UVB with cellular DNA induces damage of DNA strands [8]. UVA radiation also damages the DNA but less than UVB radiation. UVA damage is induced indirectly, through absorption by other endogenous chromophores that release reactive oxygen species, resulting in lipid peroxidation, activation of transcription factors and formation of DNA-strand breaks [8, 9]. Age-related physiological changes in elderly skin include clinical, histological, and biochemical changes, as well as changes in neurosensory perception, barrier function, wound healing and a higher incidence of benign and (pre-)cancerous diseases. Several lines of evidence suggest that the processes of intrinsic and extrinsic aging have different biologic mechanisms.
Clinical Aspects of Aging Skin The most obvious clinical impressions of the elderly skin are increased formation of wrinkles and deficits in elasticity (> Fig. 5.1). Functional changes in the skin of elderly persons include a decreased growth rate of the epidermis, hair and nails. The skin of the elderly is characterized by a decreased lipid content with a changed composition of lipids. These changes induce a rough and dry surface of the skin with tendency to irritation and redness and are likely to contribute to the increased susceptibility of aged skin to the disruption of barrier function [10]. Decrease of barrier function in the elderly skin alters penetration of contactants and primes the inflammatory response [11]. The secretion of sebaceous and sweat glands is diminished [12]. The immune response is reduced and increases the risk of suffering from age-dependent diseases [13]. Increasing degeneration and disorganization of rarefied capillaries and small vessels in elderly skin induce changes in circulation and thermoregulatory functions with predisposition to hypothermia [14]. The elderly skin is increasingly vulnerable to environmental injury by decelerated wound healing and re-epithelization, resulting in a higher
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_5, # Springer-Verlag Berlin Heidelberg 2010
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Skin Aging: A Brief Summary of Characteristic Changes
. Figure 5.1 Obvious signs of aging skin (Courtesy: Department of Dermatology, Jena)
pigmentation inducing the clinical aspect of irregular pigmentation of so-called photo-induced age spots, hyperpigmentations and hypomelanosis guttata [18].
Biochemical Changes of Aging Skin
risk of surgical procedures. The susceptibility of the skin to harmful factors is dependent on creating effective repair mechanisms and inflammatory responses [15]. The intrinsically aged skin appears dry and pale and shows an atrophic aspect with fine wrinkles, which are due to the gravitational or conformational forces. Noticeable is a transparent image of the skin with a shining through of underlying vascular structures. Intrinsically aged skin displays a certain degree of laxity and a regular pigmentation and is prone to a variety of benign neoplasms. Clinically and morphologically, the extrinsically aged skin appears in the form of two types of photoaging with either an atrophic or rather a hyperproliferational aspect [7]. The atrophic form shows small wrinkles and numerous telangiectasias on skin areas with intensive sun exposure [7]. The hyperproliferational form is thicker with a leather-like image and deeper wrinkles. Favre–Racouchot syndrome is a type of photoaging with the clinical signs of deep furrowing, nodular elastic changes, comedones and keratinous cysts tending to appear in the periorbital region of the face [16]. Erythrosis interfollicularis colli, a further example for extrinsic skin aging, shows a restricted erythema sparing the hair follicles. Lentigines solaris and seniles located on the forearms and back of the hands are typical signs of photoaging [17]. Melanocytes along the basal membrane vary in reference to size, morphology and
The surface pH of the normal skin averages a slightly acid value of pH 5.5 and increases in the aged skin [3]. A rise in cutaneous pH value increases susceptibility suffering skin damage of infection, allergy and irritation [19]. The intrinsic aging is mainly associated with decreasing levels of several hormones. The coincidence of climacteric symptoms and the beginning of intrinsic skin aging suggests that estrogen seems to play a dominant role in this process [20]. Intrinsically aged skin displays a significantly reduced expression of the extracellular matrix protein (ECM-1) in lower and upper epidermal layers inducing potential changes of the normal skin structure and function [21]. Most aging changes, intrinsically and extrinsically caused, are due to molecular damage caused by free radicals [22]. The increase in UV irradiation on the earth due to stratospheric ozone depletion may increase the risk of photooxidative damage induced by the generation of reactive oxygen species. Free radicals are highly reactive due to possession of an unpaired electron. Trans-urocanic acid is a major chromophore for UV radiation in human epidermis that undergoes isomerization to its cis-isomer due to UV exposure [23]. Aerobic metabolism generates the superoxide radical, which is metabolized by superoxide dismutase to form hydrogen peroxide and oxygen [24]. Hydrogen peroxide can rapidly generate an extremely reactive hydroxyl radical that damages DNA, proteins and lipids (> Fig. 5.2). The ultraviolet light-induced reactive oxygen species lead to induction of the transcription factor activator protein-1 (AP-1) [25]. AP-1 induces upregulation of matrix metalloproteinases (MMPs) like collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2), which specifically degrade connective tissue proteins such as collagen and elastin and indirectly inhibit the collagen synthesis in the skin, resulting in the obviously changed photodamaged skin [26–30]. Increasing age reduces the activity of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of many members of the MMPs [27]. The balance between collagen synthesis and degradation up to collagen deficiency differs between intrinsic and photoaged skin. The intrinsic aging of the skin seems to be mediated by a reduction in collagen synthesis while the damage of dermal connective tissue in extrinsically aged skin is more about the induction of matrix metalloproteinases [31]. Skin damage is mediated
Skin Aging: A Brief Summary of Characteristic Changes
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. Figure 5.2 UVA-induced generation of reactive oxygen species (Figure adapted with permission from Ziemer et al. [7])
by imperfect protection mechanisms against reactive oxygen species. The mutations of the DNA may accumulate irreversibly if repair mechanisms such as excision repair or using the enzyme photolyase fail to act [7]. The mutancy of mitochondrial DNA is multiple higher than nuclear DNA, because mitochondria do not contain any repair mechanism [32]. The synthesis of collagen types I and III, mainly structural components of the dermal connective tissue, is diminished in photoaged skin by a down-regulation of type I and III procollagen expression [33]. AP-1 has also been shown to negatively regulate type I procollagen gene expression [34]. There are lot of other environmental damages implicated in the process of extrinsic skin aging like tobacco smoke, infrared light and ozone. Tobacco smoke is an important inductor of preterm skin aging. Lahmann et al. reported significantly more MMP-1 mRNA in the skin of smokers than nonsmokers, while no difference was seen for the tissue inhibitor of metalloproteinase-1 [35].
Structural Changes of the Aging Skin Histological analysis is routinely used to diagnose structural changes of skin pathologies. Recent technical progress permits a more precise presentation of the human skin and allows detailed physiological insights into skin changes. Quantitative measurements by bioengineering allow the noninvasive studying of aging skin. Laser Doppler Velocimetry analyses the cutaneous circulation even in vessels of the deeper skin layers [36]. The optical coherence tomography is a pulsed ultrasound technique that enables the fast
analysis of the skin thickness [36, 37]. Confocal laser scanning microscopy and multiphoton laser scanning tomography are noninvasive methods for in vivo presentation of the human skin and generate horizontal images parallel to skin surface [36, 37]. Multiphoton laser scanning tomography allows analysis of skin pathologies by quantifying autofluorescent agents and presenting specific decay rates. Structurally, the intrinsically aged skin shows a thinning of all layers, the corneocytes are less adherent to one another and the dermo–epidermal junction shows a flattening aspect. Due to the reduced dermo–epidermal contact area, the involved layers may dissolve in each other, inducing an increased vulnerability of the skin with formation of ecchymosis [7]. Progressive decreases in melanocyte and Langerhans cell density occur [13, 38]. The dermis has an atrophic aspect with a loss of cells and extracellular matrix and shows a decline of the vascular network that occupies the dermal papillae [39, 40]. Dermal collagen becomes sparser and elastin is degraded slowly and accumulates in intrinsically aged skin [36]. In contrast, the extrinsically aged skin shows a completely different histological aspect (> Fig. 5.3). The epidermis becomes acanthotic and dysceratotic with a high proliferation index of keratinocytes. The epidermis of photoaged skin shows atypia and dysplasia of keratinocytes and melanocytes and a loss of Langerhans cells [41]. The number of hair follicles is reduced more than in intrinsically aged skin, inducing a thinning of the hair [7]. A loss of sebaceous and sweat glands induces increasing dryness of the skin with itching sensations [15]. The histological hallmark of dermal photodamage is the increased synthesis of abnormally structured elastin with its accumulation as elastotic
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Skin Aging: A Brief Summary of Characteristic Changes
material, termed solar or actinic elastosis. Dermal collagen becomes sparser, degenerated and changed in composition [42]. Other characteristics of extrinsically aged skin are the flattening of the dermis, a histiocyte-lymphocyte perivascular infiltrate and a loss of rete ridges [41]. Actually the dermal changes in elderly skin may be demonstrated by in vivo autofluorescence and second harmonic generation measurements using multiphoton laser scanning tomography [43].
Age-Dependent Skin Diseases The process of skin aging is not only a cosmetic problem, but also of medical-dermatological relevance due to a . Figure 5.3 Histological aspect of photoaged skin. The histological image of photoaged skin includes tight stratum corneum, discrete epidermal atypia, teleangiectasias and solar elastosis (Courtesy: Department of Dermatology, Jena)
higher incidence of age-dependent skin diseases such as bullous pemphigoid, erysipelas and herpes zoster (> Fig. 5.4). Many dermatoses of the elderly are presented completely different than in young persons. Herpes zoster is more frequently associated with severe neuralgiform pains, erysipelas appears prevalently in the absence of fever and atopic syndrome is appeared in the form of pruriginous eczema. Clinical aspects of the aging skin are pruritus senilis, age-dependent pemphigoid and atrophic balanitis and vulvitis. Intrinsically aged skin is characterized by a higher incidence of benign hyperproliferations of keratinocytes and capillary blood vessels such as seborrheic keratosis and senile hemangiomas [7]. One feature of photoaging is the higher occurrence of benign hyperproliferative skin lesions like lentigines solaris as well as seborrheic keratosis and senile hemangioma (> Fig. 5.5). Lentigines seniles are pigmented maculae in chronically sun-exposed skin, which are due to an increase in the melanin content inside keratinocytes and casual with melanotic hyperplasia [44]. There is strong evidence supporting the direct role of sunlight exposure in the development of skin cancers. The UV-induced free radicals damage the DNA with possible mutations in oncogenes and tumor-suppressor genes or activation of cytoplasmic signal transduction pathways that are related to growth differentiation, senescence, transformation and tissue degradation [45]. The matrix metalloproteinases have been associated with the process of skin aging and in particular are thought to be critical for tumor invasion and metastasis [46]. An important feature of extrinsically aged skin is the higher incidence of praecancerosa such as actinic keratosis or morbus bowen and malignoma-like basal cell carcinoma and squamous cell carcinoma (> Fig. 5.6). Black skin according to Fitzpatrick is less susceptible to sunburn, photoaging and skin carcinogenesis. A higher melanin content and a different melanosomal dispersion pattern in the epidermis are thought to be responsible for its
. Figure 5.4 Typical age-dependent skin diseases. (a) Bullous pemphigoid, (b) erysipela, (c) herpes zoster (Courtesy: Department of Dermatology, Jena)
Skin Aging: A Brief Summary of Characteristic Changes
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. Figure 5.5 Age-dependent skin tumors. (a) Seborrheic keratosis, (b) senile hemangioma, (c) lentigines solaris (Courtesy: Department of Dermatology, Jena)
. Figure 5.6 (Pre-)cancerous skin diseases. (a) Actinic keratosis, (b) basal cell carcinoma, (c) squamous cell carcinoma (Courtesy: Department of Dermatology, Jena)
more resistant behavior to UV light [47]. Sunscreens with high amounts of sun protection factors protect against solar dermatitis causing a possible decrease in the formation of UV-induced skin malignancies [48].
Conclusion Most changes that occur in the aging face are related to gravity working on skin. Exposure to sunlight hastens these changes and protection from the sun is the only proved way to delay them. Regular photoprotection by avoiding solarium, using sunscreen, which should provide broad-spectrum UVB and UVA coverage, or wearing
UV-protective clothing is an integral part in the management of photoaging. It is important to minimize sun exposure during the peak hours of 10 AM to 4 PM. Tobacco smoke is toxic to cells and should be avoided to prevent accelerating skin aging. The changes of the elderly skin are largely a normal part of aging, but may induce a higher risk for serious skin complications. An increasing part of the elderly population feels disturbed about the age-dependent uncomely signs of the skin and wish therapeutic intervention. Despite the significant improvement that can be achieved with most anti-aging therapies, no technique is perfect. Some patients may have unrealistic expectations and may be unsatisfied with the resulting outcome. However, rational behavior during the life is the
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only proved way delaying preterm uncomely signs of skin aging and may help prevent complications.
Cross-references > Degenerative
Changes in Aging Skin
References 1. Kligman AM, Koblenzer C. Demographics and psychological implications for the aging population. Dermatol Clin. 1997;15 (4):549–553. 2. Farage MA, et al. Structural characteristics of the aging skin: a review. Cutan Ocul Toxicol. 2007;26(4):343–357. 3. Farage MA, et al. Functional and physiological characteristics of the aging skin. Aging Clin Exp Res. 2008;20(3):195–200. 4. Vaillant L, Callens A. Hormone replacement treatment and skin aging. Therapie. 1996;51(1):67–70. 5. Kosmadaki MG, Gilchrest BA. The role of telomeres in skin aging/ photoaging. Micron. 2004;35(3):155–159. 6. Menon G, Ghadially R. Morphology of lipid alterations in the epidermis: a review. Microsc Res Tech. 1997;37(3):180–192. 7. Ziemer M, et al. Alterungsprozesse der haut und altersdermatosen. In: Wedding U, et al. (ed) Medizin des alterns und des alten menschen, 1st ed. Berlin: Verlag Hans Huber, Hogrefe AG, 2007, pp. 157–165. 8. Griffiths HR, et al. Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit Rev Clin Lab Sci. 1998;35(3): 189–237. 9. Berneburg M, et al. Photoaging of human skin. Photodermatol Photoimmunol Photomed. 2000;16(6):239–244. 10. Rogers J, et al. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288(12):765–770. 11. Ghadially R. Aging and the epidermal permeability barrier: implications for contact dermatitis. Am J Contact Dermat. 1998;9(3): 162–169. 12. Balin AK, Pratt LA. Physiological consequences of human skin aging. Cutis. 1989;43(5):431–436. 13. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1986;15(4 Pt 1):571–585. 14. Horvath SM, Rochelle RD. Hypothermia in the aged. Environ Health Perspect. 1977;20:127–130. 15. Harvell JD, Maibach HI. Percutaneous absorption and inflammation in aged skin: a review. J Am Acad Dermatol. 1994;31(6):1015–1021. 16. Patterson WM, et al. Favre-racouchot disease. Int J Dermatol. 2004;43(3):167–169. 17. Schafer T, et al. The epidemiology of nevi and signs of skin aging in the adult general population: results of the kora-survey 2000. J Invest Dermatol. 2006;126(7):1490–1496. 18. Ortonne JP. Pigmentary changes of the ageing skin. Br J Dermatol. 1990;122(Suppl 35):21–28. 19. Fiers SA. Breaking the cycle: the etiology of incontinence dermatitis and evaluating and using skin care products. Ostomy Wound Manage. 1996;42(3):32–34, 36, 38–40, passim. 20. Brincat MP, et al. Estrogens and the skin. Climacteric. 2005; 8(2):110–123.
21. Sander CS, et al. Expression of extracellular matrix protein 1 (ECM1) in human skin is decreased by age and increased upon ultraviolet exposure. Br J Dermatol. 2006;154(2):218–224. 22. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11(3):298–300. 23. Kaneko K, et al. Cis-urocanic acid initiates gene transcription in primary human keratinocytes. J Immunol. 2008;181(1):217–224. 24. Fridovich I. Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem. 1989;264(14):7761–7764. 25. Lo YY, Cruz TF. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem. 1995;270(20):11727–11730. 26. Angel P, et al. Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene. 2001;20(19):2413–2423. 27. Hornebeck W. Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival. Pathol Biol (Paris). 2003;51(10):569–573. 28. Varani J, et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114(3):480–486. 29. Karin M, et al. Ap-1 function and regulation. Curr Opin Cell Biol. 1997;9(2):240–246. 30. Fisher GJ, Voorhees JJ. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces map kinase signal transduction cascades that induce ap-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Invest Dermatol Symp Proc. 1998;3(1):61–68. 31. Chung JH, et al. Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J Invest Dermatol. 2001;117 (5):1218–1224. 32. Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995;27(7):647–653. 33. Talwar HS, et al. Reduced type I and type III procollagens in photodamaged adult human skin. J Invest Dermatol. 1995;105 (2):285–290. 34. Chung KY, et al. An ap-1 binding sequence is essential for regulation of the human alpha2(I) collagen (col1a2) promoter activity by transforming growth factor-beta. J Biol Chem. 1996;271(6):3272–3278. 35. Lahmann C, et al. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357(9260):935–936. 36. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res Technol. 2005;11(4):221–235. 37. Neerken S, et al. Characterization of age-related effects in human skin: a comparative study that applies confocal laser scanning microscopy and optical coherence tomography. J Biomed Opt. 2004;9(2):274–281. 38. Fenske NA, Conard CB. Aging skin. Am Fam Physician. 1988;37 (2):219–230. 39. Yaar M, Gilchrest BA. Skin aging: postulated mechanisms and consequent changes in structure and function. Clin Geriatr Med. 2001;17(4):617–630, v. 40. Yaar M, et al. Fifty years of skin aging. J Invest Dermatol Symp Proc. 2002;7(1):51–58. 41. Kerscher M, et al. Physiologie der hautalterung. In: Effendy I, Kerscher M (eds) Haut und Alter, 1st ed. Stuttgart: Georg Thieme Verlag KG, 2005, pp. 3–10. 42. Schwartz E, et al. Collagen alterations in chronically sun-damaged human skin. Photochem Photobiol. 1993;58(6):841–844.
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46. Crawford HC, Matrisian LM. Tumor and stromal expression of matrix metalloproteinases and their role in tumor progression. Invasion Metastasis. 1994;14(1–6):234–245. 47. Rijken F, et al. Responses of black and white skin to solar-simulating radiation: Differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL10 expression. J Invest Dermatol. 2004;122(6):1448–1455. 48. Elsner P, et al. Sun protection: possibilities and limitations. J Dtsch Dermatol Ges. 2005;3(Suppl 2):S40–44.
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Part 1
Basic Sciences
Histology
1 Skin Aging in Animal Models: Histological Perspective Tapan K. Bhattacharyya
"
‘‘What lies beneath this aging skin? The untold destruction stealthy creeping, The bones the organs the nerves, the brain, I am breathing, I am functioning, Am I living?’’ (Julie A. Crippin, 2008)
animal models from the mammalian kingdom. This review will explore whether intrinsic aging also affects skin histology in animal models in a comparable manner. The data are very limited, and were acquired through painstaking investigation by scientists and clinicians, dating back to several decades, using classical histological techniques; the reported quantitative data were often uncorroborated by statistical methodology.
Introduction Intrinsic aging of human skin has been a topic of interest to scientists and clinicians for over a century, and numerous studies have been published which document progressive anatomic changes of human skin with age, through various decades of human life. Human populations worldwide are living longer, and skin from older people becomes more susceptible to diseases and malformations, apart from being ravaged by environmental trauma like ultraviolet radiation. Along with diseases, natural age-induced changes of the integumentary system pose problems for the elderly population at large. The science of clinical dermatology documents hundreds of diseases of human skin, but scant attention has been paid to basic research in the microanatomy of aging skin. This is an area of research that also invokes biological and evolutionary interest about the mechanics of age-induced involution of various biological systems. Unfortunately, documentation about chronological aging of animal skin is rather limited. The main reason for this is the scant availability of animal models of skin aging with clear documentation regarding age or the stages of the life cycle. Animal husbandry for long-term maintenance of aging animals in a disease-free colony is a costly project, and is usually beyond the reach of an average laboratory. The aging human skin is sagging, with increased roughness and wrinkling, inelastic, with thinning and mottling, and histologically shows many signs of atrophy, such as epidermal thinning or abnormal collagen and elastic fibers. It is logical to ask whether similar or different conditions are observed when the analysis is extended to
The Human Scenario: Skin Aging Histology Morphologic changes of the human integument due to intrinsic aging caused by time-induced physiological changes are understandable, but can be altered due to personal and environmental factors, can vary in different anatomic sites, and are also linked to endocrine factors [1]. Despite these problems, several accounts of intrinsic aging of human skin have been published over the last few decades. Skin atrophy is marked only after the fifth decade of human life, and shows a plethora of histomorphologic changes include epidermal thinning, flattening of the dermal-epidermal junction, loss of melanocytes, and immunocompetent Langerhans cells. There are also dermal changes such as reduced fibroblast population and sebaceous glands. These histopathologic events have been reviewed recently [2]. Morphometric measurement of collagen fibers from stained human skin biopsies further showed that collagen fiber density started decreasing from around 30–40 years, with thinner and more spaced fibers [3]. Other investigators found no difference in epidermal or dermal thickness in a study of wound healing comparing skin from young and elderly volunteers [4]. The biochemical profile of collagen metabolism of human skin, however, changes with age. A steady decline in synthesis of hydroxyproline in human skin up to the fourth decade has been described [5]. Reports of dermal elastic fiber changes with age, including abnormalities and disintegration, have been
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published by some authors. As per the study of VitellaroZuccarello et al. [3], elastic fiber distribution has a different aging pattern in men and women. While the density of elastic fibers in the papillary dermis is not modified as a function of age, in the reticular dermis of both sexes the fiber density increases in the first decade, followed by a drop only in the female. Biochemically, elastin biosynthesis is stable up to approximately the fourth decade of life [5]. Although reports by different investigators vary in detail due to different kinds of sampling and histological methods employed, a general trend of attrition of the dermal connective tissue fibers is noted as a correlate of the aging process. Histopathologic changes in the epidermis and dermis of human skin in sun-exposed and sun-protected areas have been reviewed by MonteiroRiviere [6].
Aging in Rodent Models Some accounts of histological and cellular kinetic changes throughout the life span or representative stages of rodent life span are available. Most of the studies were conducted on the hairless mouse, other strains of mice (CBA, C57131/6NNia, Balb/c), and rats (Wistar rat, Fischer 344). It is difficult to make a meaningful comparison between older studies related to skin aging histopathology, as different authors have reported data on both sexes of various mouse models, which were often based upon limited sample numbers. Some of these important articles lack statistical evaluation. However, some noteworthy findings from early literature on different rodent models are summarized here. The hairless mouse owes its hairlessness to a homozygous recessive genetic condition, and structural changes of skin accompanying development of hairlessness in this animal were described in the older literature. Age-related modifications in epidermal cell kinetics in this species were described by Iverson and Schjoelberg [7]. Using autoradiography, it was shown that epidermal cell proliferation increased from birth to approximately 20 weeks of age, and remained steady. This detailed study could not confirm whether epidermal thickness or cell proliferation rate decreases systematically with increasing age. On the other hand, Haratake et al. [8] presented data showing that the thickness of the epidermis in hairless mice decreases with intrinsic aging. There was also less incorporation of tritiated thymidine in the epidermis in older mice. The tritiated thymidine technique was used in mice up to 19 months of age, and the data revealed an age-dependent decline in the cell proliferation rate [9]. The data reported from Balb/c mice [10] seemed to
indicate epidermal atrophy with age. The epidermis was thinner, with smaller nuclei, in 20-month old animals compared to 2-month animals, although there was no decrease in mitotic activity and DNA labeling index. The loss of epidermal mass was related to a decrease in protein and RNA content of the epidermis. However, similar epidermal changes were not observed in a detailed study in young and old C57B1/6NNia mice [11]. In fact, the epidermis from the ear and footpad showed a statistically significant increase in thickness and augmented cell size. The index of labeling with tritiated thymidine showed no difference between young and old mice. Moreover, in C57BL/6N mice, the number of epidermal cell layers and the epidermal thickness remained constant from 1 to 22 months of age [12]. Most of the reported studies on mouse skin were mainly concerned with the aging effect on the epidermal cell size or cell kinetics, and scarce attention has been devoted to the morphologic changes of the dermal constituents in aging animals. CBA mouse skin was investigated in three age groups (1, 6, and 27 months) from animals procured from NIH colonies [13]. As the rate of skin aging differs in different areas of the body [11], samples were studied from the dorsal, ventral, and pinna skin, and the footpad of these young, young adult, and old animals. A negative linear effect of age on epidermal depth with a significant reduction in cell count (cell/mm), and pilosebaceous unit profiles in dorsal skin samples and footpad was observed. The sebaceous glands appeared atrophied with pycnotic nuclei (> Figs 1.1 and > 1.2). No consistent change in depth of the dermis or area fraction of collagen as determined by histomorphometry was noticed. The dermal elastic fibers in the dorsal skin and footpad showed proliferation in higher age groups in this mouse model. In a study of young and old C57/B16 mice from NIH colonies, decreased dermal cellularity and thickness and decreased epidermal proliferation has been reported [14]. The aging skin in the rat shows non-uniform patterns in different strains when it comes to epidermal and dermal thickness, and the age-associated changes seem to differ from the trend noted in murine species. In Sprague-Dawley rats, the foot epidermis was explored to determine age-related changes in cell kinetics using single pulse [3H]-thymidine labeling and the percent labeled mitosis technique [15], and led to the conclusion that there is a progressive decline in the rates of cell proliferation associated with age. However, these data were presented from rats only up to the age of 52 weeks. The authors of this article referred to five reports published earlier, which showed that rodent epidermal cell proliferation decreased in middle age, and then remained
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. Figure 1.1 Histological preparation of the dorsal skin in aging CBA mice in young (a), young adult (b), and old (c) animals, showing increasing atrophy of the epidermis and shrinkage of sebaceous follicles. Dermal elastic fibers can be seen in c. Verhoeff-van Giesen stain
. Figure 1.2 Error bar chart of epidermal width measurements in three groups of CBA mice
constant, or increased in senile animals. Skin from aging Wistar rats up to the age of 34 months was studied using histomorphometric analysis by Voros and Robert [16]. Average epidermal and dermal thickness did not show appreciable change with senility in this species. In aging
Fischer 344 rats, epidermal thickness remained constant from 3 months of age onward [12]. However, increasing values in epidermal depth and nuclear population were noted in the ventral and dorsal skin and foot plate skin samples from young, 1-year-old, and 2-year-old Fischer 344 rats [17, 18]. Earlier, Lapiere [19] commented that instead of becoming atrophic, rat skin increases in size, with more collagen, although the rate of increase is greatly diminished with aging. This is due to reduced collagen biosynthesis and increased degradation of macromolecules, but the balance between synthesis and degradation remains positive in rats. Aging changes in cells other than epidermal keratinocytes, such as melanocytes or cells of Langerhans, have also been documented in some studies. Ultraviolet radiation has important health consequences on the Langerhans cells of human skin. The numerical density of Langerhans cells in aging inbred mice was studied from epidermal sheets, and showed reduction when compared to that in young animals, although cutaneous immunoreactivity was not compromised [20]. Age-related neurodegenerative changes in peripheral nerves is a widespread
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phenomenon of clinical importance, and rat studies have attested to this inhibitory pattern. Age-associated loss in size of Meissner corpuscles, with smaller and disorganized axonal processes, was reported in the digital pads of mice aged to the maximum life expectancy [21]. Interpretation of morphological changes of aging skin has many limitations, as discreet biochemical changes underlying such alterations cannot be visualized. In contrast to sparsely available accounts of dermal histochemical or morphological transformations in relation to life history in laboratory models, biochemical studies showing quantitative changes in dermal glycosaminoglycans, hydroxyproline concentration, acid mucopolysaccharides, and skin collagen and elastin changes in aging mice, rats, rabbits, and hamsters have been published. Only a few papers are cited here [22, 23]. Oxidative damage to the lipids and DNA increases with age in Fischer 344 rats, which was studied by measuring antioxidant enzyme activity [24]. In the hairless mouse, however, skin aging was not accelerated due to decreased antioxidant capacity [25]. Such molecular changes of the skin in intrinsically aged laboratory animals can only be revealed by immunohistochemistry as more suitable antibodies become available for research.
Morphologic Changes with Aging in Other Species Some sporadic accounts have also been published relating to mammals other than commonly available laboratory rodent models. Age-induced reduction in sebaceous glands was described in sheep by Warren et al. [26]. Epidermal flattening, fewer hair follicles and sebaceous glands, and a decrease in melanocytes were age-related changes in the hairless dog [27]. Veterinary textbooks describe aging changes in skin of domesticated dogs and cats. Senile changes in the skin of old cats and dogs include alopecia, callus formation over pressure points, orthokeratic hyperkeratosis of the epidermis, and atrophied hair follicles [28]. Due to certain structural similarities with human skin, the pig skin model has been used in many experiments for studying responses to surgical and physiological manipulations [29], but no account is available on aging morphologic changes in this species.
Calorie Restriction (CR) and Skin Aging Calorie restriction (CR) can reverse age-associated alterations in many organs like the heart, the liver, and
the brain [30], and has been shown to exert beneficial effects on many skin disorders, although its morphologic effect on the aging skin has not been thoroughly explored. Whether CR can modify age-related histomorphologic features of skin in colony-raised rats was evaluated with morphometric procedures in Fischer 344 rats in two aforementioned studies [18, 19]. Three age groups (young, adult, old) of this strain belonging to ad libitum and CR feeding regimens were obtained from NIH colonies, and skin samples from the dorsum, footpad, and abdominal skin were analyzed with morphometric procedures to evaluate various skin compartments (thickness of stratum corneum, epidermis, dermis, fat layer, percentage fraction of dermal collagen, elastic fibers, fibroblast density, capillary profiles, and staining intensity of dermal glycosaminoglycans). The Fischer 344 rat showed many age-related skin changes, and these were prevented or delayed by CR, presumably due to metabolic alterations imposed by the dietary regimen (> Fig. 1.3). CR reduces cell proliferation in some tissues, with inhibited pace of DNA replication, and this makes those tissues less susceptible to DNA damage by carcinogens. Epidermal cell proliferation as quantified by immunohistochemistry was also correlated to age-related changes in epidermal thickness in these colony-raised Fischer 344 rats. Just as CR somewhat inhibited the trend of increasing epidermal width in aging rats, the keratinocyte proliferation rate as measured by staining of proliferating cell nuclear antigen (PCNA) was correspondingly lower in aging CR rats. This trend was observed in the epithelium of the dorsal skin as well as the foot plate (> Figs. 1.4 and > 1.5) [31]. In C57BL/6J mice, epidermal cell proliferation was reduced by CR and alternate day fasting regimens [32]. A recent study in SENCAR mice also shows that dietary calorie restriction may inhibit gene expression in skin tissues relevant to cancer risks [33]. CR has been reported to reduce the level of free radicals and prevent accumulation of advanced glycation products, and thus may be beneficial to aged skin. On the other hand, loss of subdermal adipose tissue stores, with restricted feeding, may accentuate fine wrinkles in human facial skin. CR can diminish subdermal adipose tissue [18]; therefore, its esthetic effect on the profile of the aging human face remains to be seen.
Conclusion Due to the ease of studying skin aging phenomena and age-associated progressive changes in short-lived mammals, published studies were mostly confined to
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. Figure 1.3 Bar graph representation of the dermis depth from Fischer 344 rats in CR study. Three age groups of ad libitum (AL) and calorie-restricted animals (CR) are represented
laboratory rodent models. Unfortunately, many earlier descriptions of the histopathologic changes in aging skin in such models did not consider old or senile specimens, and therefore lack a proper population sampling. Thus, the story is somewhat incomplete when compared to gerontologic cutaneous changes studied from skin biopsy samples of 90-year old human patients! It should be noted that rodent life span is also highly variable in different strains. Early literature has recorded that, among inbred mice strains used for aging research, a mean life span can vary from a low of 276 days in the AKR/J strain to a maximum of 799 days in the LP/J strain [34]. The lack of availability of suitably aged rodent models is a handicap to such research. This review, however, shows that the mouse model may be more suitable for documenting age-related histological changes that can be compared to human data, despite the caveat that comparing skin from laboratory-grown rodents with a short life span with the human skin assaulted by years of disease and environmental challenge is a rather risky endeavor. Histological deficits in intrinsically aged human skin affect the epidermis, dermal thickness, cellularity, and the elastic fiber system. Efforts should be made to search for analogous chronologic changes in animal models that can be utilized for studies aimed at rejuvenation of the aged skin. Similarities between human and murine skin have been reported in
many studies, making mouse skin a suitable material for studying inflammatory skin diseases. Apart from certain similarities in basic molecular and physiological processes between the mouse and the human [35], the mouse can be maintained under proper husbandry conditions, and may be a more suitable animal for studying the process of senescence of the integumentary system. Virtually no data are available on histological changes from intrinsic aging in other animals like the dog, the domestic pig, or the rabbit, although such species are extensively used for biomedical and physiological studies. Wrinkle formation is a vexing problem of aging human facial skin, and there is little information about skin wrinkles in any animal model at the senile state. Suitable models for studying the histophysiology of wrinkles or their reversal have not been reported, except for one study. Cross-breeding between the Wistar and the wild rat was reported to generate a rat model (the Ishibashi rat) in which skin aging, with wrinkles and furrows, appeared at 12 weeks. Wrinkle formation in this new model was due to a reduction in elastin and collagen contents of the aging skin [36]. This kind of experimental model would prove useful to define the underlying anatomic correlates of skin wrinkle formation, to study its profilometry, and to test the effect of topical anti-wrinkle products for its amelioration [37].
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. Figure 1.4 Representation of epidermal width and PCNA-index from foot plate in rats from CR study. Three age groups from AL and CR animals are represented
. Figure 1.5 PCNA staining in foot plate epidermis from adult (a) and old CR (b) rats. (a) CR rat, adult group, foot plate section. A broad epidermis with a thick cornified layer, and brown stained PCNA reactive cells in the basal layer can be observed. (b) CR rat, old group. The foot plate section shows regression of the epidermis and fewer PCNA positive cells
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In recent years, stem cells in skin have attracted a lot of scientific introspection due to their potential clinical application in wound healing, burns, and alopecia [38], and these cells can go through self-renewal and terminal differentiation, and may regulate tissue aging [39]. It remains to be determined whether stem cells in aging mouse skin can show temporal regression. In vitro studies have shown aged mouse epidermal keratinocytes can function comparably as those cells from young mice [40]. Keratin 15 (K15) promoter activity, which is specific for stem cells, was shown to be active in the hair follicle bulge in murine skin, and the basal epidermal expression of K15 gradually decreased with age [38]. On the other hand, in C57/BI6 mice from NIH colonies, despite a loss in dermal cellularity and thickness in the dorsal skin associated with skin aging, epidermal stem cells were maintained at normal levels throughout life [14]. Further research will elaborate how stem cells can respond to intrinsic aging in different mouse models, and whether they can be pharmacologically stimulated to reverse the process of cutaneous aging.
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Acknowledgment The kind support and encouragement provided by Dr. J. Regan Thomas, MD, Chairman, Department of Otolaryngology-Head & Neck Surgery, UIC is greatly appreciated. Some of the cited work was partly supported by the Bernstein grant from the AAFPRS foundation.
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References 1. Farage MA, Miller KW, Elsner P. Functional and physiological characteristics of the aging skin. Aging Clin Exp Res. 2008;20:195–200. 2. McCullough JL, Kelly KM. Prevention and treatment of skin aging. Ann N Y Acad Sci. 2006;1067:323–331. 3. Vitellaro-Zuccarello L, Cappelletti S, Rossi VDP, et al. Stereological analysis of collagen and elastic fibers in the normal human dermis: variability with age, sex, and body region. Anat Rec. 1994;238: 153–162. 4. Thomas DR. Age-related changes in wound healing. Drugs Aging. 2001;18:607–620. 5. Uitto J. The role of elastic and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol. 2008;7(2):Suppl. S 12–16. 6. Monteiro-Riviere NA. Anatomical factors affecting barrier function. In: Zhai H, Wilhelm K-P, Maibach HI (eds) Dermatoxicology. New York: CRC Press, 2008, pp. 39–49. 7. Iversen OH, Schjoelberg AR. Age related changes of epidermal cell kinetics in the hairless mouse. Virchows Arch B Cell Pathol Incl Mol Pathol. 1984;46:135–143. 8. Haratake A, Uchida Y, Mimura K, et al. Intrinsically aged epidermis displays diminished UVB-induced alterations in barrier function
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associated with decreased proliferation. J Invest Dermatol. 1997; 108:319–323. Cameron IL. Cell proliferation and renewal in aging mice. J Gerontol. 1972;27:162–172. Argyris TS. The effect of aging on epidermal mass in Balb/c female mice. Mech Ageing Dev. 1983;22:3437–3354. Hill MW. Influence of age on the morphology and transit time of murine stratified squamous epithelia. Arch Oral Biol. 1988;33: 221–229. Monteiro-Riviere NA, Banks YB, Birnbaum LS. Laser Doppler measurements of cutaneous blood flow in ageing mice and rats. Toxicol Lett. 1991;57:329–338. Bhattacharyya TK, Thomas JR. Histomorphic changes in aging skin. Obervations in the CBA mouse model. Arch Facial Plast Surg. 2004;6:21–25. Giangreco A, Qin M, Pinter JE, et al. Epidermal stem cells are retained in vivo thoughout skin aging. Aging Cell. 2008;7: 250–259. Morris GM, Van den Aardweg GJMJ, Hamlet R, et al. Age-related changes in cell kinetics of rat foot epidermis. Cell Tissue Kinet. 1990;23:113–123. Voros E, Robert AM. Changements histomorphometriques de la peau Rat en fonction de l’age. C R Soc Biol. 1993;187:201–209. Bhattacharyya TK, Merz M, Thomas JR. Modulation of cutaneous aging with calorie restriction in Fischer 344 Rats. Arch Facial Plast Surg. 2005;7:12–16. Thomas JR. Effects of age and diet on rat skin histology. Laryngoscope. 2005;115:405–411. Lapiere CM. The ageing dermis: the main cause for the appearance of ‘‘old’’ skin. Br J Dermatol. 1990;122(35):5–11. Choi KL, Sauder DN. Epidermal Langerhans cell density and contact sensitivity in young and aged BALB/c mice. Mech Ageing Dev. 1987;39:69–79. Nava PB, Mathewson RC. Effect of age on the structure of Meissner corpuscles in murine digital pads. Microsc Res Tech. 1996;34: 376–389. Prodi G. Effect of age on acid mucopolysaccharides in rat dermis. J Gerontol. 1964;19:128–131. Murai A, Miyahara T, Shiozawa S. Age-related variations in glycosylation of hydroxylysine in human and rat skin collagens. Biochim Biophys Acta. 1975;404:345–348. Tahara S, Matsuo M, Kaneko T. Age-related changes in oxidative damage to lipids and DNA in rat skin. Mech Ageing Dev. 2001; 202:415–426. Lopez-Torres M, Shindo Y, Packer L. Effect of age and molecular markers of oxidative damage in murine epidermis and dermis. J Invest Dermatol. 1994;102:476–480. Warren GH, James PJ, Neville AM. A morphometric analysis of changes with age in skin surface wax and the sebaceous gland area of Merino sheep. Aust Vet J. 1983;60:238–240. Kimura T, Doi K. Age-related changes in skin color and histologic features of hairless descendents of Mexican hairless dogs. Am J Vet Res. 1994;55:480–486. Scott DW, Miller WH, Griffin CE. Small Animal Dermatology. New York: W.B. Saunders, 2001, pp. 64–65. Alex JC, Bhattacharyya TK, Smyrniotis G, et al. A histologic analysis of three-dimensional vs. two-dimensional tissue expansion in the porcine model. Laryngoscope. 2001;111:36–43. Spindlor SR. Rapid and reversible induction of the longevity, anticancer and genomic effects of caloric restriction. Mech Ageing Dev. 2005;126:960–966.
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31. Bhattacharyya TK, Jackson P, Thomas JR. Epidermal proliferating cell nuclear antigen in aging rats. Otolaryngol Head Neck Surg. 2008;139:178 (Abs.). 32. Varady KA, Roohk DJ, McEvoy-Hein BK, et al. Modified alternateday fasting regimens reduce cell proliferation rates to a similar extent as daily calorie restriction in mice. FASEB J. 2008;22:2090–2096. 33. Lu J, Xie L, Sylvester J, et al. Different gene expression of skin tissues between mice with weight controlled by either calorie restriction or physical exercise. Exp Biol Med (Maywood). 2007;232:473–480. 34. Walford RL. When is mouse ‘‘old’’? J Immunol. 1976;117:352–353. 35. Demetrius L. Aging in mouse and human systems. Ann N Y Acad Sci. 2006;1067:66–82.
36. Sakuraoka K, Tajima S, Seyama Y, et al. Analysis of connective tissue macromolecular components in Ishibashi rat skin: role of collagen and elastin in cutaneous aging. J Dermatol Sci. 1996;12:232–237. 37. Bhattacharyya TK, Linton J, Thomas JR, et al. Profilometric and morphometric response of murine skin to cosmeceuticals. Arch Fac Plast Surg. 2009;11:332–337. 38. Roh C, Lyle S. Cutaneous stem cells and wound healing. Pediatr Res. 2006;59:100R–103R. 39. Rando TA. Stem cells, aging and the quest for immortality. Nature. 2006;441:1080–1086. 40. Stern MN, Bickenbach JR. Epidermal stem cells are resistant to cellular aging. Aging Cell. 2007;6:439–452.
44 Skin Photodamage Prevention: State of the Art and New Prospects Denize Ainbinder . Elka Touitou
Introduction Human skin aging is caused by a number of factors. One of the most important and influential factors is the exposure of the skin to UV radiation, which leads to the damage of the skin’s structure and integrity. UV radiation is responsible for up to ninety percent of visible skin aging. However, the effects of the sunlight on the skin include not only dryness, loss of elasticity, wrinkles, discoloration and changes in texture, but also increased incidence in various precancerous conditions and skin malignancies. The prominent dermatologist Prof. Albert Kligman, once said, ‘‘Wear a sunscreen every day of your life, or live as shady a life as possible.’’ In this chapter the state of the art in skin photodamage prevention by the use of sunscreens and the latest technologies in this field will be reviewed.
The Mechanism of Skin Photodamage In order to understand the need for preventing skin damage, which is a result of exposure to UV radiation, one must first explore the underlying mechanisms of UVinduced skin damage. UV radiation is composed of three main wavelength ranges: UVC (100–290 nm) which is largely blocked by the ozone layer and has little impact on skin; UVB (290– 320 nm) which penetrates mostly into the dermis and is responsible for both severe acute sunburn damage and keratinocyte mutations; and UVA (320–400 nm), which for years was considered to be irrelevant to skin damage but is now held responsible for most of the skin damage in the dermis causing skin aging and prolonged pigmentation [1] (> Fig. 44.1). The molecular mechanisms underlying the UVinduced human skin aging process have been extensively investigated in the last two decades. It is now clear that UV irradiation invokes a complex cascade of molecular responses which eventually alter the structure of dermal
extracellular matrix causing wrinkle formation, loss of skin elasticity, increased skin fragility and impaired barrier function of the skin. Upon exposure to UV, as a primary event, the light interacts with a suitable chromophore, which could be either an exogenous agent or an endogenous compound, including porphyrins, flavins, DNA bases, amino acids and their derivatives such as urocanic acid. As a result, the chromophore may be damaged directly or may act as a photosensitizer, leading to the generation of reactive oxygen species (ROS) in the presence of oxygen. Increased ROS concentration initiates a number of signal transduction pathways through activation of cell surface receptors, including receptors for epidermal growth factor (EGF), interleukin -1 (IL-1), insulin, keratinocyte growth factor (KGF) and tumor necrosis factor-a (TNF-a). Activated cell surface receptors stimulate intracellular kinases (p38, c-Jun) leading to up-regulation of the expression and functional activation of the nuclear transcription factor, AP-1 (composed of Jun and Fos proteins). AP-1 activation blocks the effect of transforming growth factor-b (TGF-b) resulting in reduced collagen gene transcription. Moreover, activation of AP-1 stimulates transcription of genes for matrix-degrading enzymes such as matrix metalloproteinase 1 (MMP-1 - collagenase), MMP-3 (stromelysin 1), and MMP-9 (92-kd gelatinase). These MMPs together can degrade collagenous and noncollagenous molecules in the extracellular matrix, thereby impairing the structural integrity of the skin. Following repeated exposure to UV irradiation, MMP-mediated collagen damage accumulates and contributes excessively to the phenotype of photoaged human skin [2, 3]. Ultraviolet irradiation also activates the transcription factor NF-Κb which positively regulates gene transcription of 92k-gelatinase and proinflammatory cytokine genes, including IL-1b, TNF-a, IL-6, and IL-8. UVinduced cytokine gene products act to trigger AP-1 and NF-Κb, thereby amplifying the UV damage. Additionally, stimulation of the expression of the proinflammatory cytokine genes IL-1 and TNF-a is partly responsible for the recruitment of inflammatory cells from the circulation
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. Figure 44.1 The effects of UV radiation on the skin
into the skin, notably neutrophils. Infiltration of neutrophils into the UV-damaged skin is accompanied by an increase in MMP-8 (neutrophil collagenase) protein levels, which in turn contribute to dermal degradation in addition to the AP-1 regulated MMPs. Collagen degradation as a result of exposure to UV irradiation is generally incomplete, leading to the accumulation of partially degraded collagen fragments in the dermis. These large collagen fragments reduce the structural integrity of the skin and negatively regulate new collagen synthesis [3]. The result is formation of sagging skin and wrinkles. Besides the deteriorating effect of UV on skin elasticity, it also decreases the number of Langerhans cells present in the dermis and induces photodermatoses such as lupus erythematosus and polymorphous light eruption (PMLE) [4] (> Fig. 44.2). UV radiation is also responsible for cellular damage resulting in precancerous and cancer skin conditions. UVA toxicity to the cells depends mainly on indirect mechanisms through generation of ROS, which in turn cause the destruction of DNA and other cellular components. UVB light causes DNA damage by the formation of dimmers between adjacent pyrimidine bases on the same strand, generating the cyclobutyl pyrimidine dimmer (CPD)
and pyrimidine (6–4) pyrimidinone photoproducts. This dimerization of pyrimidines leads to the distortion of DNA structure, accounting for about 95% of all DNA lesions. The result is a blockage of DNA replication, cell division, and DNA transcription needed for messenger RNA synthesis. Moreover, as a consequence of repeated exposure to UV light, the number of DNA distortions increases so that the normal repair mechanisms of the cells are unable to correct them. Thus, continuous exposure to UV irradiation generates DNA mutations in the epidermal cells, which cause the development of skin cancer [5, 6]. Recent studies have shown that one of the targets of UV-induced DNA damage is the p53 tumor suppressor gene. Fifty percent of epidermal tumors exhibit DNA mutations at bipyrimidine sites in the p53 gene. Under normal circumstances, p53 is activated as a transcription factor in response to accumulation of DNA alterations. It can then cause cell cycle arrest or apoptosis of the damaged cell. When p53 gene undergoes mutation, the ability to discharge these cells is lost and a tendency toward the selection of highly damaged cells with p53 gene mutations occurs. Eventually, this increasing mass of mutated cells progresses into cancer [5, 6].
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. Figure 44.2 The mechanism of skin photodamage
Photodamage Prevention Sunscreens as the Gold Standard for Photodamage Prevention Although it is currently impossible to prevent or reverse the genetic processes responsible for intrinsic skin aging and cutaneous cancers, skin changes associated with extrinsic aging and photocarcinogenesis are largely avoidable. Protection from UV rays decreases photoaging and reduces the risks of age-related skin diseases. The ‘‘gold standard’’ for skin protection from UV radiation is topical application of sunscreens. The history of sunscreens began around 1928, when the first commercial sunscreen became available as a formulation of benzyl salicylate and benzyl cinnamate. Later, in the 1940s, the FDA (Food and Drug Administration) began to regulate sunscreen formulations. From the 1980s until now, the development of sunscreens has focused on finding broader spectrum sunscreens with higher stability and minimal toxicity [4]. In 1993, a sunscreen active ingredient was defined by the FDA as: ‘‘an active ingredient that absorbs at least 85%
of radiation in the UV range at wavelengths from 290 to 320 nm (UVB), but may or may not transmit radiation at wavelengths longer than 320 nm’’ [7]. Later, due to the acknowledgment of the harmful effects of UVA radiation on the skin, this definition was modified to include sunscreen active ingredients whose absorption maxima is within the UVA range of 320–400 nm [8]. So what should the requirements of an optimal modern sunscreen be? It should absorb and/or reflect UV radiation and provide absorption over a wide spectrum of UV. Protection of the skin against both UVA and UVB is crucial in order to prevent sun-induced damage, including erythema and sunburn, photoaging, the possibility of carcinogenesis, and immunosuppression. It must be stable on the human skin despite exposure to heat and sunlight. Decomposition of the UV-filter decreases the expected UV-protective capacity. Moreover, photodegradation could result in toxic byproducts. It must remain on the skin surface without penetrating into and through the skin, exposing the whole body to the active agent.
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Skin Photodamage Prevention: State of the Art and New Prospects
It should also be water resistant, colorless and suited for formulation in cosmetic preparations [9]. Currently, no sunscreen, exists which encompasses all the above requirements, exists. Therefore, to enable better coverage of both UVA and UVB wavelength range, sunscreen formulations usually combine a number of UVfilters, selecting those with favorable stability and safety properties. The number of approved sunscreen active ingredients and their concentrations in use differs considerably around the world. In Europe, 27 active sunscreen agents are permitted, while only 16 UV-filters are approved for use by the FDA. This discrepancy could be explained by the fact that sunscreen agents are treated like drugs in the US, but as cosmeceutical products in Europe. The sunscreens can be subdivided according to whether they are organic or inorganic agents, as well as by the range of their UV absorption and by their chemical structure. > Table 44.1 lists all the organic FDA approved sunscreens and their properties. Unlike organic sunscreens which absorb UV radiation, inorganic sunscreens mainly reflect/scatter UV, but depending on the particle size may also absorb UV. Titanium dioxide (TiO2) and zinc oxide (ZnO), the most commonly used inorganic sunscreens, are regarded as photostable, nontoxic and non-allergenic. They are used at a concentration of up to 25% to protect the skin in the UVA and UVB region, with maximum absorption at around 400 nm, depending on particle size [4, 9–11]. Until recently, metal oxides have been promoted as safer alternatives to organic sunscreens, based on the belief that they do not penetrate beyond the stratum corneum of the skin thus negating the possibility of systemic absorption. Effective protection by inorganic sunscreens requires thick coating of the systems on the skin. As a consequence of large particle size, topical application of systems containing metal oxides resulted in an opaque whitish layer on the skin. Reduction of the size of metal oxides particles to nanoscale was developed in order to improve their undesirable cosmetic appearance. The advantages and drawbacks of this technology will be discussed later in this chapter.
Innovations in Sunscreen Delivery In spite of all the benefits, the use of sunscreens is not without drawbacks. One of the major concerns, besides skin reactions, is the possibility of skin penetration and as a result of that, toxic systemic effects. This alarm should
be considered heavily, especially in view of the fact that sunscreen active ingredients are available in a range of different cosmetic products such as shampoos, conditioners, skin moisturizers, and lipsticks and may be applied over large skin areas repeatedlyas in the case of beach-type application, or may be applied on a daily basis to the face and other more restricted areas of skin when using non-beach products. It has been suggested that the organic UV sunscreens could be absorbed by the skin because of their lipophilic nature. Their hydrophobic character ensures their ability to resist removal by water and tends to reduce diffusion of the active into the viable epidermis and deeper skin layers. However, as for other lipophilic substances applied topically it should be anticipated that sunscreens will eventually enter the systemic circulation. Watkinson and his group predicted skin absorption of various sunscreens 12 h following application and found that some of the sunscreens might undergo systemic absorption which could be significant if the sunscreen is to be applied over large surface areas for prolonged periods of time [12]. Thus, there is clearly a need for new strategies to ensure sunscreen safety by minimizing its penetration into viable tissues. Another issue associated with the use of sunscreens is their photostability. Upon exposure to UV radiation some of the organic sunscreens can undergo structural transformation or degradation, while the filter looses its absorption capacity. Other, even worse scenarios, could include interactions of the excited molecules with other ingredients of the sunscreen product or skin components leading to the production of undesirable toxic reactive species [9]. Several methods for increasing the efficiency, preventing penetration into the skin and increasing the photostability of sunscreen agents have been developed and tested over the last few years: encapsulation of sunscreens has been applied to both decrease their possible skin absorption and increase their photostability; sunscreen particle size reduction to nanoscale has been used to increase the appearance of metal oxide sunscreen formulations and improve the efficacy of existing sunscreen actives; new combinations of sunscreens with antioxidants were developed to prevent photoaging and to decrease the incidence of photocarcinogenesis.
Encapsulation of UV Absorbers in Particles Various methods based on particulate systems have been used to increase the efficiency and stability of the
Skin Photodamage Prevention: State of the Art and New Prospects
44
. Table 44.1 Currently Food and Drug administration (FDA) approved organic sunscreens and their properties [4, 10, 11]
Chemical group
Sunscreen active
Synonyms and abbreviations
Maximum approved concentration (%)
Absorption (nm)
Comments
Aminobenzoates PABA
4-aminobenzoic acid
15
283–289
Nearly insoluble, highly substantive, photoallergic reactions in 4% of the population, skin discoloration
Pamidate O
2-Ethylhexyl 4-dimethylaminobenzoate, octydimethyl PABA
8
290–310
Possible mutagenicity and carcinogenicity
Meradimate
Methyl-2-aminobenzoate, methyl anthranilate
5
286, 335
Octinoxate
Octyl methoxycinnamate, ethylhexyl methoxycinnamate, Parsol MCX, OMC
7.5
311
Cinoxate
2-ethoxyethyl p-methoxycinnamate
3
289
Octisalate
2-ethylhexyl salicylate, octyl salicylate
5
307
Homosalate
Homomethyl salicylate, HMS
15
306
Trolamine salicylate
Triethanolamine salicylate
12
260–355
Benzopehnone-3, BENZ-3, Eusolex 4360
6
288, 325
Cinnamates Most commonly used, low skin irritancy potential
Salicylates Weak UVB absorbers, highly substantive, safe and photostable
Benzophenones Oxybenzone
Sulisobenzone 2-Hydroxy-410 methoxybenzophenone-5sulfonic acid, Benzophenone-4, BENZ-4
288, 366
Dioxybenzone Benzophenone-8
3
288, 352
3
360
Highest percents of contact dermatitis, low photostability. Presence in blood and urine after topical application (up to 10%).
Dibenzoylmethane Avobenzone
1-(4-tert-butylphenyl)-3(4methoxyphenyl)propane-1,3dione, butyl methoxy dibenzomethane, BMDBM, Parsol 1789, Eusolex 9020
Broad UV absorption spectrum (up to 380nm), highly photounstable, combined with other sunscreens for increased photostability
Miscellaneous Octocrylene
2-cyano-3,3-diphenyl acrylic 10 acid, 2-ethyl hexyl ester, Eusolex OCR
303
Ensulizole
2-phenylbenzimidazole-54 sulfonic acid, PBSA, Eusolex 232, Parsol HS
310
Ecamsule
Terephthalylidene dicamphor 10 sulfonic acid, TDSA, Mexoryl SX
345
Broad UV absorption spectrum (290-390nm), photostable, low systemic absorption
433
434
44
Skin Photodamage Prevention: State of the Art and New Prospects
sunscreens and to decrease skin penetration of the existing sunscreen actives. In 2003, Sol-Gel Technologies Ltd published their new technology for reducing skin absorption, improving safety profile, and increasing the Sun Protection Factor (SPF) of the sunscreen. They have used the sol–gel silica glasses to function as an entrapping matrix. In this system, the sunscreen, which is usually a lipophilic compound, composes the core that is entrapped within a silica shell. The sol–gel sunscreen containing systems are then incorporated into a suitable cosmetic vehicle, enabling high UV protection together with reduced penetration into the skin. The investigators have tested systems containing combinations of OMC, benzophenone-3, butyl methoxy dibenzomethane (BMDBM) and methyl benzylidene camphor to achieve systems with a broad UV absorption spectrum. OMC encapsulated into sol–gel microcapsules showed a close to zero percent leaching profile, while other OMC systems, like OMC absorbed to cosmetic grade silica showed a leaching profile of more than fifty percent. Skin absorption of free OMC formulation was about three times higher than that of encapsulated OMC system. The sol–gel technology enabled encapsulation of high levels of sunscreen actives resulting in high SPF values. Reduction of the contact between the actives and the human tissue resulted in decreased skin absorption of the sunscreen and thus decreased possibility of toxic effects [13]. Wissing and Mu˝ller have proposed a novel sunscreen system based on tocopherol acetate incorporated into solid lipid nanoparticles (SLNs). SLNs have been shown to form a protective and occlusive film on the skin. Previous studies have shown that tocopherol acetate has a UV-blocking efficacy in the UV-B region. Moreover, it reduces the skin damage caused by UV radiation and increases both cell proliferation and moisture content of the skin. SLNs with tocopherol acetate were prepared and tested against placebo SLNs, tocopherol acetate emulsion, and placebo emulsion. Investigation of the UV-blocking capacity showed that tocopherol SLNs were at least twice as effective as the reference emulsions. Placebo SLNs showed even greater blocking efficiency than tocopherol acetate emulsions. Thus incorporation of tocopherol acetate into SLNs prevented chemical degradation of the active and increased its UV-blocking capacity [14]. Scalia et al. prepared 4-methylbenzyldiene camphor (4-MBC) solid lipid microparticles in order to prevent systemic absorption of the sunscreen after topical application. Systemic absorption of 4-MBC has raised safety issues based on in vitro and animal studies showing that 4-MBC has estrogenic activity; moreover, permeation of
the sunscreen leaves the skin surface unprotected. Microparticles containing various lipids (tristearin, cetyl palmitate, glyceryl behenate) and surfactants (hydrogenated phosphatidylcholine, polysorbate 60) were prepared and tested by the researchers. The results of the study showed that the use of microparticles resulted in a decreased release rate of the sunscreen compared to its dissolution rate. In vivo tape-stripped human skin penetration experiments showed that emulsion containing the 4-MBC microparticles decreased, by 33%, the amount of the sunscreen penetrating into the stratum corneum when compared with the use of emulsion alone [15]. Another technique for improved efficacy of UV-filters, the SunSpheres, was introduced by adding nonabsorbing material to sunscreens. The nonabsorbing material, developed by Rohm and Haas, is a styrene/acrylates copolymer manufactured by emulsion polymerization. The polymer does not absorb UV light and is designed to scatter the UV-light on the skin surface thereby increasing its probability of coming into contact with the sunscreen active. SunSpheres are filled with water, which migrates out of the spheres when the system is applied on the skin, leaving microscopic hollow beads. As UV radiation hits these hollow beads, it is scattered on the skin surface traveling sideways, resulting in the increased probability of the sunscreen reacting with UV radiation. The SunSpheres technology is claimed to increase the SPF of sunscreen by 50–70%, by thus enabling the use of a decreased amount of active in the formulation without compromising its photoprotective effect [16].
Sunscreen Particle Size Reduction to Nanoscale In order to overcome the whitening effect associated with topical application of metal oxides for sun protection, micronized powders of both titanium dioxide and zinc oxide were produced with an average particle size of 10–50 nm. Decreasing the particle size into the micronized or ultrafine form has considerably improved cosmetic acceptability by reducing the scattering of visible light and making the new formulations transparent; it is important to take into consideration that it has also shifted the protection toward shorter wavelengths (> Fig. 44.3). An additional disadvantage of micronized sunscreens was found in their tendency to agglomerate because of the electrostatic effects, and thus losing their dispersive properties and becoming opaque once again. A solution to this problem was found by coating the microparticles with dimethicone or silica to keep them in dispersion [4].
Skin Photodamage Prevention: State of the Art and New Prospects
44
. Figure 44.3 Influence of primary particle size on absorption spectra of water in oil emulsions containing 4% titanium dioxide (Forestier S. [9], with permission)
Although, until recently metal oxides have been promoted as safe sunscreens, in vitro studies showed that titanium dioxide nanoparticles can induce free radical formation in the presence of UV. Incubation of titanium dioxide solutions with pyrimidine and purine bases, together with DNA and RNA, under artificial mercury light exposure produced damage to DNA and RNA through free radical generation [17]. The possibility of systemic toxicity of metal oxides and their reduced particle size have raised concerns about the safety of their use. In a recent review by Nohynek et al, a comprehensive assessment of metal oxide nanoparticle skin absorption and safety was carried out. Most of the studies evaluating skin absorption of metal oxide nanoparticles have shown no or negligible penetration beyond the stratum corneum layer of the skin. However, several works have shown the presence of nanoparticles in the hair follicles. Based on the current knowledge and on the results of various works, Nohynek and his group concluded that there is no evidence that metal oxide nanoparticles penetrate into or through human skin [18]. While accessing the skin absorption of metal oxide nanoparticles, it is important to bear in mind that skin absorption of actives can be influenced by system composition. Bennat and his colleagues evaluated the skin absorption of microfine titanium dioxide from oily and aqueous dispersions. They found that titanium dioxide penetrated deeper into the skin from an oily dispersion
with octyl palmitate than from an aqueous one. Further, the penetration depth of microfine titanium dioxide from an oil-in-water emulsion containing carboxymethylcellulose sodium to stabilize the micropigment was tested by tape-stripping technique. The results showed that after nine strips titanium dioxide was no longer detectable. Since liposomal vesicles have the possibility to stabilize titanium dioxide formulation and prevent agglomeration of the micropigments, micropigments incorporated into liposomes were also investigated. The amount of titanium dioxide in the strips following application of liposomal formulation was higher compared to the emulsion, with penetration depth comparable to that from oily dispersion (up to 15 strips) [19]. Despite these encouraging reports on low skin absorption of metal oxide nanoparticles, concerns regarding the safety of their use in sunscreen products have been raised. Just recently, a number of environmental and consumer oriented organizations have urged the FDA and other regulatory authorities worldwide to re-evaluate thoroughly the safety of sunscreens containing nanoparticles. In 2007, the Scientific Committee on Consumer Products (SCCP) published an opinion on the safety of nanomaterials in cosmetic products [20]. The committee concluded that based on the current knowledge there is no conclusive evidence for skin penetration into viable tissues by 20 nm or larger nanoparticles, which are used in metal oxide sunscreens. However, they emphasize that the
435
436
44
Skin Photodamage Prevention: State of the Art and New Prospects
current validated methods are unable to detect small quantities of nanoparticles in the skin and there is an urgent need for new methodologies to assess skin absorption. Further, it is important to note that the studies for the evaluation of metal oxide nanoparticle skin absorption were performed on healthy skin and no information is present for skin with impaired barrier function as in the case of atopic or sunburn skin. The 2007 SCCP opinion concludes that there is a need to review the safety of the insoluble nanomaterials (i.e.; metal oxides) used in sunscreen formulations. Reduction of particle size to nanoscale is applied not only for inorganic sunscreens. In the endless search for a better UV-filter, a system enabling the combination of both organic and inorganic sunscreen properties was developed [21]. In this system, Methylene-bis-benzotriazolyl tetramethylbutylphenol (MBBT or Tinosorb™), a UVfilter, is presented as microfine organic particles ( Fig. 44.4) [28]. The concept of skin Non-Permeating SUNscreens (NPSUN) was to immobilize UV-absorbing moieties in the structure of a jojoba backbone. Jojoba oil is an ester of polycarbonous fatty acids and alcohols (C18–22), widely used as an ingredient in cosmetic and pharmaceutical formulations. It was chosen as the backbone structure because of its high molecular weight (600–700) and high lipophilicity; the two characteristics, which ensure accumulation of the molecule in the outermost upper layers of the skin where the sunscreen is aimed to act. Due to its structure two to four UV-sunscreen actives could be linked to the backbone via ester bonds, and thus enabling the design of UVA, UVB or UVA-UVB systems (> Fig. 44.5). Methoxycinnamate (MC) was chosen as a model sunscreen active. NPSUNs containing two or four MC units were synthesized and characterized by 1H-NMR, UVspectroscopy, FTIR, mass spectroscopy, and elemental analysis. The UV absorption spectrum of the MC-NPSUNs was similar to OMC and the modified sunscreen could be easily formulated in standard cosmeceutical and pharmaceutical topical products. In vitro permeation experiments showed
. Figure 44.4 The dual approach for efficient protection from UV induced skin damage
437
438
44
Skin Photodamage Prevention: State of the Art and New Prospects
. Figure 44.5 Structure of NPSUN derivatives with two (a) and four (b) UV absorbing units (Reprinted with permission from Venditi E. et al. [27])
. Figure 44.6 Permeation profile of OMC and NPSUN-MC across nude mice skin after application of 10 mg of the sunscreens on skin surface (Reprinted with permission from Venditi E. et al. [27])
absolutely no permeation of MC-NPSUN across the skin in 24 h (> Fig. 44.6). Moreover, MC-NPSUNs were found to exhibit high substantivity on the skin and thereby decreasing the need for repetitive applications.
Results obtained so far point toward a high applicability of NPSUNs given the significant advantage of skin nonpenetrability. The nonpenetrating characteristics of NPSUNs are a major improvement over the currently
Skin Photodamage Prevention: State of the Art and New Prospects
used sunscreens and can be seen as meeting one of the most important safety requirements of sunscreens. Another arm of this dual approach is an antioxidant delivered into the deep skin, intracellularly by an efficient carrier. Vitamin E (a-tocopherol) was chosen since it is the major lipid phase antioxidant of the body and has a number of activities. It acts as a free radical scavenger, enables photoprotection due to UV-light absorption with a maximum at 295 nm and suppression of the risk of skin cancer, and inhibits the activity of cyclooxygenase (COX). Vitamin E has a lipophilic nature that makes it attractive for topical application. The effectiveness of topically applied vitamin E was tested in various studies. It was shown to protect rabbit skin against UVinduced erythema and mice against UV-induced skin lipid peroxidation, photoaging, immunosuppression, and photocarcinogenesis. Studies on the mechanism of photocarcinogenesis prevention by vitamin E have revealed that it inhibits pyrimidine dimmer formation in mouse skin cells. Vitamin E has also shown a protective effect against melanogenesis by inhibition of melanin formation in human melanoma cells. As with other nonenzymatic antioxidants, vitamin E’s primary site of action is within the cells, especially in the cellular membrane. Thus, intracellular uptake of tocopherol is necessary for optimal photoprotection effect and prevention of malignant processes [25]. Tocopherol carrier systems were tested for skin penetration and intracellular delivery. Skin penetration experiments showed that 55% of the applied a-tocopherol accumulated in full thickness skin after 24 h. Quantification of the intracellular delivery of a-tocopherol has showed an efficient intracellular delivery of the vitamin. These results demonstrate that the proposed carriers are able to efficiently entrap a-tocopherol and deliver it into the skin as well as through cellular membranes into the cells, pointing toward a high potential of this delivery system to enhance skin photoprotective effects of the antioxidant. A new skin non-permeating sunscreens based on OMC combined with jojoba oil backbone was synthesized and a dual approach for increased skin photoprotection which encompasses the use of skin non-penetrating sunscreens together with intracellularly delivered antioxidants from carriers proposed. In the proposed approach, prevention of systemic absorption of sunscreens will diminish the possible toxic side effects of these compounds, while powerful delivery of a-tocopherol to the site of its action, in the deep dermal layers and inside the cells, will increase its efficiency in inhibiting UV-induced carcinogenic processes.
44
Conclusion A good clinical strategy would be the use of nonpenetrating sunscreens to prevent UV-radiation damages upon exposure to sunlight followed by application of a-tocopherol carrier systems after short or long term solar radiation in order to prevent the UV-induced cellular damage.
Cross-references > Effect
of Ozone on Cutaneous Tissues Warming and its Dermatologic Impact on Aging Skin > In Vitro Method to Visualize UV-induced Reactive Oxygen Species in a Skin Equivalent Model > Global
References 1. Farage M, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 2. Pillai S, et al. Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation – a review. Int J Cosmet Sci. 2005;27:17–34. 3. Fisher G, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138:1462–1470. 4. Palm M, O’Donoghue M. Update on photoprotection. Dermatol Ther. 2007;20:360–376. 5. Marrot L, Meunier JR. Skin DNA photodamage and its biological consequences. J Am Acad Dermatol. 2008;58:s139–s148. 6. Matsumura Y, Ananthaswamy, H. Toxic effects of ultraviolet radiation on the skin. Toxicol Appl Pharmacol. 2004;195:298–308. 7. Federal Register. Sunscreen drug products over-the-counter human use; Tentative final monograph; Proposed rule. Fed Register. 1993;58:28194–28320. 8. Federal Register. Sunscreen drug products over-the-counter human use; Amendment to the tentative final monograph. Fed Register. 1996;61:48645. 9. Forestier S. Rationale for sunscreen development. J Am Acad Dermatol. 2008;58:S133–138. 10. Gonzalez S, et al. The latest on skin photoprotection. Clin Dermatol. 2008;26:614–626. 11. Nash JF. Human safety and efficacy of ultraviolet filters and sunscreen products. Dermatol Clin. 2006;24:35–51. 12. Watkinson AC. Prediction of the percutaneous penetration of ultraviolet filters used in sunscreen formulations. Int J Cosmet Sci. 1992;14:265–275. 13. Lapidot N, et al. Advanced sunscreens: UV absorbers encapsulated in Sol-Gel glass microcapsules. J Sol-Gel Sci Technol. 2003;26:67–72. 14. Wissing SA, Muller RH. A novel sunscreen system based on tocopherol acetate incorporated into solid lipid nanoparticles. Int J Cosmet Sci. 2001;23:233–243. 15. Scalia S, et al. Influence of solid lipid microparticle carriers on skin penetration of the sunscreen agent, 4-methylbenzylidene camphor. J Pharm Pharmacol. 2007;59:1621–1627.
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16. Jones CV. Use of SunSpheres™ technology to increase the effective SPF and UVA absorbance of personal care products containing UV actives. Available at: www.rohmhaas.com/assets/attachments/ business/pcare/formulations/SunSpheres_%20PCIA-Bangkok.pdf (2005). 17. Serpone N, et al. Deleterious effects of sunscreen titanium dioxide nanoparticles on DNA: efforts to limit DNA damage by particle surface modification. Proc SPIE. 2001;4258:86–98. 18. Nohynek GJ, et al. Grey Goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol. 2007;37:251–277. 19. Bennat C, Mu¨ller-Goymann CC. Skin penetration and stabilization of formulations containing microfine titanium dioxide as physical UV filter. Int J Cosmet Sci. 2200;22:271–283. 20. SCCP (Scientific Committee on Consumer Products), 18 December 2007. Safety of nanomaterials in cosmetic products. SCCP/1147/ 07,1–63. 21. Muller S, et al. Microfine organic particles – A new type of ‘‘physical’’ sunscreen actives. Presented at the 63th Annual Meeting of the American Academy of Dermatology, New Orleans, February 18–23, 2005. doi:10.1016/j.jaad.2004.10.171
22. Vettor M, et al. Poly(D,L-lactide) nanoencapsulation to reduce photoinactivation of a sunscreen agent. Int J Cosmet Sci. 2008;20:219–227. 23. Yang J, et al. Influence of hydroxypropyl-b-cyclodextrin on transdermal penetration and photostability of avobenzone. Eur J Pharm Biopharm. 2008;69:605–612. 24. Pinnell SR. Cutaneous photodamage, oxidative stress, and topical antioxidant protection. J Am Acad Dermatol. 2003;48:1–19. 25. McVean M, Liebler DC. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake. Mol Carcinog. 1999;24:169–176. 26. Dreher F, Maibach H. Protective effects of topical antioxidants in humans. In: Thiele J, Elsner P (eds). Oxidants and Antioxidants in Cutaneous Biology., Basel, Karger; 2001. vol 29, pp. 157–164. 27. Venditti E, et al. In vitro photostability and photoprotection studies of a novel ‘multi-active’ UV-absorber. Free Radic Biol Med. 2008;45:345–354. 28. Touitou E, Godin B. Skin nonpenetrating sunscreens for cosmetic and pharmaceutical formulations. Clin Dermatol. 2008;26: 375–379.
17 Skin Reactivity of the Human Face: Functional Map and Age Related Differences Slaheddine Marrakchi . Howard I. Maibach
Introduction One of the first reports of age- and anatomical-related variations in percutaneous absorption were presented by Feldman and Maibach [1] more than 40 years ago. Additionally, in a series of articles, Montagna described anatomical variations in the histological structure of various sites of the facial skin [2]. More recently, bioengineering methods allowed accurate and objective measurements of skin reactivity to chemicals in order to establish a map of the human face by focusing on regional variation, age-related differences in terms of biophysical parameters, and reactivity to chemicals [3–7]. Among these bioengineering tools, capacitance meters usually evaluate stratum corneum (SC) hydration. Transepidermal water loss (TEWL), which evaluates the SC barrier function, is measured by tewameters. Skin pH, skin temperature, and lipid content of the skin surface are measured by appropriate instruments. Laser blood flow monitors are used to evaluate dermal blood flow. Aging skin is under the influence of various factors. Intrinsic influences on skin aging might include genetic (i.e., racial) and hormonal factors, and normally involve the entire integument. Extrinsic influences, including environmental and ultraviolet (UV) exposure, involve mainly the exposed facial skin. This area has to be better studied in order to try to explain the various modifications that occur with aging. Furthermore, only a few reports in the literature extensively evaluated the effect of various chemicals on the areas of the face. Some of these studies focused on the age and regional differences of reactivity to chemicals between different areas of the face. Correlation studies were also undertaken to try and understand how the skin reacts to chemicals, and which intrinsic and extrinsic factors could influence skin reactivity [3–5, 7]. This chapter will focus on the few studies that tried to establish a map of the human face based on the objectively measured biophysical parameters. These studies
demonstrated regional and age-related differences and correlations between some measured parameters. Various chemicals experimentally used to induce irritant dermatitis or contact urticaria also showed age- and regionrelated differences as well as significant correlations in aged persons, between reactivity of the skin and some measured biophysical parameters.
Biophysical Parameters of Skin: Map of Human Face As anatomical variations have been reported in facial skin, an attempt was made to establish a map of the human face for 6 biophysical parameters used to explore various components of the skin: transepidermal water loss (TEWL), skin hydration (capacitance), upper dermis vascularization by laser doppler flowmeter (LDF), skin surface temperature, skin surface pH, and lipid content of the skin surface (sebum). Stratum corneum turnover (dansyl chloride test) was also studied. These parameters were studied in ten aged (66–83 years) Caucasian and Hispanic men and women volunteers. This group was compared to a young group, aged 29.8 3.9 years, ranging from 24–34 years. Nine regions – forehead (FH), upper eyelid (UE), nasolabial area (NL), perioral area (PO), nose, cheek, chin, neck, and volar forearm (FA) – were studied. TEWL was measured using an evaporimeter. The results were expressed as gram per square meter per hour. The electrical capacitance of the SC was measured with a capacitance meter. The results were expressed in arbitrary units. An infrared pyrometer was used to measure skin temperature. Skin pH was recorded using a skin surface pH meter. Lipid content of the skin surface (sebum) was determined by using a sebumeter. The measurements were expressed in microgram per square centimeter. Stratum corneum turnover was studied in all the areas cited except the UE. A concentration of 5% dansyl chloride
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_17, # Springer-Verlag Berlin Heidelberg 2010
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Skin Reactivity of the Human Face: Functional Map and Age Related Differences
in petrolatum was applied on the eight studied skin sites for 16 h, using patch tests. Stratum corneum turnover was determined by detecting fluorescence on each skin site every day, using a UV lamp. The time for the disappearance of fluorescence was considered the SC turnover. All parameters were correlated with each other without considering the regions separately.
Anatomical Variations in the Human Face: SC Turnover The SC turnover, as determined by the disappearance time of dansyl chloride, was slower in the FA (18.3 3.6 days) and NL areas (15.5 3.9 days) than in the chin (10.4 2.8 days), the FH (11.1 2.4 days), the cheek (11.2 2.6 days), and PO areas (11.2 3.8 days), the neck (11.5 2.3 days), and the nose (11.8 2.7 days) [5]. Compared with the young group, the aged individuals showed a trend to a faster SC turnover in almost all the areas studied, with statistically significant values in the nose and the neck (> Fig. 17.1). Grove and Kligman found a significant correlation between SC transit time and corneocyte size in the volar forearm [8]. Plewig and Jansen [9] found corneocytes of the face to be larger than those of the extremities, suggesting that epidermal proliferation in the face was more active than in the extremities. They found corneocyte size in the aged subjects larger
than in the young people, suggesting that proliferation activity in aged people diminishes with age. This was confirmed by Kobayashi and Tagami [7]. The authors focused on the perioral area and demonstrated that corneocyte size increases with age in the cheek, the nasolabial fold, and the chin.
LDF Measurements Blood flow in the nose area was significantly higher than in all the other areas except the chin [6] (> Table 17.1). This was followed by volume of blood flow in the chin, PO, and NL areas, and in the cheek. These areas showed higher blood flow values than the neck and forearm. FH and neck blood flow was higher than only the FA. Correlation between sebum and LDF was relatively strong (r = 0.65, P < 0.01). In concordance with the Shriner classification [3], the nose area showed the highest blood flow levels. A great spatial and temporal intra-individual heterogeneity of LDF measurements has been demonstrated by Braverman [10]. This variation could be related to anatomical organization of the microcirculation units of the upper dermis. The vascular unit was compared to an umbrella, with the handle representing ascending arterioles, and the umbrella proper representing the arteriolar and veinular branches forming the upper vascular plexus. The
. Figure 17.1 Stratum corneum turnover. Regional variation in the young and aged groups, and age-related differences FH: Forehead; NL: Nasolabial area; PO: Perioral area; FA: Forearm *Regions where the difference between the two age groups was significant (p < 0,05)
Skin Reactivity of the Human Face: Functional Map and Age Related Differences
. Table 17.1 LDF (expressed in arbitrary units) (Marrakchi S et al. [6])
17
. Table 17.2 TEWL (expressed as g/m2 h) (Marrakchi S et al. [6]) Young group (Mean SD)
Aged group (Mean SD)
Forehead
6.9 3.4
7.6 3.2
Area
Young group (Mean SD)
Aged group (Mean SD)
Area
Forehead
96.50 115.18
98.60 37.70
Nose
147.87 78.87
204.57 80.07
Upper eyelid
9.5 2.5
11.6 4.0
Cheek
132.96 145.96
129.77 80.81
Nose
9.4 3.8
9.8 4.3
94.71 45.66
118.78 54.38
Cheek
7.1 2.8
7.1 3.3
Perioral
136.14 87.99
127.59 75.03
Nasolabial
14.0 4.4
18.6 12.2
Chin
134.94 69.53
167.71 121.69
Perioral
15.8 11.6
12.3 6.3 10.9 4.2
Nasolabial
Neck
75.51 86.36
73.88 25.89
Chin
9.5 6.1
Forearm
18.13 11.66
24.51 24.95
Neck
9.6 6.5
7.9 2.6
Forearm
3.9 2.7
2.7 1.3
ascending arterioles are spaced at intervals of 1.5–7 mm and give high erythrocytes flux and high concentration of moving red blood cells. Areas of relative avascularity give low erythrocytes flux and low concentration of moving red blood cells. Thus, LDF depends upon the position of the LDF probe on the skin. The moderate correlation found between LDF and sebum content is not surprising, since arterioles and capillaries are organized in vascular units around the appendages of the face, and an increase in sebaceous glands number and sebum production might require more vascularization [11].
Transepidermal Water Loss The highest values were found in the NL area and the lowest values in the FA area [6] (> Table 17.2). There were significant differences between the NL area and all the other areas studied, except the PO area. In the PO area, the chin, and the UE area, TEWL was significantly higher than the FH, cheek, FA, and neck areas. In the nose, neck, FH, and the cheek areas, TEWL was higher than in the FA area. It is clear that water retention is less efficient in the areas surrounding the mouth and the nose. This is confirmed by Lopez [12]. The author divided three areas of the face (cheek, forehead, and chin) into 90 small segments, measured TEWL in each segment, and established a map for these three areas. Conclusions were that locations surrounding the orifices (mouth and nose) showed higher TEWL values. Although the study concerned five young women, it reinforces the results of regional variations of TEWL in the human face. When TEWL in aged people was compared with a young group, no statistical differences were found
between the groups. However, conflicting results have been published concerning the effect of aging on TEWL. Some studies reported a significant decrease in TEWL with age, mainly after the 6th decade [7, 13]. Others reported a trend to decrease in TEWL with age, without reaching statistical significance [3, 14]. The results reported in the literature are difficult to compare, because only a few studies involving various areas of the face in aged people have been published [3, 7]. Therefore, more studies are needed to evaluate age-related changes in TEWL. In the future, the studied groups should be more homogeneous as race, gender, exposure to sunlight, and other environmental factors could induce the SC changes that usually occur with age. Various factors could influence water retention in the SC. In the same study [6], TEWL was found to be moderately correlated to skin temperature (r = 0.44, P < 0.01) in aged individuals. The NL and PO areas showed the highest skin temperature values. This could partly explain the high TEWL values in these areas. As described earlier [14, 15], skin temperature and TEWL are correlated when sweat glands are inactivated. Corneocyte size has been reported to be correlated to stratum corneum renewal [8] and inversely correlated to TEWL [16]. Plewig [9] found that the cornocytes of the face were smaller than those of the extremities and corneocytes in the aged were much larger than in young individuals, which could partly explain the tendency to higher TEWL in aged individuals. However, the influence of various factors on TEWL in aged people should be considered as a complex phenomenon, with the
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Skin Reactivity of the Human Face: Functional Map and Age Related Differences
intervention of various factors that could interact with each other to determine a final TEWL value.
Stratum Corneum Hydration (Capacitance) The neck was the most hydrated area, and the nose area the least hydrated [6] (> Table 17.3). The FA area was more hydrated than the nose and the cheek areas. The remaining areas (UE, FH, PO, NL) had higher capacitance values than the nose, but without reaching statistical significance. As reported by Shriner [3] and Tagami [17], the neck has to be considered as the most hydrated area of the skin, independent of the age. When compared to a young group, the aged group showed more hydrated FA skin, but exposed areas of the face like UE and PO area were less hydrated in aged individuals. However, conflicting results were published with regard to the influence of age on facial skin hydration. Tagami [17] reported that only the perioral areas (nasolabial fold and the chin) are influenced by aging or photoaging. These areas showed an increased hydration state with age. The author related the hydration state to lipid content of the skin. Therefore more data are needed to determine the influence of age on skin hydration. Study of more homogeneous groups should be considered in order to reduce the influence of race and gender on the hydration state of facial skin. Considering two parameters (capacitance and TEWL), the facial skin may be subdivided into three areas, based on the dynamic characteristics of water motion within the epidermis:
. Table 17.3 Capacitance (expessed in arbitrary units) (Marrakchi S et al. [6])
i. areas with a lack of influx of water in the epidermis: low capacitance and low TEWL values (nose and FH areas), ii. areas with excess of water evaporation: high TEWL and low capacitance (NL and PO areas), iii. areas with high water-holding capacity: high capacitance and low TEWL (neck area).
Skin Temperature The NL area, followed by the PO area and the chin, showed the highest skin temperature [6] (> Table 17.4). Skin temperature of the NL area was significantly higher than the skin temperature in three areas (the FH, UE, and FA areas). The PO area temperature was higher than that of the FH and FA areas. Skin temperature in the chin area was almost significantly higher than the temperature in the FH and FA areas. Temperature was similar in the remaining six areas: the neck, cheek, FH, UE, FA, and nose areas. As reported previously [3], skin temperature in the aged people was higher than in the young individuals, although statistical significance was not reached. As reported by Kobayashi [7], moderate to weak correlations were found between skin surface temperature and TEWL (r = 0.44, P < 0.01) and between skin temperature and surface sebum content (r = 0.29, P < 0.01). The correlation with TEWL has already been discussed (TEWL section). Correlation between sebum levels and skin temperature was confirmed by the previous report demonstrating that increase in skin temperature by 1 C
. Table 17.4 Skin temperature (expressed in degrees Celsius) (Marrakchi S et al. [6]) Young group (Mean SD)
Aged group (Mean SD)
Forehead
33.6 0.9
34.0 1.1
72.80 14.86
Upper eyelid
33.6 1.1
34.0 1.3
67.20 21.36
Nose
32.7 2.9
33.5 3.6
87.40 12.44
84.00 16.86
Cheek
32.9 2.0
34.1 1.3
Nasolabial
77.10 14.37
75.60 15.22
Nasolabial
34.6 1.7
35.3 1.4
Perioral
88.90 12.55
72.30 17.31
Perioral
34.1 1.7
35.2 1.3
Chin
88.50 7.90
78.80 29.62
Chin
34.1 1.6
35.0 1.5
Neck
109.60 9.95
Neck
34.6 1.1
34.8 1.5
Forearm
32.8 1.7
33.6 1.6
Area
Young group (Mean SD)
Aged group (Mean SD)
Area
Forehead
89.33 12.72
76.90 18.24
Upper eyelid
89.66 15.97
Nose
56.20 15.61
Cheek
Forearm
81.70 11.14
106.60 6.93 95.10 7.10
Skin Reactivity of the Human Face: Functional Map and Age Related Differences
increases sebum excretion by 10% [18]. Also, as reported by Lopez [12], (using young subjects, however) sebum levels and skin temperature showed comparable patterns, the distribution occurring in a T-shaped pattern, with highest values in the FH area and central part of the face for both parameters.
Sebum Skin Content It is well known that sebum production follows a T-shaped pattern in the human face [6, 12] (> Table 17.5). However, few studies investigated facial sebum excretion in aged individuals in detail. The highest values were in the chin area, followed by the nose, NL, and PO areas, and then the FH area. These areas showed significantly higher values than the UE, neck, and FA areas. The cheek demonstrated higher sebum content than the UE and FA areas. No statistical differences were found in the sebum content of the skin between the aged and the young individuals. Sebum content was correlated with LDF (r = 0.65, P < 0.01), skin temperature (r = 0.29, P < 0.01), and TEWL (r = 0.39, P < 0.01). Correlation with LDF has been previously discussed (LDF measurements section) as has correlation with temperature (Skin Temperature section). Correlation with TEWL could be an indirect relationship, linked to the well-known and demonstrated correlation between TEWL and skin temperature. However, sebum excretion could be a more complicated process than expected. Le Fur [19] showed chronological variation in sebum production in the human face. Other factors beside age (hormonal, gender, and race)
. Table 17.5 Sebum content (expressed in g/cm2) (Marrakchi S et al. [6]) Young group (Mean SD)
Aged group (Mean SD)
Forehead
62.9 28.8
74.7 51.9
Upper eyelid
46.8 25.9
29.3 22.4
Nose
65.2 39.1
Area
17
could influence sebum secretion, rendering the need for standardized protocols and experimental conditions mandatory.
Skin pH Skin pH in the chin area was higher than the pH in four areas: the FH, UE, nose, and PO areas [6] (> Table 17.6). In the FA, neck, and NL areas, skin pH was higher than the pH in the FH and UE areas. The FH and UE areas demonstrated the most acidic skin pH in the face. Compared to the young group, skin pH was significantly higher in the aged individuals in four areas: the FH, UE, neck, and FA. In concordance with these data, Wilhelm’s [13] comparison of the skin pH of the FA and FH areas with nine other skin areas in two age groups found that the pH in the FH area was among the most acidic, while the pH of the FA area was among the most alkaline. In the same study, the pH of the FH area was higher in the elderly compared to young individuals. Zlotogorski [20], who studied the skin pH distribution in the FH and cheek areas, found that the pH was lower in the FH area in 89% of the subjects, and that the pH in these two areas was significantly higher in individuals more than 80 years of age. In a chronobiologic study of biophysical parameters of the skin, Le Fur [19] found a circadian rhythm of pH in facial skin, but not in FA skin, suggesting that structural specificities of the epidermis might account for the arearelated variability of skin pH measurements.
. Table 17.6 Skin pH (Marrakchi S et al. [6]) Young group (Mean SD)
Aged group (Mean SD)
Forehead
4.43 0.44
5.19 0.44
Upper eyelid
4.62 0.40
5.13 0.49
93.6 65.2
Nose
5.23 0.55
5.39 0.50
Area
68.0 46.7
65.3 48.9
Cheek
5.07 0.45
5.47 0.52
147.9 80.6
92.3 63.2
Nasolabial
5.17 0.58
5.59 0.51
81.4 46.9
84.4 65.8
Perioral
5.05 0.48
5.35 0.56
Chin
123.6 87.5
108.5 60.8
Chin
5.55 0.57
5.86 0.31
Neck
33.5 27.4
32.2 26.6
Neck
5.20 0.43
5.67 0.49
0.5 1.6
1.7 2.3
Forearm
5.30 0.32
5.75 0.43
Cheek Nasolabial Perioral
Forearm
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Skin Reactivity of the Human Face: Functional Map and Age Related Differences
Although few studies focused on the skin pH in terms of regional variations and age-related differences [13, 20], Marrakchi and Maibach confirmed that facial skin pH is more alkaline in aged subjects and that regional differences exist among the various areas of the face. A moderate and negative correlation was found between skin pH and sebum content in two areas (which is not surprising with regard to the fatty-acid content of sebum): the chin (r = 0.49, P = 0.03) and the neck (r = 0.53, P = 0.02).
Skin Reactivity Nonimmunologic Contact Urticaria Induced by Hexyl Nicotinate: Functional Map of the Human Face and Age-Related Differences Beside age-related and regional variation studies of the biophysical parameters of the human face, a few studies evaluated the effect of chemicals on facial skin [3–5]. Data of skin reactivity, obtained after hexyl nicotinate (HN) was applied to eight areas of the skin (the FH, nose, cheek, NL, PO, chin, neck, and FA areas), is reported here. Hexyl nicotinate, a lipophylic compound used in cosmetic products, is known to induce nonimmunologic contact
urticaria in the skin. The regional and age-related blood flow changes induced by HN were investigated. The vasodilation induced by HN was recorded by a Laser Doppler Flowmeter. Two age groups were studied: ten healthy young volunteers, aged 29.8 3.9 years, ranging from 24 to 34 years, and ten older volunteers, aged 73.6 17.4 years, ranging from 66 to 83 years. Baseline cutaneous blood flow was monitored at one measurement per second for 30 s and the values were averaged. Subsequently, using a saturated absorbent filter paper disc (0.8 cm diameter) (Finn Chamber1; Epitest Ltd Oy), 5 mM HN in ethanol was applied on the eight skin areas for 15 s to elicit NICU. Following this, the blood flow measurements were taken every 10 min for 1 h, in order to detect the maximum vascular response of the skin to HN (peak value, considered as the response of the skin to HN). In the aged group, the chin, followed by the cheek and the NL areas, showed higher mean peak values (> Fig. 17.2). However, no statistically significant differences were found between the various areas of the face. The FA was found to be the less sensitive area. Peak values were higher in the aged group in three areas: the FH (P = 0.047), cheek (P < 0.001), and NL (P = 0.012) areas. The higher reactivity in the aged group could be explained by the enlargement of sebaceous glands in the elderly [21]. UVA light has been reported to induce sebaceous gland hyperplasia [21], which may lead to the
. Figure 17.2 Baseline LDF to peak changes. Regional variation in the young and aged groups and age-related differences FH: Forehead; NL: Nasolabial area; PO: Perioral area; FA: Forearm *Regions where the difference between the two age groups was significant (p < 0,05)
Skin Reactivity of the Human Face: Functional Map and Age Related Differences
enlargement of sebaceous glands of the face when compared to other areas [2], and in the elderly when compared to the younger subjects [21, 22]. Appendages may be an important factor in HN absorption, because the areas in the aged group where peak values were significantly higher than those in the young group are known to have a high appendage density [23]. Hueber [24] demonstrated that the appendageal route accounts for the transport of hydrocortisone and testosterone, but it is more important for the latter, which is the more lipophilic compound. More recently, vellus hair follicle density has been measured at different body sites: the back, the thorax, the upper arm, the forearm, the thigh, and the calf [25]. The highest density was found in facial skin, the forehead showing an average follicular density of 292 follicles/cm2. The diameters of the follicular orifices showed great variations on the forehead. The authors estimated that this could be explained by the seborrheic nature of this area as compared to the other areas. Another aspect could explain the variations in skin penetration of chemicals: the lipid contents of the skin. Morganti [26] estimated that a lipophilic vehicle can easily penetrate facial skin where the SC is lipid rich (10–20% by weight), compared to palmoplantar skin (2% by weight). In a similar study, Shriner [3] found regional and agerelated differences in facial skin reactivity to benzoic acid. The neck was the most reactive area, and the young individuals reacted more than the aged group. Based on these conflicting results, one should consider that skin penetration might be dependent on the nature of the compound (more or less lipophilic), which penetrates more or less easier in some specific areas of the skin, depending on hydration status, density of follicles, and the composition of the lipids. Moreover, reactivity of the skin to chemicals depends not only upon their transcutaneous penetration, but could be the expression of variability in the vascularization of the epidermis. Vascular anatomy of facial skin was reported to change with age [27]. Chung studied 21 healthy volunteers age-distributed from the 3rd to 9th decade. He found that the intrinsically aged skin (buttock) and photoaged skin (face) showed a reduction in the cutaneous vessel size, with a diminished dermal area covered by the vessels. However, only photoaged skin showed significantly reduced density of dermal vessels. Thus age-related variability of the facial skin to HN could be due to a combination of multiple factors that need more extensive studies, which should take into account racial and gender differences, as well as environmental influences.
17
Sodium Lauryl Sulfate-Induced Irritation in the Human Face: Regional and Age-Related Differences Sodium lauryl sulfate (SLS) (C12H25SO4Na) is an anionic surfactant used for its thickening effect in toothpastes, shampoos, and shaving foams [4]. However, it has a potential irritant effect on the skin. It has been extensively studied in dermatological research. TEWL was considered a predictive parameter for skin susceptibility to SLS [28]. With regard to the lack of investigations exploring its irritant action on human facial skin, a study that investigated regional and age-related differences in skin reactivity to SLS when applied to various areas of the human face was conducted [4]. The potential role of TEWL as a predictive parameter of skin response to SLS was also investigated. Various protocols (concentration, application time) used SLS in a water solution to induce skin irritation. In this study, as the face was suspected to be more sensitive to irritants than the remaining integuments of the human body, SLS 2% was only applied for 1h, under occlusion. This protocol was sufficient to induce subclinical irritation in most of the areas of the face, but not in the FA area, confirming that the face is more sensitive than the FA area. Two age groups were included: ten young subjects, aged 25.2 4.7 years (range 19–30 years) and ten elderly subjects, aged 73.7 3.9 years (range 70–81 years). Twelve Caucasian and eight Hispanic volunteers of both sexes were included in the study. Eight areas of the skin (FH, nose, cheek, NL, and PO areas; chin, neck, and volar FA) were studied. Baseline TEWL and capacitance were measured before a 2% SLS w/v solution was applied for 1 h under occlusion to each of the eight areas. On the contralateral side, water was applied in the same condition as control. To evaluate skin irritation, TEWL was measured 23 h after removal of SLS. TEWL values of the areas tested were corrected according to the changes in the control areas DTEWL ¼ TEWL23 h ðTEWLH2 O 23h TEWLH2 O 0h Þ TEWL0h: Where TEWL 23h is the measured TEWL in the tested area at 23 h, TEWL 0h. is the baseline TEWL in the tested area, TEWL H2O 23h is the measured TEWL in the control area at 23 h, and TEWL H2O 0h is the baseline TEWL in the control area. DTEWL expresses the skin reactivity to SLS. Little is known about susceptibility of the facial skin to the irritant potential of SLS [29]. With age, there is a decrease of irritation induced by SLS in at least some areas of the facial skin, as
179
180
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Skin Reactivity of the Human Face: Functional Map and Age Related Differences
demonstrated earlier. This is in concordance with the overall decrease in sensitivity in the elderly [29, 30]. Skin reactivity to SLS is summarized in > Table 17.7. In the young group, all the areas except the FA area reacted to SLS. In the aged group, all regions reacted except the nose, PO, and FA areas. The cheek and chin showed the highest DTEWL mean values in the aged group. These two areas showed significantly higher reactivity when compared to the FA area, and the chin area demonstrated higher reactivity when compared to the FH area. Although the cheek and chin areas showed the highest DTEWL mean values, statistically significant regional differences in facial skin reactivity to SLS were not detected. This is probably related to the high SD observed in DTEWL values (> Table 17.7). Skin sensitivity to water-soluble irritants has also been explored by the stinging test [31, 32]. Marked regional variation in the intensity of stinging was found [31]: nasolabial fold area > cheek area > chin area > retroauricular area > forehead. A stinging test probably expresses more than just percutaneous diffusion of the irritant compound; it may also express the magnitude of sensory nerve response, which may depend upon nerve density in the skin, which was found to be variable in human epidermis [33]. This was a regional variability, with a higher number of epidermal nerves in facial skin (upper eyelid and preauricular areas) compared to truncal skin. The authors also demonstrated a trend toward the decrease of innervation of the facial skin with increase in age [33]. Skin irritation expressed by DTEWL is the result
of percutaneous penetration of the compound and the changes made to the skin barrier. This could explain why both methods (stinging test and SLS-induced irritation) might give different results in regional variation of sensitivity in the human face, although the cheek and the chin areas were demonstrated as among the most sensitive by both methods. Comparison between both groups showed that facial skin reactivity to SLS in the young group was higher, but the differences were significant only in the chin area (P = 0.035) and the NL area (P = 0.005). Correlation between baseline TEWL and DTEWL revealed that baseline TEWL is a predictive factor of facial skin reactivity to SLS in five areas: cheek, FH, neck, NL, and PO areas (> Table 17.8). This comparative study used SLS to induce an acute irritation. However, repeated or cumulatively induced irritation should better reflect the common use of potential irritants on the skin. This was done by Schwindt,
. Table 17.8 Correlations in each area between baseline TEWL (BTEWL) and reactivity of the skin to SLS, 23 h after patch removal (DTEWL) (Marrakchi S et al. [4]) BTEWL (Mean SD)
. Table 17.7 Reactivity of regions in the young and aged groups (Marrakchi S et al. [4]) Area
DTEWL (Mean SD) g/m2 h
TEWL 23H (Mean SD)
DTEWL (Mean SD)
r
p
Cheek*
15.63 6.70
26.63 15.30
10.96 11.01
0.4616 0.040
Chin*
20.87 6.37
30.47 12.08
9.77 8.13
0.3535 0.126
Forearm
8.64 3.97
9.70 4.92
1.51 1.83
–
Forehead*
14.10 5.71
20.40 14.96
6.39 10.53
0.6474 0.002
Neck*
11.55 4.35
16.63 8.54
5.18 5.12
0.6273 0.003
p value
–
Young group
Aged group
Cheek
15.1 12.8
6.8 7.3
0.093
Chin*
13.5 9.9
6.0 3.3
0.035
Forearm
1.9 2.1
1.1 1.5
0.354
10.4 13.9
2.3 2.3
0.086
Nasolabial* 28.74 8.56
36.93 13.44
8.40 6.78
0.4831 0.031
Forehead
Nose*
19.04 6.03
25.27 11.15
6.77 6.92
0.3218 0.166
Perioral
24.25 8.93
29.98 14.6
7.47 8.17
0.4547 0.044
Neck
6.8 6.0
3.6 3.7
0.165
Nasolabial area*
12.4 6.3
4.4 4.8
0.005
Nose
8.6 7.6
5.0 6.0
0.251
Perioral area
10.7 10.0
4.2 4.1
0.074
DTEWL = TEWL 23 h after patch test removal corrected to the control – baseline TEWL * Difference between the young and aged group statistically significant (p < 0.05)
Areas which reacted to SLS: statistically significant (p < 0.05) difference baseline TEWL and TEWL 23 h after patch removal r coefficient of correlation p significance (significant correlation when p < 0.05)
*
Skin Reactivity of the Human Face: Functional Map and Age Related Differences
who compared the effect of SLS on the skin of the back in young and aged humans [34]. In the skin of the aged group repeated application of SLS induced less pronounced irritation than in the young group. This was believed to be related to a decrease in percutaneous absorption and an altered inflammatory skin reaction. The process by which irritants induce skin modifications is complex and still under investigation. Several mechanisms seem to be implicated. SLS induces modification in the proteins of the SC [35] as well as in the lipid bilayers [36], leading to modifications in the skin barrier function. There are also inflammatory changes secondary to the toxic effect of SLS on keratinocytes. These changes involve cytokines that induce infiltration of the epidermis by inflammatory cells and vasodilation in the dermis [37]. The correlation study showed that skin reactivity to SLS (DTEWL) and baseline TEWL values were correlated in five facial areas (> Table 17.8). The correlations between baseline TEWL and TEWL measured 23 h after SLS removal were stronger. The facial areas that reacted to SLS showed strong correlation coefficients, varying from 0.76 to 0.88, with a high significance (P < 0.001). However, correlation between basal TEWL and the absolute value measured after irritation (TEWL 23h) does not imply that higher basal TEWL values predispose to higher skin sensitivity, but that only the correlation between baseline TEWL and the changes in TEWL after irritation (DTEWL) may have this significance. Conflicting results have been published with regard to this aspect. Some authors correlated absolute TEWL values before and after induction of irritation [28], while others [38] correlated basal TEWL and changes to TEWL (DTEWL). Agner [38], studying healthy and atopic subjects, reported a positive correlation between baseline TEWL and the increase in TEWL induced by SLS only in the healthy group. Although basal TEWL was significantly higher in the atopic group, the changes in TEWL detected after exposure to the irritant were not significantly different between the two groups. These findings are in concordance with Marrakchi and Maibach’s results, where some areas of the face (NL area) showed higher basal TEWL values than others (the cheek), but failed to induce higher sensitivity (> Table 17.8). Therefore, beside basal TEWL, each region of the skin face probably has its own characteristics that influence the skin sensitivity to irritants. Using a tape-stripping method, de Jongh [39] evaluated penetration parameters of SLS in the SC. The partition coefficient of SLS between water and SC, K, determines the amount of SLS that enters the SC. The diffusion coefficient, D, gives the rate by which
17
SLS moves into the SC. Both parameters, K and D, constitute the penetration parameters of SLS. When associated with baseline TEWL and SC thickness, the penetration parameters demonstrated a better predictive value of impairment of the skin water barrier function induced by SLS than baseline TEWL and SC thickness alone [39].
Conclusion A map of the human face based on objectively measured biophysical parameters was established. Various chemicals experimentally used to induce irritant dermatitis or contact urticaria also showed age- and region related differences as well as significant correlations in aged persons, between reactivity of the skin and some measured biophysical parameters.
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Skin Reactivity of the Human Face: Functional Map and Age Related Differences
women’s facial skin follow characteristic patterns. Skin Res Technol. 2000;6:31–36. Wilhelm K-P, Cua AB, Maibach HI. Skin aging: Effect on transepidermal water loss, stratum corneum hydration, skin surface pH, and casual sebum content. Arch Dermatol. 1991;127:1806–1809. Roskos KV, Guy RH. Assessment of skin barrier function using transepidermal water loss: Effect of age. Pharmacol Res. 1989;6: 949–953. Mathias CGT, Wilson DM, Maibach HI. Transepidermal water loss as a function of skin surface temperature. J Invest Dermatol. 1981;77: 219–220. Marks R, Nicholls S, King CS. Studies on isolated corneocytes. Int J Cosmet Sci. 1994;3:251–258. Tagami H. Location-related differences in structure and function of the stratum corneum with special emphasis on those of the facial skin. Int J Cosm Sc. 2008;30:413–434. Cunliff WJ, Burton JL, Shuster S. The effect of local temperature variations on the sebum excretion rate. Br J Dermatol. 1970;83: 650–654. Le Fur I, Reinberg A, Lopez S, Morizot F, Mechkouri M, Tschachler E. Analysis of circadian and ultradian rhythms of skin surface properties of face and forearm of healthy women. J Invest Dermatol. 2001;117:718–724. Zlotogorski A. Distribution of skin surface pH on the forehead and cheek of adults. Arch Dermatol Res. 1987;279:398–401. Kligman AM, Balin AK. Aging of human skin. In: Balin AK, Kligman AM (eds) Aging and the Skin. New York: Raven, 1989, pp. 1–42. Smith L. Histopathologic characteristics and ultrastructure of aging skin. Cutis. 1989;43:419–424. Blume U, Ferracin I, Verschoore M, Czernielewski JM, Schaefer H. Physiology of the vellus hair follicle: hair growth and sebum excretion. Br J Dermatol. 1991;124:21–28. Hueber F, Wepierre J, Schaefer H. Role of transepidermal and transfollicular routes in percutaneous absorption of hydrocortisone and testosterone: In vivo study in the hairless rat. Skin Pharmacol. 1992;5:99–107. Otberg N, Richter H, Schaefer H, Blume-Peytavi U, Sterry W, Lademann J. Variations of hair follicle size and distribution in different body sites. J Invest Dermatol. 2004;122:14–19. Morganti P, Ruocco E, Wolf R, Ruocco V. Percutaneous absorption and delivery system. Clin Dermatol. 2001;19:489–501.
27. Chung JH, Yano K, Lee MK, Youn CS, Seo JY, Kim KH, Cho KH, Eun HC, Detmar M. Differential effects of photoaging vs intrinsic aging on the vascularization of human skin. Arch Dermatol. 2002;138: 1437–1442. 28. Tupker RA, Coenraads P-R, Pinnagoda J, Nater JP. Baseline transepidermal water loss (TEWL) as a prediction of susceptibility to sodium lauryl sulfate. Contact Dermatitis. 1989;20: 265–269. 29. Cua AB, Wilhelm KP, Maibach HI. Cutaneous sodium lauryl sulfate irritation potential: age and regional variability. Br J Dermatol. 1990;123:607–613. 30. Elsner P, Wilhelm D, Maibach HI. Irritant effect of a model surfactant on the human vulva and forearm. J Reprod Med. 1990;35: 1035–1039. 31. Frosch PJ, Kligman AM. A method for appraising the stinging capacity of topically applied substances. J Soc Cosmet Chem. 1977;28:197–209. 32. Seidenari S, Francomano M, Mantovani L. Baseline biophysical parameters in subjects with sensitive skin. Contact Dermatitis. 1998;38:311–315. 33. Besne´ I, Descombes C, Breton L. Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. 2002;138:1445–1450. 34. Schwindt D, Wilhelm K-P, Miller DL, Maibach HI. Cumulative irritation in older and younger skin: a comparison. Acta Derm Venereol (Stockh). 1998;78:279–283. 35. Faucher JA, Goddard ED. Interaction of keratinous substrates with sodium lauryl sulfate. J Soc Cosmet Chem. 1978;29:323–337. 36. Jiang SJ, Zhou XJ, Sun GQ, Zhang Y. Morphological alterations of the stratum corneum lipids induced by sodium lauryl sulfate treatment in hairless mice. J Dermatol Sci. 2003;32:243–246. 37. de Jongh CM, Verberk MM, Spiekstra SW, Gibbs S, Kezic S. Cytokines at different stratum corneum levels in normal and sodium lauryl sulphate-irritated skin. Skin Res Technol. 2007;13:390–398. 38. Agner T. Susceptibility of atopic dermatitis patients to irritant dermatitis caused by sodium lauryl sulphate. Acta Derm Venereol (Stockh). 1990;70:296–300. 39. de Jongh CM, Jakasa I, Verberk MM, Kesic S. Variation in barrier impairment and inflammation of human skin as determined by sodium lauryl sulphate penetration rate. Br J Dermatol. 2006;154: 651–657.
37 Stratum Corneum Cell Layers Hachiro Tagami
Introduction Despite its most important role in the skin as a barrier membrane, the structural intactness of the stratum corneum (SC) has rather been neglected in histological examination because of its disordered features when prepared in ordinary histological specimens. Although the SC is only an extremely thin membrane covering the skin surface, it plays the most important and vital role of the skin in maintaining life, a skin barrier preventing loss of water from the underlying fully hydrated living tissue even in an extremely dry atmosphere. Hence, it is natural that the SC can also protect the body from invasion by various injurious exogenous chemicals and microorganisms from the environment [1]. Moreover, it keeps skin surface smooth and soft by binding water even under very dry condition. These characteristics definitely require the structural intactness of the SC in vivo on the skin surface. The SC produced under pathologic conditions such as various lesional skins is not only deficient in the barrier function, but also presents a dry, scaly clinical appearance. Functional studies of the SC have demonstrated that even normal healthy skin shows a great difference in its water-holding capacity as well as in the barrier function depending on the anatomical locations [2]. For example, the face is often affected by steroid-induced dermatitis after inadvertent prolonged usage of potent topical steroids. In fact, the barrier function of the facial skin is much less capable than that of abdominal skin [3, 4]. However, it is hard to demonstrate these differences in an ordinary histologic specimen except for that of the palmoplantar skin, which shows closely packed layers of corneocytes even in the ordinary histologic preparations because of the strong adhesiveness of the corneodesmosomes between the corneocytes to construct a tough and thick SC structure.
The Uniqueness of the Palmoplantar Stratum Corneum The palmoplantar SC is remarkably thick to be able to resist even a strong external force. However, it exhibits
unbelievably poor barrier function as compared with the SC of other locations [5]. Recently developed Raman spectroscopy has revealed that, except for the narrow, deepermost portion close to the viable epidermis, most of its thick outer and mid portions show a lower hydration state in contrast to the thin SC found in other bodily locations [6]. Electron microscopic study demonstrated that, in contrast to the SC of other bodily portions, the plantar SC is accompanied by unique intercellular spaces occupied by corneodesmosomes rather than by the intercellular lipids that play an important role in providing the SC with its barrier function [7]. The palmoplantar skin is covered by a uniquely thick SC distinct from that of other locations to execute its specific function, to withstand the strong external physical forces even that which is required to sustain heavy body weight with their small surface areas. The mechanical role is more important than the biological role of barrier function here. Its barrier function should rather be poor enough to allow sufficient water from the deeply located viable epidermis to the skin surface to make the latter soft and flexible.
Differences in Barrier Function of the SC at Various Anatomical Locations Although the SC covering most parts of the skin surface is extremely thin, in general less than15 mm thick, they are far more efficient in its barrier function as well as in waterholding capacity than the palmoplantar SC [2, 5, 8]. In contrast to the latter, the SC covering most part of the body is highly rich in its unique intercellular lipids crucial for the barrier function [1]. However, they are easily extracted during the preparatory process required for the production of ordinary histologic specimens. The data obtained measuring percutaneous permeation of chemicals demonstrated a parallel variation with those data recorded in vivo measuring transepidermal water loss (TEWL). Such data strongly supports that TEWL can be employed as a parameter of skin barrier function [4]. TEWL takes place between the watersaturated living epidermis and the dry environmental
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_37, # Springer-Verlag Berlin Heidelberg 2010
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atmosphere through the SC under non-sweating condition, namely at the ambient condition of 20 C and 40–50% relative humidity. It can be used as an endogenous standard for the SC barrier function. Various types of scaly skin lesions such as those found in atopic dermatitis and psoriasis covered by pathological SC always reveal higher TEWL due to impaired barrier function [8]. However, even on the normal skin, in vivo measurement of TEWL clearly demonstrates great variations among various body regions, reflecting the differences in thickness as well as the degree of the structural integrity due to maturity of the SC. The facial SC shows the feature of immaturity in their composing corneocytes [9]. Usually, freshly produced immature corneocytes existing in the deepest SC portion are covered by a cornified envelope composed of protein components such as involucrin, loricrin, and small proline-rich proteins. However, as they become maturated with upward movement, they acquire hydrophobicity, because of covalent attachment of omegahydroxyceramides of the intercellular lipids to the extracelluar surface of CE. In fact, the SC covering normal facial skin shows rather rapid turnover similar to that noted in mildly irritated hyperproliferative epidermis. Scattered immature corneocytes can be detected even in the superficial SC cell layer of normal facial skin, but not on the normal skin covering the trunk and the limbs, where slow turnover of the SC takes place. These findings suggest that the corneocytes of the facial skin tend to desquamate even in the immature state to maintain its thin SC cell layer as compared to those of the volar forearm. Reflecting such immaturity of the corneocytes composing the facial SC, the TEWL values measured on the facial skin is more than 10 g/m2/h, being much higher than those of the forearm and upper arm whose TEWL is around 5 g/m2/h similar to those measured on the trunk and the lower extremity [5, 8]. Likewise, TEWL measured on the scalp [10] or on the axilla [11] is around 7 g/m2/h, while that on the female vulva reaches 25 g/m2/h, because the SC of the genital skin is the thinnest on the body [12]. Such a high level of TEWL corresponds to that of the acutely inflamed, eczematous facial lesions of atopic dermatitis [8]. The nape (12 g/m2/h) and elbow (11 g/m2/h) reveal unexpectedly high TEWL values, even though they are regarded to constitute a part of the trunk and the extremities. Both are the body locations characterized by frequent bending and stretching movement. In contrast, the skin of the knee covering another highly movable joint does not show any such high TEWL values. Likewise, the antecubital fossa or the popliteal fossa, the flexor surfaces
of the joint regions, does not show any specifically higher TEWL than other sites of the extremities, although they constitute one of the predilection sites for atopic dermatitis like the neck [8]. In contrast, despite the relatively high TEWL observed in the facial skin, its SC lipid content is not lower than that of the SC of other anatomical locations. Thus, it is presumed that their poor barrier function reflects their extremely thin SC structure [13, 14], as well as the unique rapid turnover speed that does not allow the sufficient maturation process for the corneocytes [9].
Differences in the Hydration State of the Stratum Corneum Composing the Skin Surface Because the lower portion of the SC can receive an ample supply of water from the underlying hydrated living epidermal tissue, it is the water content in the superficial portion of the SC, only several microns in depth, which keeps the skin smooth and soft. To evaluate such a skin surface hydration state, the high-frequency impedance measurement of the skin [15] is used conventionally. Between the two components of high-frequency impedance, i.e., conductance and capacitance, conductance is more suitable for the measurement of the hydrated state of the skin surface [15]. Thus, it is also fitted for the measurements of the efficacy of various moisturizers and skin care cosmetics. The pattern of the increase in highfrequency conductance with the depth of the SC also corresponds to the water distribution in the SC directly estimated by recently developed in vivo Raman spectroscopy [6]. There exists an exponential increase in water concentration from the skin surface to the fully hydrated viable epidermis [6]. Such a water gradient in the SC can also be observed with conductance measurements after serial stripping of the SC, but is difficult to demonstrate with capacitance measurements [15, 16]. In contrast, while capacitance measurements are less sensitive to evaluate the hydrated skin surface, they are more sensitive for the evaluation of dry skin conditions as noted in various skin diseases. Among various anatomical locations of the adult Japanese, the measurements of the skin surface hydration state demonstrated that high-frequency conductance values were highest on the anterior neck, amounting to 225 micro-Siemens (mS) on average, being followed by the nape of the neck with 123 mS as compared to 108 mS measured on the cheek [9]. While the seborrheic areas
Stratum Corneum Cell Layers
such as the cheek in adults definitely showed high skin surface lipid levels (117 AU) when measured with a Sebumeter (Courage & Khazaka, Cologne, Germany), the lipid levels were much lower on the frontal neck (44 AU) or the nape (21 AU). Such moderate sebum excretion alone does not seem to account for the remarkably high hydration state of the neck skin. Other flexural areas of the extremities such as the antecubital fossa with 91 mS and popliteal fossa with 85 mS were also relatively well hydrated. In fact, they rarely develop dry skin even in winter. Their hydration levels were significantly higher than those of the volar forearm with 47 mS or the calf with 33 mS (P < 0.001). In contrast, the extensor surfaces of the joint regions such the elbow (28 mS) and the knee (25 mS) were demonstrated to be poorly hydrated sites even compared with the volar forearm. In fact, they easily develop dry skin in children and elderly in dry and cold winter.
Materials and Methods for Visualizing the SC Cell Layer in Normal Skin Specimens For the demonstration of the cellular layer nature of the SC of other skin areas, Chistophers and Kligman [17] utilized frozen sections expanded in alkaline solutions, which allow such counting of the SC cell layer. However, in the past available data were based only on the findings in a small number of skin specimens that were obtained from a limited area of the body such as the trunk and the proximal extremities [18, 19]. Ya-Xian et al. [20] have also tried to use biopsy specimens of normal skin from various parts of the body including the face, scalp, genitals, and acral regions of the extremities, the locations where biopsy is not easy to perform in normal individuals. Hence, an uninvolved area of the surgical margin of excised skin tumors was used at various locations of the body of 301 Japanese patients, who had complaints of either benign or malignant tumors; the complaints led to an analysis of the relation of the SC cell layer to age, sex, and anatomical locations. In addition to these study samples, a small portion of normal skin specimens was included, which was obtained from certain areas such as the abdomen and anterior aspect of the thigh that frequently served as a donor site for a skin graft. As the nature of the sampling method depended totally on the chance of surgery, sufficient numbers of samples could not be collected for all the locations
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of the body to analyze the SC. However, the counting of the cell layers in the SC in a large numbers of samples was successfully completed; skin specimens were obtained from a total of 158 males and 143 females, ranging from 1 to 97 years of age with the mean age of 42 26 years. Preoperative procedures of the skin consisted of applying povidone-iodine with gauze, which was subsequently wiped off with gauze soaked in 0.02% chlorhexidine gluconate solution. The skin samples were frozen quickly and 6-mm-thick cryostat sections were prepared by cutting them in a plane perpendicular to the skin surface. The method to determine the number of cell layers in the SC is based on that reported by Christophers and Kligman [17] in principle and modified from that described by Blair [18]. In short, a cryostat section was at first stained with 1% aqueous solution of safranin for 1 min and then flooded with 2% potassium hydroxide (KOH) aqueous solution. The safranin produces clear reddened intensification of the intercellular portions of the SC even in the presence of the KOH solution. The number of swollen corneocyte layers over the epidermis was counted at several spots avoiding the sites of sweat pores and follicular ostium, to obtain the mean value [20]. The obtained data was statistically analyzed with Sheffe’s F procedure for comparisons of the SC cell layers at various locations, Mann-Whitney test for comparisons of functional data of the SC, and Fisher’s r to z transformations to check the correlation between age and the number of corneocyte layers.
Anatomical Differences in SC Cell Layer The typical features of histologic specimens consisted of a compact feature of the SC, which helped in easy counting of the number of flattened cell layers in the SC (> Fig. 37.1). The number of cell layers was smallest in the genital (both for the penis and the scrotum of the male genitalia and the vulva of the female genitalia) skin (6 2; n = 9), whereas it was between 10 and 20 in most locations of the trunk and the extremities. The obtained data are summarized for various anatomical locations of the body. In general, the SC of the face, neck, and scalp skin tended to be smaller than that of the trunk. Although they look similar, the SC of the extremities showed higher number of cell layers than the trunk (P < 0.01). The facial skin showed statistically significant smaller numbers of cell layers (9 2; n = 84) than those of the
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. Figure 37.1 Stratum corneum of the back (¥ 400)
. Table 37.1 Comparison of the number of cell layers of the SC at various anatomical locations Location Face
9 ± 2 (n = 84)
Forehead
9 ± 1 (n = 8)
Eyelid
8 ± 2 (n = 16)
Cheek
10 ± 3 (n = 43)
Nose
10 (n = 2)
Nasolabial fold Lip Ear Periauricular region
extremities (15 4; n = 55) (P < 0.05). From the smallest number found in the genital skin (6 2; n = 9), they could be put in order by placing the face (9 2; n = 84) next and then from the neck (10 2; n = 5), scalp (12 2; n = 12), trunk (13 4; n = 94), extremities (15 4; n = 55) to the acral regions, namely the palms and the soles (47 24; n = 42). Among them, the heel showed the largest numbers (86 36; n = 5) (> Table 37.1). The SC cell layers of the face, neck, and scalp skin tended to be smaller than that of the trunk. The facial skin showed statistically significant smaller numbers of cell layers (9 2; n = 82) than those of the extremities (15 4; n = 55) (P < 0.05). Even among the facial skin, there were some differences depending upon the location. The eyelid (8 2; n = 8), nasolabial fold (7; n = 2), and the ear lobe (7 2; n = 8) showed lower numbers. In contrast, the forehead (9 1; n = 8), nose (10; n = 2), cheek (10 3; n = 43), preauricular region (10 3; n = 30, and the lip (10; n = 2) exhibited somewhat higher numbers. The skin of the trunk showed similar SC cell layer numbers ranging from 12 4 (n = 20) on the buttock to 14 4 on the abdomen (n = 44). On the other hand, the extremities revealed some differences between those of the extensor upper arm (13 4; n = 13), flexor upper arm (14; n = 2), flexor forearm (16 4; n = 4), and extensor thigh (16 4; n = 31), and the flexor surface of the leg (18 5; n = 5). In contrast, it was remarkably thick in the distal portion of the extremities, such as the dorsa of the hands (25 11; n = 10) and those of the feet (30 6; n = 7) and particularly the palms (n = 50 10; n = 8) and the soles (55 14; n = 12). As mentioned above, the heel showed the largest numbers (86 36; n = 5).
Number of cell layers (mean ± SD)
7 (n = 2) 10 (n = 2) 7 ± 2 (n = 8) 10 ± 3 (n = 3)
Scalp
12 ± 2 (n = 12)
Neck
10 ± 2 (n = 5)
Trunk
13 ± 4 (n = 94)
Shoulder
13 ± 2 (n = 3)
Chest
13 ± 4 (n = 9)
Back
13 ± 3 (n = 18)
Abdomen
14 ± 4 (n = 44)
Buttock
12 ± 4 (n = 20)
Genital Extremities
6 ± 2 (n = 9) 15 ± 4 (n = 55)
Extensor surface, upper arm
13 ± 4 (n = 13)
Flexor surface, upper arm
14 (n = 2)
Flexor surface, forearm
16 ± 4 (n = 4)
Thigh
16 ± 4 (n = 31)
Flexor surface, leg
18 ± 5 (n = 5)
Acral region
47 ± 24 (n = 42)
Dorsum of the hand
25 ± 11 (n = 10)
Dorsum of the foot
30 ± 6 (n = 7)
Palm
50 ± 10 (n = 8)
Sole
55 ± 14 (n = 12)
Heel
86 ± 36 (n = 5)
Differences in SC Cell Layer due to Age, Sex, and Race In regard to age, their numbers could be compared at four different skin regions, i.e., the cheek, back, abdomen, and the anterior surface of the thigh, where sufficient numbers of samples were available for such analysis. The results
Stratum Corneum Cell Layers
. Figure 37.2 The number of corneocyte layers in the stratum corneum of the check versus age (Cited from [20])
showed a significant increase in the SC cell layers with increasing age only in the cheek skin of the males with the correlation coefficient of 0.67 (P < 0.05). Such a tendency was not found in the females. There is no clear-cut explanation for these findings. In the skin of the back, the SC cell layers showed an increased with age both in male and female individuals (r = 0.63; P < 0.05). However, the increase was much more prominent in males than in females (> Fig. 37.2). By contrast when the skin from elderly individuals with senile xerosis was studied mainly on the extensor surface of the legs in winter, there was a significant increase in the SC cell layers in those with senile xerosis versus the young healthy individuals [21]. A comparative study was conducted between different sexes at the sites where such comparison was possible. Surprisingly, there was no significant difference in the number of corneocyte layers due to sex. Although there was a great individual difference in the number of cell layers in the SC even from the same location, in general, the skin of the trunk and that of the extremities of the Japanese are covered by the SC with relatively similar numbers of cell layers ranging from 10 to 20. These numbers generally agree well with those reported previously using a small number of samples from other countries [17–19], ruling out the influence of racial difference.
Comparison of the Number of SC Cell Layer with Functional Properties of the SC In Vivo Finally, when a comparison of these histologic data of the SC with its functional data, the values of in vivo
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measured TEWL, was made, the latter seemed to correlate with the number of the SC cell layer except for the palmoplantar SC. The obtained TEWL values were close to each other in representative areas of the trunk and the extremities, where the numbers of cell layers of the SC were also close to each other. By contrast, they were remarkably high on the face as compared with the trunk and limbs, being double to triple on the cheek and the eyelid. In contrast, using high-frequency conductance, the parameter of the water content in the outer SC did not show any correlation with the number of the SC cell layers. There is a water concentration gradient from the skin surface exposed to the dry atmosphere to the lowermost layer of the SC facing the hydrated epidermal tissue [6]. Thus, when the SC is serially removed with adhesive cellophane tape stripping, there occurs a gradual increase in high-frequency conductance [15]. Although their SC cell layers were almost comparable in number, the hydration values were statistically higher on the back than on other portions of the trunk and limb (P < 0.05) [20]. Hence, it is likely that the water content of the outer SC is influenced not only by the thickness of the SC, but also by other factors such as sweat and sebum secretion, as well as by the physicochemical properties of the SC themselves [22]. It is well known that even a simple application of effective moisturizing agents produces a prominent increase in water content of the SC without any change in the number of SC cell layers. In fact, on the facial skin covered with thin SC layers, the site rich in skin surface lipids such as the nose tends to show significantly higher conductance levels than other regions [23]. Moreover, the areas covered with larger mature corneocytes such as the eyelid skin reveal higher conductance than those sites covered by smaller immature corneocytes such as the cheek, nasolabial fold, and forehead [24]. The size of the corneocytes conversely correlates with the turnover speed of the SC [25].
Clinical Implications From the obtained data, it is clear that the genital skin and certain parts of the facial skin such as the eyelid, ear, and nasolabial portion showed small numbers of SC cell layers. These findings seem to explain well that these skin regions are sensitive to topical applications of irritants or lipid solvents. These areas are also the sites often affected by contact dermatitis [26]. The frequently experienced, but poorly understood intolerance of the face or
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neck skin to various topical formulations [27, 28] can also be in part understood from their relatively thin corneocyte layers. Such skin areas with thin SC are also the predilection sites for atopic dermatitis, contact dermatitis, and seborrheic dermatitis, because they are not only rich in skin appendages but also are more permeable to various environmental substances including irritants, haptens, aeroallergens, and microorganisms. Moreover, these findings also correspond well to the experimental observations that the barrier function of the scrotal skin, face, and neck is much less effective than that of abdominal skin [3], although no obvious histological differences can be found in ordinary histology specimens. The face is also the site where the development of steroidinduced dermatitis is often observed clinically, because even uninvolved skin allows penetration of ample amounts of potent steroids as compared to that of the trunk and extremities.
Conclusion To count the number of cell layers in the SC of normal skin taken from different anatomical locations of the body, normal skin samples were collected from 301 individuals with various ages. Frozen sections of 6 mm thickness that were stained with 1% safranin aqueous solution and observed under microscope after application of 2% KOH solution showed that there were great variations in the SC cell layers (mean SD) according to the location and among different individuals. The smallest number was found in the genital skin (6 2), being followed in order by the face (9 2), neck (10 2), scalp (12 2), trunk (13 4), extremities (15 4), and the acral regions (47 24). Among them, the heel showed the largest numbers (86 36). No definite correlation of the number of corneocyte layers was found with sex of the individuals, whereas there was a slight increase in the number with age in the skin of the cheek and back, particularly in males. Comparison of these data with those of functional assessment of the SC showed that TEWL, a parameter of SC barrier function, reflects the number of corneocyte cell layers. In contrast, high-frequency conductance, a parameter for the hydration state of the outer SC, does not seem to be influenced only by it. As compared with other methods that have been utilized to measure the thickness and numbers of cell layers in the SC, the technique used in the present study is simple and not time-consuming, that enables the study of a large number of skin samples. It does not require any
wide laboratory space or special facilities except for an ordinary microscope and cryostat.
Cross-references > Corneocyte
Size and Cell Renewal: Effects of Aging and Sex Hormones > The Stratum Corneum and Aging
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Stratum Corneum Cell Layers 16. Hashimoto-Kumasaka K, Takahashi K, Tagami H. Electrical measurement of the water content of the stratum corneum in vivo and in vitro under various conditions. Comparison between skin surface hygrometer and corneometer in evaluation of the skin surface hydration state. Acta Dermatol Venereol (Stockh). 1993;73:335–339. 17. Christophers E, Kligman AM. Visualization of the cell layers of the stratum corneum. J Invest Dermatol. 1964;42:407–409. 18. Blair C. Morphology and thickness of the human stratum corneum. Br J Dermatol. 1968;80:430–43. 19. Holbrook KA, Odland GF. Regional differences in the thickness (cell layers) of the human stratum corneum: an ultrastructural analysis. J Invest Dermatol. 1974;62:415–422. 20. Ya-Xian Z, Suetake T, Tagami H. Number of cell layers of the stratum corneum in normal skin – relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291:555–559. 21. Hara M, Kikuchi K, Watanabe M, Denda M, Koyama J, Nomura J, Horii I, Tagami H. Senile xerosis: functional, morphological, and biochemical studies. J Geriatr Dermatol. 1993;1:111–120. 22. Egawa M, Tagami H. Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm
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skin: effects of age, seasonal changes and artificial forced hydration. Br J Dermatol. 2008;158:251–260. Kobayashi H, Tagami H. Distinct locational differences observable in biophysical functions of the facial skin: with special emphasis on the poor functional properties of the stratum corneum of the perioral region. Int J Cosmet Sci. 2004;26:91–101. Pratchyapruit W, Kikuchi K, Gritiyarangasan P, Aiba S, Tagami H. Functional analyses of the eyelid skin constituting the most soft and smooth area on the face: contribution of its remarkably large superficial corneocytes to effective water-holding capacity of the stratum corneum. Skin Res Technol. 2007;13:169–175. Ho¨lzle E, Plewig GJ. Effects of dermatitis, stripping, and steroids on the morphology of corneocytes. A new bioassay. J Invest Dermatol. 1977;68:350–356. Ockenfels HM, Seemann U, Goos M. Contact allergy in patients with periorbital eczema: an analysis of allergens. Dermatology. 1997;195:119–124. Shriner DL, Maibach HI. Regional variation of nonimmunologic contact urticaria. Functional map of the human face. Skin Pharmacol. 1996;9:312–321. Berardesca E, Fluhr JW, Maibach HI. Sensitive Skin Syndrome. New York: Taylor & Francis, 2006.
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8 The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function Sandy Sercu . Noritaka Oyama . Joseph Merregaert
Introduction Historical Background: Discovery of the Extracellular Matrix Protein 1 Gene and its Transcripts The extracellular matrix protein 1 (Ecm1) was identified in 1994 as a novel glycosylated 85-kDa protein secreted in the conditioned medium of the murine osteogenic stromal cell line, MN7 [1]. It was discovered amidst various connective tissue proteins including collagens, osteonectin, and bone sialo protein, and was therefore named Ecm1, although its potential relevance to extracellular matrix physiology was not immediately apparent. The mouse Ecm1 gene has been characterized further by cloning and sequencing of its cDNA, analysis of its expression pattern, and its genomic localization [2]. The 5 kb long Ecm1 gene maps to chromosome 3 and encodes for two distinct splice variants: a complete cDNA clone, Ecm1a, with an open reading frame of 1,677 bp that encodes for a protein of 559 amino acids (aa) (11 exon gene) and a shorter alternatively spliced Ecm1b (lacks exon 8) mRNA of 1.5 kb, coding for a protein of 434 aa [2, 3]. Thereafter, the human ECM1 gene was isolated in 1997 and mapped to chromosome 1q21 [4, 5]. Comparison in the plane structure between the mouse and the human ECM1 gene [4] reveals that the human gene contains one exon less than the mouse gene, that is, the sequence homologous to the sixth and shortest mouse exon [4]. The 5’-upstream regulatory sequences of the mouse Ecm1a gene contains putative binding sites for GATA, Sp1, AP1, and the Ets family of transcription factors. Comparing this region to the equivalent portion of the human gene reveals a strong conservation. The potential AP1 and Sp1 sites are perfectly conserved, while the potential Ets site only differs in one nucleotide between both species, with the human sequence conforming even better to the consensus sequence for this transcription factor family.
The Sp1, AP1, and Ets factor-binding regions are necessary for the expression of Ecm1 in the MN7 cell line, while the potential GATA site on the other hand is not conserved between human and murine ECM1 genes and is therefore functionless in the former [3]. The human ECM1 gene (> Fig. 8.1) encodes for four splice variants: the full-length transcript ECM1a (1.8 kb, 540 aa), which is widely expressed in liver, small intestines, lung, ovary, prostate, testis, skeletal muscle, pancreas, kidney, placenta, heart, basal keratinocytes, dermal blood vessels, and adnexal epithelia including hair follicles and sweat glands [4, 6–9]; ECM1b (1.4 kb, 415 aa), which lacks exon 7 and is detectable in tonsils and the spinous and granular layers of the epidermis [6]; ECM1c (1.85 kb, 559 aa), which has an extra exon 5a within intron 5 and is known to be expressed in the basal layer of the epidermis [9]; ECM1d, the fourth splice variant, results in a truncated protein of 57 amino acids for which the biological relevance is still unclear (> Fig. 8.1a) [10].
The Protein Structure of ECM1 The ECM1 protein has abundant cysteine residues (4.8%) with a specific distribution according to the CC-(X7–10)-C pattern of six cysteine doublets [2]. This cysteine arrangement, which is also found in serum albumin and the sea urchin Endo16 protein, may determine the formation of double-loop structures, specifying putative, important biological protein–protein interactions [2]. ECM1 comprises five different regions: a 19 aa signal sequence for extracellular secretion, an NH2-terminal cysteine-free domain that is rich in prolines and glutamines, two tandem repeats, and a COOH-terminal region [2, 4] (> Fig. 8.1a). ECM1a contains three double-loop domains, one present in each of the two central tandem repeats and one in the COOH-terminal domain. It has been hypothesized that the tandem repeat structure could allow the ECM1
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_8, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 8.1 Gene and domain organization of the human ECM1 gene: (a) Schematic representation of ECM1 and its four splice variants. Full-length ECM1 is divided in a signal sequence (black box) and four different domains based on the presence or absence of cysteines: an N-terminal cysteine-free domain (white box), two tandem repeats (green and gray box), and a C-terminal region (blue box). Full-length ECM1c differs from ECM1a containing 19 aa encoded by an additional exon 5a, ECM1b results from an alternative transcript caused by splicing out exon 7 (shaded black), and ECM1d encodes for a truncated protein composed of 57 amino acids containing exon 1, exon 2, and a part of exon 3. The amino acid positions that mark the ends of each region are indicated. (b) A schematic representation of the ECM1 genomic organization. Boxes represent exons and thin lines represent introns. The positions of exon 1 (aa1–aa24), exon 2 (aa25–aa41), exon 3 (aa42–aa75), exon 4 (aa76–aa102), exon 5 (aa103–aa129), exon 6 (aa130–aa236), exon 7(shaded gray: aa237–aa361), exon 8 (aa362–aa434), exon 9 (aa435–463), and exon 10 (aa464–aa540) of ECM1 in the domain structure are indicated by dotted lines. (c) A computationally predicted three-dimensional structure of ECM1a. Based on the third serum albumin domain ECM1a can be divided into four domains. The first domain containing a-helices (aD1) and three serum albumin subdomain-like domains (SASDL 2–4), each of three sequences comparable with a complete subdomain of the third serum albumin domain. aD1 exists only of a-helices, whereas SASDL2 and 3 are capable of binding most of the extracellular matrix proteins (Adapted with permission from Sercu, S. et al. [39])
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function
protein to function as a transport protein or to be involved in binding of various regulatory factor(s) [2]. The three cysteine-rich domains (two tandem repeats and COOH-terminus) are the most conserved among species; the human pattern is 75% identical to that of the mouse, whereas the overall identity between the two species reaches 69.4%. This indicates that this unique pattern of cysteine arrangements is critical in maintaining the in vivo structural stability of the molecule. More recently, a rudimentary three-dimensional model was predicted using the third human serum albumin domain as template [11], and divided the ECM1a protein into four distinct domains: an NH2-terminal domain forming probably a-helical structures (aD1: aa20–aa176), followed by three domains, whose amino acid sequences were highly comparable with the third domain of human serum albumin (Serum Albumin Subdomain Like 2 [SASDL2]: aa177–aa284; SASDL3:aa284–aa431, and SASDL4: aa432–aa540] (> Fig. 8.1c) [11]. More specifically, the last three domains contain a number of typical C–CC–C motifs, which could form disulfide bonds, sharing putative double loops. It is possible that these motifs also give rise to ‘‘finger-like’’ structures, which provide the fatty acid-binding clefts in serum albumin [12]. This characteristic configuration of ECM1a is conserved between species [2, 4], and also between the splice variants ECM1a–c. This emphasizes the importance of the overall structure and its putative significance in protein–protein interaction. Furthermore, a striking observation for the biological importance of the SASDL subdomains in protein interaction is supported by loss-of-function mutations in the ECM1 gene as the cause of a rare autosomal recessive genodermatosis lipoid proteinosis also known as hyalinosis cutis et mucosae or Urbach-Wiethe disease (OMIM 247100)[13, 14] (see Sections ‘‘Binding Partners of ECM1’’ and ‘‘Human Disease Model for Skin Aging’’). Interestingly, most of the pathogenic mutations are nonsense changes and occur frequently within exon 6 and the alternatively spliced exon 7 of ECM1 (reviewed in [15]), coding for SASDL2 and a part of the SASDL3 domain(> Fig. 8.1b).
Biological Functions of ECM1 The biological function of ECM1 is not elucidated yet, but indications for its involvement in important biophysiological processes, like skin differentiation [6], endochondral bone formation [16], and angiogenesis [17] have now emerged the following multipotent actions.
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Role of ECM1 in Endochondral Bone Formation The first indication of a putative role of ECM1 during endochondral ossification was provided by a study of Deckers and coworkers [16]. In situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) analysis on 16 dpc old murine, embryonic metatarsals, which still consist of cartilage, revealed that the Ecm1a transcript and protein were present in the connective tissue surrounding the cartilage, but not in the cartilage itself. Exogenously added human recombinant ECMla had a biphasic effect on cultured 15-day-old fetal mouse metatarsals and stimulated alkaline phosphatase activity, a marker of chondrocyte differentiation, without stimulation of the mineralization when present at low concentration (0.25 ng/ml). In contrast, higher concentrations of recombinant ECM1a protein inhibited both alkaline phosphatase activity and mineralization in a dose-dependent manner [16]. It is therefore probable that ECM1a has a paracrine and/or juxtacrine action in the regulation of endochondral bone formation. These observations have recently been confirmed by the identification of Cartilage Oligomeric Matrix Protein 1 (COMP) as a binding partner of ECM1 (Dr. C. Liu, personal communication 2009). COMP was able to overcome the inhibitory effect of ECM1 on matrix mineralization of mouse metararsals in vitro. This effect is interaction dependent since COMP largely fails to overcome the ECM1 inhibition in the presence of the epidermal growth factor (EGF) domain of COMP that disturbs the association between COMP and ECM1. These findings provide the first evidence that endochondral bone formation is mediated by a functional interaction between both molecules. Furthermore, both in vitro [18] and gene targeting studies have shown that a major heparin sulfate proteoglycan perlecan affects chondrogenesis and endochondral ossification. Perlecan null mice develop exencephaly and chondrodysplasia with severe disorganization of the columnar structures of the chondrocytes and defective endochondral ossification. Part of the morphological changes observed in the perlecan null mice has been ascribed to extracellular matrix disorganization [19]. Because disruption of these two molecules leads to similar effects, it is possible that the interaction of perlecan with ECM1 is also important for the process of biomineralization. All together, these findings support the notion that the interaction of ECM1 with extracellular matrix proteins like COMP and perlecan are vital in the spaciotemporal regulation of endochondral bone growth.
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The Role of ECM1 in Angiogenesis and Tumor Progression At this moment, the exact role of ECM1 in tumor development and progression is not known. Addition of purified, recombinant human ECM1a/b stimulates the proliferation of endothelial cells in vitro and promotes the formation of new blood vessels in the chorio-allantoic membrane of chicken embryos [17]. Since ECM1 is capable of binding with domain V of perlecan (endorepellin), an angiogenesis inhibitor, it is possible that the binding of ECM1 with endorepellin inhibits the anti-angiogenic function of this protein (> Fig. 8.2). Another possibility is that binding of ECM1 with matrix-metalloproteinase-9 (MMP-9) inhibits the proteolytic function of this enzyme
on perlecan interaction so that endorepellin cannot be released into the skin matrix [20]. On the other hand, binding of ECM1 with MMP-9 can also inhibit its proteolytic function on tumstatin and TGF-b. This could lead to a decrease in circulating tumstatin, which accelerates tumor growth (> Fig. 8.2). It is well known that MMP family members play a critical role in cancer invasion and metastasis. ECM1 might be a novel target to prevent overproduction of MMP in cancers. Another binding partner of ECM1 is fibulin-1C and fibulin-1D [21]. It is possible that binding of ECM1 with fibulin-1D can inhibit its decelerating action on tumor transformation of fibrosarcoma cells, which will strengthen their invasive potential (> Fig. 8.2). Although to date there have been no clear
. Figure 8.2 The role of ECM1 in angiogenesis and tumor growth: Binding of ECM1 with domain V of perlecan could inactivate endorepellin, which would stimulate angiogenesis. Binding of ECM1 with MMP-9 might inactivate the proteolytic activity of MMP-9 [20], which could inhibit the release of endorepellin, tumstatin, and TGFb resulting in activation of an angiogenic and anti-angiogenic pathway. Binding with fibulin-1D results in the feedback deactivation of this protein, which will lead to the activation of angiogenesis and cell proliferation [21]. ! Binding with, ! activation, ! inhibition, – -> possible activation (From Sercu, S. et al. [22]. With permission)
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function
correlation between ECM1 expression and the degree of angiogenesis, the association of ECM1 with different tumor origins has a number of implications. Firstly, the biological activity of ECM1 suggests that endothelial cells may have ECM1 receptors on their cell surface. Secondary, ECM1 may represent an easily accessible marker for the presence of tumors. Because ECM1 is a secretory glycoprotein, the concentrations of this protein in the patients’ serum may be correlated with the presence of certain types of tumors, like breast, lung, laryngeal, colon, or thyroid cancers. Such a marker could prove to be beneficial for assessing tumor load and may be of prognostic value in determining tumor recurrence after surgery and during follow-up stage. The role of ECM1 in cancer biology has recently been reviewed by Sercu and coworkers [22].
Role of ECM1 in Skin The human ECM1 gene maps to a region on chromosome 1q21.2 centromeric to the epidermal differentiation complex (EDC), containing genes that fulfill important functions in epidermal differentiation. ECM1 may thus also have a role in terminal keratinocyte differentiation. The full-length ECM1a transcript is expressed in cultures of normal human keratinocytes irrespective of their differentiation state. In contrast, the expression of the shorter, alternatively spliced ECM1b transcript is restricted to keratinocyte cultures undergoing late phase of differentiation, as assessed by the early (keratin 10) and late (involucrin) markers of epidermal differentiation [6]. In line with these observations, it was seen that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which efficiently induces terminal keratinocyte differentiation in vitro, is a potent inducer of ECM1b mRNA expression. TPA activates the protein kinase C (PKC) signaling pathway that has been shown to mediate cell-density-induced keratinocyte differentiation. It is interesting to note here that the PKC pathway leads, via action of MAP kinases, to the activation of the AP1 and/or the Ets family of transcription factors. Functional binding sites for AP1 and Ets transcription factors are present in the mouse Ecm1 promoter region [3]. The activation of these transcription factors by stimulation of the PKC pathway, either by increased cell density or TPA, is most probably responsible for the increased ECM1a expression in the keratinocyte cultures. The appearance of the ECM1b transcript in the stimulated cultures also indicates that the PKC pathway might result in the induction or activation of differentiation-dependent splicing factors of the ECM1 gene, because of the biphasic expression of ECM1a/b in
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human skin. ECM1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers and low expression in terminally differentiated keratinocytes, while ECM1b mRNA is present in the differentiated keratinocytes [6].
ECM1 is a Protein Expressed in the Basement Membrane and the Epidermal Basal Layer in Skin Extracellular matrix proteins, like ECM1, play an important role in the extracellular matrix formation, cell adhesion, cell signaling, and regulation of tissue differentiation and/or maturation. This is exemplified by the identification of loss-of-function mutations in the ECM1 gene as the cause of lipoid proteinosis [13]. This disease is characterized by generalized thickening of the skin and mucosal infiltration with scarring. Histologically, these patients show vascular anomalies, which represent severe functional defects, caused by an excessive deposition of hyaline-like (glassy) material (like collagen IV and laminin), that presumably lead to the disruption/reduplication of the basement membrane and the underlying dermal blood vessels [23]. These abnormalities suggest that ECM1 might have a regulatory role in maintaining skin integrity. Autoantibodies against ECM1 protein were detected in the inflammatory mucocutaneous disorder lichen sclerosus, characterized by fragility and hyalinization of the upper dermis [7]. Further clues to the physiological role of ECM1 in skin have been suggested by the discovery of interactions with the proteins; perlecan [9], MMP-9 [20], fibulin-1C/1D [21], fibulin-3 [11], laminin 332 and collagen IV [24], and polysaccharides (e.g., HA, heparin, and CSA), using different binding sites (> Fig. 8.3) [24].
Binding Partners of ECM1 ECM1–protein interaction partners have been identified by functional genetic screens based on the yeast twohybrid methodology or enzyme-linked immunosorbent assay (ELISA)-based methods, while ECM1–carbohydrate interactions were established through in vitro binding experiments.
Perlecan The COOH-terminus of ECM1 has been shown to bind specifically with epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V [9].
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. Figure 8.3 Summary of the different binding partners of ECM1 in the dermal–epidermal junction: (a) Site-specific binding of ECM1a to different hemidesmosomal and extracellular matrix molecules. ECM1a is capable of binding different extracellular matrix proteins with different binding sites. It is known that domain V of perlecan interacts with the C-terminus of ECM1 (aa424– aa540), while fibulin-1C/D and MMP-9 bind to the tandem repeat region of ECM1 (aa236–aa361). Also laminin 332 and fibulin-3 are capable of binding ECM1 at the tandem repeat region (aa207–aa340) within ECM1. Collagen IV that interacts with the NH2-domain of ECM1 (aa14–aa207) interacts also with laminin 332, and these interactions could be enhanced by ECM1. (b) The basal keratinocytes in human skin are finely anchored to the underlying superficial dermal components, forming hemidesmosomes. Some of these anchoring proteins are secreted by keratinocytes as well as fibroblasts like the heparinbinding perlecan or fibronectin. Other ECM1-binding proteins are specifically localized in the lamina lucida (like laminin 332, fibulin-1, and fibulin-3) or in the lamina densa (like collagen type IV) of the basement membranes. Typical dermal proteins are MMP-9, chondroitin sulfate A and hyaluronic acid. The remaining proteins that are thought to be currently uninvolved in ECM1 interaction(s), that is, bullous pemphigoid antigens I and II, and integrins, were omitted (Adapted from Sercu, S. et al. [24])
Perlecan is an intrinsic component of basement membranes and is also involved in the binding with interstitial dermal components, such as fibronectin, laminins, collagen IV, fibulin-2, and heparin, which with the exception of fibulin-2 are reported binding partners of ECM1. These interactions may play a role in the extracellular matrix assembly around the dermal–epidermal junction.
MMP-9 MMP-9 is a proteolytic enzyme, which has a catalytic activity against several extracellular matrix components. The tandem repeat domains of ECM1 can bind with MMP-9 in vitro resulting in a negative regulatory effect on the enzymatic activity, as assessed by a gelatin-based
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function
ELISA [20]. The formal proof whether these interaction is physiologically relevant awaits further in vivo experimentation.
The Fibulins Domain III of fibulin-1C/-1D is capable of binding with the second tandem repeat of ECM1 [21], while fibulin-3 binds with low affinity to the tandem repeat region of ECM1a [11]. Fibulin-1 and fibulin-3 are members of an extracellular matrix protein family, participating in embryonic development, wound repair, and carcinogenesis. Fibulin-1 is expressed in various basement membranes in many organs, as was found in skin. Based on the expression pattern, the binding of both proteins will be restricted to the epidermal basal layer. Interaction problems between both proteins could contribute to the pathophysiological phenotype of lipoid proteinosis, characterized by reduplication of the basal lamina in skin basement membrane. Recently, an angiogenic role of fibulin-3 has been suggested by inhibition of endothelial cell proliferation, invasion, and angiogenic sprouting, as well as p38 MAPK activation in vascular endothelial growth factor (VEGF)stimulated endothelial cells [25].
Laminin 332 and Collagen IV The tandem repeat and the NH2-terminal parts of ECM1a interact with the b3 chain of laminin 332 and collagen IV, respectively [11]. Laminin 332 is a large heterotrimeric glycoprotein consisting of three chains (a3b3g2). It is a component of the epithelial cell basement membrane, which functions as a ligand for the a3b1 and a6b4 integrins to regulate cell adhesion, migration, and morphogenesis. It forms a building block of the anchoring filaments, connecting hemidesmosomes with lamina densa in skin basement membrane zone. Laminin 332, broadly expressed in skin and most other epithelia, plays a tissue-specific role in the regulation of tumors derived from different tissues. Downregulation of laminin 332 may contribute to the structural failure in forming anchoring filaments and hemidesmosomes, resulting in disturbance of a stable epithelial–stromal junction, possibly allowing a local invasion of tumor cells into the adjacent tissues. A functional loss of laminin 332 has been known to cause junctional epidermolysis bullosa, a lethal and/or incurable blistering genodermatosis [26]. In contrast, circulating autoantibodies against laminin 332 also cause
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an immuno-bullous disease, mucous membrane pemphigoid, that primarily affects mucous membranes leading to a scarring phenotype [27]. These findings indicated that laminin 332 plays a vital role in epidermal–dermal assembly[28]. The results from the different localization studies support the possible in vivo interrelationship between collagen IV and laminin 332. Surprisingly, most signals for ECM1 colocalized with those for collagen IV or laminin 332, as well as perlecan, suggesting that ECM1 is exclusively involved in part of two independent suprastructure networks, containing either laminin 332 or collagen IV, at the basal lamina. Laminin 332 and collagen IV are two pivotal structural proteins in the skin basement membrane, facilitating the epidermal–dermal assembly; the laminin 332 is a major anchoring molecule at the base of anchoring filaments in the upper lamina densa/lamina lucida of the basement membrane, while the basal membrane is mainly composed of collagen IV within the basal lamina (> Fig. 8.4). Interestingly, binding experiments reveal that laminin 332 interacts with collagen IV and that ECM1a is able to enhance this binding [24]. Recently, McKee and colleagues showed that type IV collagen recruitment into the laminin 111 extracellular matrices appears to be mediated through a nidogen bridge and a lesser contribution arising from a direct interaction with laminin 111 [29]. They also suggested that collagen IV binds other laminins. Since collagen IV is linked to laminin 332 through ECM1a, which binds to the two proteins and facilitate their interaction, it is possible that ECM1a functions as a bridging core stabilizing these two hemidesmosomal molecules, thereby linking them to each other. However, definitive biochemical proof of a bridging mechanism is still lacking.
ECM1 is Not Essential for Epidermal Differentiation The expression of ECM1a was found in the epidermal basal layer, in dermal blood vessels, the outer root sheath of hair follicles, and in sebaceous lobules and epithelium of sweat glands, while ECM1b was expressed in the suprabasal layers of the epidermis. ECM1 is also capable of binding with perlecan, an important intrinsic molecule in skin organogenesis [9]. These observations suggest a crucial role for ECM1 in epidermal differentiation [6]. However, in vitro and in vivo experiments revealed no essential role for ECM1 in the terminal differentiation process of epidermal keratinocytes [30]. Presence or absence of ECM1a/b in keratinocytes from patients with
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. Figure 8.4 Colocalization of ECM1 with laminin 332 and collagen type IV in the basal layer of the epidermis: 2-week-old air-exposed organotypic keratinocyte monocultures (a), or cocultures (b–d), were stained for antibodies against ECM1 (OAP 12516). Colocalization (orange, arrows) of ECM1(green) with laminin 332 (red), and collagen type IV (red) in the basal cell layers. Scale bar: 50 mm (Adapted from Sercu, S. et al. [24])
lipoid proteinosis did not lead to differences in epidermal differentiation. In addition, overexpression of Ecm1a in vivo did not affect the stratified epidermal structure [30]. It is therefore feasible that ECM1 is dispensable for terminal differentiation of keratinocytes in unstressed conditions. However, it remains possible that in skin under certain exogeneous stress (UV irradiation or traumatic injury), ECM1 will still be necessary for the maintenance of skin homeostasis. Instead of a role in ECM1 splicing-dependent differentiation processes in the skin, differences in expression pattern of ECM1b compared with ECM1a could affect a more structural function. The ECM1a protein contains a typical CC-(X7–10)-C arrangement, which is responsible for the generation of ‘‘double-loop’’ domains that are involved in ligand
binding. Full-length ECM1a has three serum albumin subdomains, while ECM1b contains only two subdomains [11]. The elimination of one serum albumin subdomain in ECM1b may be responsible for exposing different binding regions, capable of binding other extracellular matrix molecules than ECM1a, necessary for the integrity of the suprabasal layers of the skin. Another indication for a putative structural role of ECM1b is the presence of two N-glycosylation sites (aa338 and aa410) modified with high mannose-type oligosaccharides [31]. The evidence that murine epidermal glycoproteins are actually modified with high mannose-type oligosaccharides in contrast with dermal glycoproteins indicates that epidermal cells project high mannose glycans to the cell surface and thus may be involved in molecular recognition events. This might be related to the role of lysosomal
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function
enzymes and/or high abundance of lamellar granule in the epidermis or other processes involved in the terminal differentiation of keratinocytes with a possible participation of Ecm1b. Ecm1b was found to be upregulated by a diffusible factors secreted by stromal cells. Furthermore, the induction and upregulation of Ecm1b RNA expression is dependent on the capacity of the cells to terminally differentiate [6].
ECM1 Plays an Important Role in the Maintenance of the Dermal–Epidermal Communication After cloning of the ECM1 gene, its expression in human tissues and its pathogenic role in relevant human diseases such as lipoid proteinosis and lichen sclerosus have been demonstrated [4, 7, 13]. However, the underlying mechanisms through which the ECM1 dysfunction leads to a warty epidermal thickening, scarring, and dermal hyalinosis are not well understood. Recently, it has been shown that ECM1a/c expression, in addition to its expression in the epidermal basal layer, was also found in the network-like suprastructures of the skin basement membrane, containing laminin 332 and collagen type IV [24]. The skin basement membrane at the dermal–epidermal junction has an important function in tightly linking the epidermis to the underlying dermis, and providing a barrier to epidermal migration. Once the basement membrane has been assembled, stratification of the epidermis proceeds, with the proliferating cells attached to the basement membrane and the daughter cells migrating into the upper layers. The importance of ECM1 in linking the dermal–epidermal junction has been emphasized by its ability to bind important extracellular molecules of the basement membrane zone (> Fig. 8.3). ECM1 is capable of binding laminin 332 and collagen IV and it also accelerates the binding between laminin 332 and collagen IV themselves. Therefore, ECM1 was suggested to act as ‘‘biological glue’’ to maintain the integrity of the skin [24]. However, the molecular mechanism underlying interactions of these molecules would open a new view in understanding the skin pathophysiology relevant to ECM1 function, and further encourages to study these aspects in more detail. The ‘‘biological glue’’ theory has been supported by evidence that the different binding regions within ECM1 can interact with various extracellular matrix molecules in the skin. The importance of these binding regions was further illustrated by the presence of autoantibodies in the sera from lichen sclerosus patients, whose circulating IgGs are reactive with
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NH2- and COOH-ECM1 [32]. These antibodies may attack the interaction of ECM1 with its interacting partners, which could be the cause of further intense damage to ECM1–matrix assembly in the skin. The discovery of the important binding regions of ECM1 might be a starting point of understanding the heterogeneous reactivity of antibodies with ECM1 in lichen sclerosus sera. Furthermore, the role of ECM1 in dermal–epidermal expression has also been demonstrated by organotypic keratinocyte monocultures treated with diffusible growth factors, like epidermal growth factor (EGF) and granulocyte-monocyte-colony stimulating factor (GM-CSF) secreted by stromal cells (authors’ unpublished data). Upregulation of ECM1 expression in the epidermis after the addition of these growth factors indicates dermal–epidermal interaction through dermal fibroblasts, a source for the synthesis and deposition of this molecule. Interaction can be based upon (1) production of soluble factors by either epithelial (after being activated by, e.g., growth factors) or mesenchymal cells that exhibit autocrine and/or paracrine activities, (2) cell–matrix interactions, or (3) signaling by direct cell–cell contact. Through the release of interleukin 1 (IL-1), keratinocytes can enhance release of growth factors such as GM-CSF, [32] IL-6, or IL-8 [33, 34] in dermal cells, which in turn stimulate basement membrane formation. EGF is a strong stimulus for MMP-9 production, which plays an important role in keratinocyte migration and granulation tissue remodeling. It is possible that EGF stimulates the induction of MMP-9, which is capable of interacting with ECM1. This interaction could be necessary for a normal wound healing. Absence or even disturbance of ECM1 action, like in lipoid proteinosis patients and lichen sclerosus patients, causes the ablation of this sequential molecular binding, leading to the skin pathology exposed to mechanical friction [15].
Lessons from Lipoid Proteinosis and Lichen Sclerosus in Skin Aging Human Disease Model for Skin Aging Generally, skin is the best approachable organ for investigating both intrinsic (i.e., chronogenic) and extrinsic (i.e., photo-induced) aging. In aging animal models established by transgenic or knock-down approaches, however, some inevitable problems arise: (1) because of the entirely hairy skin, photo-induced aging phenotype is masked, and (2) species-specific hair cycles and/or turnover time of the stratified epithelia considerably differ from those in the human skin. For this clue, certain human diseases
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regarding skin aging may simply provide insight for better understanding of intrinsic and extrinsic aging properties. One plausible disease candidate is lipoid proteinosis [13]. Clinicopathologically, the disease may represent the prematurely aged condition in various organs; for example, in early childhood the disease is characterized with a hoarse cry or voice and generalized skin scarring, and later develops persisted infiltration and thickening of the skin and mucosa during the first decade of life (> Fig. 8.5). Deposits of hyaline material can also occur abruptly on the conjunctivae, cornea, and other ocular components, being similar to the clinical sign of senile cataracts. Other extracutaneous symptoms may include epilepsy and neuropsychiatric abnormalities, sometimes in
association with calcification in the central nervous system. Otherwise, fatal manifestations are less remarkable throughout life. Among a variety of these clinical features, the hallmarks for the skin aging condition include waxy and yellowish papulo-nodules, varicelliform (pox-like) or acneiform scar formation, and profound wrinkling, with generalized thickening [14, 15] (> Fig. 8.5). Hyperkeratosis and pigmentation may appear in area exposed to mechanical friction, such as extremity joints, buttocks, and axillae. Scalp involvement may cause loss of hair, although alopecia and baldness are not of clinical significance in lipoid proteinosis. Moreover, the patients’ skin may be easily traumatized, a condition resembling common skin aging
. Figure 8.5 Clinical features of lipoid proteinosis: Thickening and hardening of vocal cords (a), lips and tongue (b), axillae (c), caused by persistent infiltration; abrupt skin scarring on the trunk and extremity joints (d) or acneiform scarring on the face (e)
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function
in human. By adolescence, some of these changes become obvious, particularly in sun-exposed skin.
ECM1 and its Related Skin Disorders Recent molecular-based investigation has demonstrated loss-of-function mutations in the gene encoding ECM1 in patients with lipoid proteinosis [13, 15]. Accumulating reports for the discovery of various typed mutations in other cohorts further strengthens the monogenic trait of the disease, although there seems no critical association between ECM1 genotype and disease phenotype in individual patients thus far reported [14, 35]. Apart from the precise genomic structure and transcriptional events in ECM1, four currently known splice variants, ECM1a–c,
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and the newly identified transcript encoding a truncated 57 amino acids, each displays a characteristic distribution in the skin (see the first section), and this leads to image ECM1 isoform-specific organization in the skin pathophysiology. Collectively, ECM1 protein(s) widely distribute to the whole epidermis, blood vessel walls in the dermis, and to some extent with dermal collagen fibers around (> Fig. 8.6). In spite of extensive clinical studies including molecular genetics, there is still a very limited knowledge on the actual molecular interactions and their consequences on the basement membrane or cell and tissue biological properties in lipoid proteinosis. As described precisely in Section ‘‘Binding Partners of ECM1,’’ a series of experimental genetic screens for the identification of ECM1-binding partner(s) have revealed multilaterally interactions with structural and extracellular matrix
. Figure 8.6 Localization of ECM1 protein in human skin and presumable histological changes relevant to ECM1 damage: ECM1 is ubiquitously expressed in most of the skin components, including the whole living layers of the epidermis (ECM1a/c in the basal layer and ECM1b in the suprabasal layers, blood vessel walls, and interstitial collagens in the dermis, thus contributing to the overall skin homeostasis. Once in vivo ECM1 is damaged by genetic (lipoid proteinosis) and autoimmune (lichen sclerosus) basis, then it affects the biological interaction between ECM1 and its relevant binding partner(s), such as laminin 332, collagen IV, perlecan, fibulin-1C/D, fibulin-3, fibronectin, MMP-9, and polysaccharides (> Fig. 8.3), thereby resulting in the establishment of the skin pathology characteristic for both diseases, such as dyskeratinization and epidermal atrophy, thickening basement membrane at the epidermal–dermal junction and around blood vessels, dilated blood vessels (teleangiectasiae), homogenization of collagen bundles, and hyaline change in the upper dermis
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molecules, including perlecan [9], MMP-9 [20], fibulin1C/D [21] and fibulin-3 [11], laminin332, collagen IV, fibronectin, and major polysaccharides [24], some of which are essential for maintaining comprehensive skin homeostasis. These are all regular basement membrane constituents, which possess a large regulatory potency for enhancing or depressing a variety of functions, depending on the cellular or tissue context [23]. Like lipoid proteinosis, therefore, once ECM1 is mutated, the micro skin components can be affected by decrease of ECM1 function/expression or even dominantly increase of alternative functionless ECM1 (i.e., truncated form). This causes subsequent instability of extracellular matrix and basement membrane assembly in conjunction with decreased enzymatic activity of MMP-9 – via aberrant increase of collagen IV and laminins, which are both ECM1binding partners – thereby resulting in the establishment of the skin pathology characteristics for lipoid proteinosis, such as epidermal atrophy, thickening of the basement membrane at the epidermal–dermal junction and around blood vessels, dilated blood vessels, and hyaline change in the upper dermis [15, 23] (> Fig. 8.6). This unique molecular-based scenario is further emerged by subsequently established evidence that lichen sclerosus, an acquired chronic inflammatory skin disorder characterized by circulating IgG autoantibodies directed against ECM1, has the pathological features seen in the lipoid proteinosis skin. In this context, lichen sclerosus and lipoid proteinosis are immunogenetic counterparts in terms of targeting ECM1 (> Fig. 8.7). Indeed, more than 70% of lichen sclerosus patients had IgGs reactive with the COOH-terminal part of ECM1a (340–540 aa), and interestingly, the same patients’ sera frequently contained IgGs against the NH2-termial part (32–203 aa), suggesting heterogeneous IgG reactivity to spared antigenic epitopes within ECM1 [32]. In contrast, most ECM1 gene mutations in lipoid proteinosis patients have been detected in exon 7, a region spliced out in ECM1b, while their nonsense or out-of-frame mutations have been found in exon 6, causing deletion of exon 7 and its downstream region [15] (> Fig. 8.1b). Combining clinical and the basic research data, one may speculate that anti-ECM1 IgGs in lichen sclerosus interfere with the functional binding of ECM1 with collagen IV and perlecan, whereas lipoid proteinosis mutations mainly collapse the binding of ECM1 with laminin 332, MMP-9, fibulin-1C/D, fibulin-3, and perlecan [15, 24]. The reaction chain may cause further damages in the interaction between ECM1 and other extracellular matrix components, ultimately contributing to the similar clinicopathology in both disease conditions. To date, there have
been no convincing evidence for other functionally compensative molecule(s) equivalent to ECM1, and therefore, ECM1 action is indispensable for maintaining the structural and biological integrity in the skin components.
ECM1 Function in Skin Aging ECM1 has now been considered to play some pivotal roles in cell signaling, tumor growth and metastasis via angiogenesis, endochondral bone mineralization, skin differentiation, and photoreactivity [8, 16, 17]. Nevertheless, lipoid proteinosis skin never shows any delayed wound healing, and more interestingly, mortality with specific organ failures, growth retardation (i.e., lower statue), shortened life span, or obvious carcinogenic potential are not found in vast majority of the patients. This clinical setting is considerably different from other disorders mimicking prematurely aged condition, such as Werner syndrome, Rothmund–Thomson syndrome, and Alport syndrome. The former two syndromes are rare autosomal recessive ‘‘progeroid’’ disorders caused by loss of WRN [36] and RECQL4 [37]. These are RecQ-like enzymatic nuclear protein family members, which possess helicase and exonuclease activities in repair and replication of DNA double-strand break and telomere maintenance. The absence of both proteins directly leads to genetic instability and cancer predisposition that display many symptoms of premature aging, such as hoarse voice, atrophy (poikiloderma-like), and photosensitivity in skin, baldness or sparse hair, delayed wound healing, cataracts, as well as growth disturbance, part of which resembles the clinical manifestations of lipoid proteinosis. Both syndromes are currently considered the most proper human models for premature skin aging. Contrary to lipoid proteinosis, most patients with Werner syndrome develop normally until the third decade of life, and thereafter gradually establish the mimicry of age-related phenotypes such as atherosclerosis, occasional neoplasia, diabetes mellitus, and osteoporosis by the fifth decade. Another aged condition, Alport syndrome, is an X-linked inheritance albeit much lesser with the autosomal dominant or recessive form, characterized by nephritis progressing to end-stage kidney failure, a high-tone sensorineural deafness, and retinopathy/corneal dystrophy, by adolescence age [38]. The causative gene is the one coding for the collagen IV family members, a structural axis in the basement membrane of kidney glomerulus, certain ocular components, as well as epidermal–dermal junction and blood vessels in the skin. The clinical symptoms normally show mild skin scarring with occasional pigmentation.
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. Figure 8.7 Lipoid proteinosis and lichen sclerosus are immunogenetic counterparts targeting ECM1: Genetic mutation of ECM1 gene causes lipoid proteinosis (left column), whereas development of humoral autoimmunity to ECM1 causes lichen sclerosus (right column). Both diseases have similar skin pathology; hyperkeratosis, epidermal atrophy, dilated blood vessels, less lymph vascularity, and hyaline (glassy) changes in the upper dermis (From Chan I. [40]. Reprinted with permission)
Despite the potential impact of collagen IV–ECM1 network in the skin structural components [24], patients with Alport syndrome do not fulfill the overt skin changes seen in lipoid proteinosis. From the clinicopathological overview between these comparative aging disorders, therefore, ECM1 is not seemingly responsible for dynamics of cell cycle and DNA metabolism, but is explicitly associated with tissue remodeling and integrity via protein–protein interaction as a binding core molecule. This notion is also true regarding the interrelationship between ECM1 and
laminin 332, a causative basement membrane structural molecule for an autosomal recessive genodermatosis ‘‘junctional epidermolysis bullosa’’ [26] and an autoimmune mucocutaneous blister and scarring condition ‘‘mucous membrane pemphigoid,’’ otherwise termed as ‘‘antiepiligrin cicatricial pemphigoid’’ [27]. Both diseases genetically and immunologically target laminin 332, but do not clearly represent any of premature and photo-induced aging phenotypes. It remains an attractive question how much the functional disturbance of ECM1 binding
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partners, like collagen IV or laminin 332, can indeed affect the in vivo biological action of ECM1, and vice versa. The molecular-based orchestration in common skin aging properties mediated by ECM1 needs to be further updated. In this context, it is important to note that ECM1 expression is exclusively downregulated in intrinsically aged skin protected from chronic UVexposure [8], whereas the decreased ECM1 expression in chronologically aged skin may have profound effects on dermal and epidermal homeostasis, leading to the clinical features of skin atrophy, including the loss of adnexal epithelia. However, the photoaging pathway(s) mediated by ECM1 remains unclear. The promoter region of mouse Ecm1 gene contains a functional binding site for the transcription factor AP1 (composed of the Jun and Fos protein family) well known for its role in skin aging. However, the c-Fos protein expression in young and aged skin is unaltered, while c-Jun is increased in aged skin. ECM1 is capable of binding with MMP-9, thereby restricting its bioactivity [20]. If ECM1 function/expression is reduced, then there will be increased activation of MMP-9 leading to an enhanced breakdown of interstitial and basement membrane collagen (e.g., collagen type IV) and hence to signs of skin aging. As already noted, ECM1 mutations cause the characteristic dermatopathology in lipoid proteinosis and the patients from sunny regions have a more severe phenotype (i.e., more scarring and photoaged appearance), compared with nonexposed areas [35]. These findings suggest that lack of ECM1 may predispose to increased or accelerated signs of photoaging. Recently, a higher expression of ECM1 was found in the whole layers of the human epidermis, induced by chronically UVexposure [8]. UV irradiation stimulates the production of matrixmetalloproteinases, like MMP-1, 3, and 9, and it subsequently increases the presence of mixed inflammatory cell infiltrates and decreases the number of irregularly dilated blood vessels. Furthermore, MMP-3 degrades important ECM proteins like collagen IV, laminin-332, fibronectin, and proteoglycans (i.e., perlecan), which are all binding partners of ECM1. Combining all, ECM1 might thus act as an internal sunscreen via comprehensive regulation with multiple binding partners in skin aging properties.
Acknowledgments We thank Professor Dr. J. Lambert for critically reading this manuscript. The authors would also like to thank all investigators who contributed to ECM1 research and due to word limitations are not cited in the references.
References 1. Mathieu E, Meheus L, Raymackers J, et al. Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing. J Bone Miner Res. 1994;9:903–913. 2. Bhalerao J, Tylzanowski P, Filie JD, et al. Molecular cloning, characterization, and genetic mapping of the cDNA coding for a novel secretory protein of mouse. Demonstration of alternative splicing in skin and cartilage. J Biol Chem. 1995;270:16385–16394. 3. Smits P, Bhalerao J, Merregaert J. Molecular cloning and characterization of the mouse Ecm1 gene and its 5’ regulatory sequences. Gene. 1999;226:253–261. 4. Smits P, Ni J, Feng P, et al. The human extracellular matrix gene 1 (ECM1): genomic structure, cDNA cloning, expression pattern, and chromosomal localization. Genomics. 1997;45:487–495. 5. Johnson MR, Wilkin DJ, Vos HL, et al. Characterization of the human extracellular matrix protein 1 gene on chromosome 1q21. Matrix Biol. 1997;16:289–292. 6. Smits P, Poumay Y, Karperien M, et al. Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes. J Invest Dermatol. 2000;114: 718–724. 7. Oyama N, Chan I, Neill SM, et al. Autoantibodies to extracellular matrix protein 1 in lichen sclerosus. Lancet. 2003;362:118–123. 8. Sander CS, Sercu S, Ziemer M, et al. Expression of extracellular matrix protein 1 (ECM1) in human skin is decreased by age and increased upon ultraviolet exposure. Br J Dermatol. 2006;154: 218–224. 9. Mongiat M, Fu J, Oldershaw R, et al. Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis. J Biol Chem. 2003;278: 17491–17499. 10. Horev L, Potikha T, Ayalon S, et al. A novel splice-site mutation in ECM-1 gene in a consanguineous family with lipoid proteinosis. Exp Dermatol. 2005;14:891–897. 11. Sercu S, Lambeir AM, Steenackers, et al. ECM1 interacts with fibuline-3 and the beta 3 chain of laminin 332 through its serum albumin subdomain-like 2 domain. Matrix Biol. 2009;28:160–169. 12. Kragh-Hansen U. Structure and ligand binding properties of human serum albumin. Dan Med Bull. 1990;37:57–84. 13. Hamada T, McLean WH, Ramsay M, et al. Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1). Hum Mol Genet. 2002;11:833–840. 14. Urbach EWC. Lipoidosis cutis et mucosae. Virchows Arch Pathol Anat. 1929;273:285–319. 15. Chan I, Liu L, Hamada, et al. The molecular basis of lipoid proteinosis: mutations in extracellular matrix protein 1. Exp Dermatol. 2007;16(11):881–890. 16. Deckers M, Smits P, Karperien M, et al. Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro. Bone. 2001;28:14–20. 17. Han Z, Ni J, Smits P, et al. Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells. FASEB J. 2001;15:988–994. 18. French MM, Gomes RR, Jr., Timpl R, et al. Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I. J Bone Miner Res. 2002;17:48–55.
The Importance of Extracellular Matrix Protein 1 as Basement Membrane Protein in Maintaining Skin Function 19. rikawa-Hirasawa E, Wilcox WR, Le AH, et al. Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the perlecan gene. Nat Genet. 2001;27:431–434. 20. Fujimoto N, Terlizzi J, Aho S, et al. Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high-affinity protein/protein interactions. Exp Dermatol. 2006; 15:300–307. 21. Fujimoto N, Terlizzi J, Brittingham R, et al. Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2. Biochem Biophys Res Commun. 2005;333:1327–1333. 22. Sercu S, Zhang L, Meregaert J. The extracellular matrix protein 1: its molecular interaction and implication in tumor progression. Cancer Invest. 2008;26(4):375–384. 23. Mirancea N, Hausser I, Beck R, Metze D, Fusenig NE, Breitkreutz D. Vascular anomalies in lipoid proteinosis (hyalinosis cutis et mucosae): basement membrane components and ultrastructure. J Dermatol Sci. 2006;42:231–239. 24. Sercu S, Zhang M, Oyama N, et al. Interaction of extracellular matrix protein 1 with extracellular matrix components: ECM1 is a basement membrane protein of the skin. J Invest Dermatol. 2008;128 (6):1397–1409. 25. Albig AR, Neil JR, Schiemann WP. Fibulins 3 and 5 antagonize tumor angiogenesis in vivo. Cancer Res. 2006;66:2621–2629. 26. Pulkkinen L, Uitto J. Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol. 1999;18:29–42. 27. Bekou V, Thoma-Uszynski S, Wendler O, Uter W, Schwietzke S, Hunziker T, Zouboulis CC, Schuler G, Sorokin L, Hertl M. Detection of laminin 5-specific auto-antibodies in mucous membrane and bullous pemphigoid sera by ELISA. J Invest Dermatol. 2005; 124:732–740. 28. McMillan JR, Akiyama M, Shimizu H. Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions. J Dermatol Sci. 2003;31:169–177. 29. McKee KK, Harrison D, Capizzi S, Yurchenco PD. Role of laminin terminal globular domains in basement membrane assembly. J Biol Chem. 2007;282:21437–21447.
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30. Sercu S, Poumay Y, Herphelin F, Liekens J, Beek L, Zwijsen A, et al. Functional redundancy of extracellular matrix protein 1 in epidermal differentiation. Br J Dermatol. 2007;157(4):771–775. 31. Uematsu R, Furukawa J, Nakagawa H, Shinohara Y, Deguchi K, Monde K, et al. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis. Mol Cell Proteomics. 2005;4:1977–1989. 32. Oyama N, Chan I, Neill SM, et al. Development of antigen-specific ELISA for circulating autoantibodies to extracellular matrix protein 1 in lichen sclerosus. J Clin Invest. 2004;113:1550–1559. 33. Werner S, Smola H. Paracrine regulation of keratinocyte proliferation and differentiation. Trends Cell Biol. 2001;11:143–146. 34. Boxman IL, Ruwhof C, Boerman OC, Lowik CW, Ponec M. Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. Arch Dermatol Res. 1996;288:391–398. 35. van Hougenhouck-Tulleken W, Chan I, Hamada T, et al. Clinical and molecular characterization of lipoid proteinosis in Namaqualand, South Africa. Br J Dermatol. 2004;151:413–423. 36. Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, Martin GM, Mulligan J, Schellenberg GD. Positional cloning of the Werner’s syndrome gene. Science. 1996;12(272):258–262. 37. Kitao S, Shimamoto A, Goto M, Miller RW, Smithson WA, Lindor NM, Furuichi Y. Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nat Genet. 1999;22:82–84. 38. Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR, Gerken SC, Gregory MC, Skolnick MH, Atkin CL, Tryggvason K. Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science. 1990;248:1224–1227. 39. Sercu S, Oyama N, Merregaert J. Importance of extracellular matrix protein 1 (ECM1) in maintaining the functional integrity of the human skin. Open Dermatol J. 2008;2:98–105. 40. Chan I. The role of extracellular matrix protein 1 in human skin. Clin Exp Dermatol. 2004;29:52–56.
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53 The New Face of Pigmentation and Aging John Nip . S. Brian Potterf . Sheila Rocha . Shilpa Vora . Carol Bosko
Introduction Pigmentation is a universal physiological process that occurs in all organisms from bacteria, fish, and amphibians to birds, mammals, and humans [1]. Pigmentation provides camouflage and protection from UV, but in some lower organisms pigmentation is also involved in wound healing [2–4]. In humans, the major determinant of skin color is the pigment/complex polymer, melanin. The variation in human skin color is striking, and has great physiological and sociological implications. The color of one’s skin is a strong predictor of social interactions. That skin color has immense psychosocial impacts is evidenced by the billions of dollars spent annually in search of the perfect skin color. Tanning beds and artificial tanners are used to achieve a bronzed glow, while fairness creams and bleaches are used to lighten skin color and achieve an even skin tone. The concept of the ideal skin color varies across cultures and geographies, and has great significance on the perception of beauty. With age, changes in the amount and distribution of melanin are evident. Increases in skin pigmentation as well as the appearance of mottled and discrete hyperpigmented lesions are a hallmark of photoexposure and advancing age. To better understand the changes that may occur in aging skin, a closer look at the pigmentation system and its components is needed. The chapter provides an overview on melanogenesis, from the production of melanin to its transfer to keratinocytes, as well as the genetic and biological pathways that regulate pigment production. Studies on the clinical and biological manifestations of hyperpigmentation in Asian populations will be presented. Melanin is one of the major determinants of human skin color. It is produced by neural crest-derived melanocytes, which are found in many sites throughout the body including the skin, hair, eye, ear, and the central nervous system (CNS) [5–7]. Extracutaneous melanocytes do not produce melanin throughout their lifespan and, unlike epidermal melanocytes, they do not transfer their melanosomes and melanin to neighboring cells [6].
Besides imparting color to the skin, melanin serves diverse other functions. Undoubtedly, its most important function is protection from the damaging effects of solar UV radiation [8], the most dire consequence of which is the development of cancer [9, 10]. The photoprotective properties of melanin have been well documented [11]. Melanin protects the cells of the skin by shielding the cell nuclei, thus preventing DNA damage in the form of cyclobutane pyrimidine dimers and 6–4 DNA photoproducts [12]. In addition to photoprotection, melanin gives skin, hair, and eyes their color and is also involved in hearing [5–7]. Impaired hearing in individuals with vitiligo may be associated with lack of these melanocytes [13]. Melanins may also serve a unique function in the central nervous system. In fact, neuromelanins found in the dopaminergic neurons in the substantia nigra of the brain have been implicated in the pathology of Parkinson’s disease [7].
Melanin Synthesis Within the melanocyte, melanin is synthesized in specialized organelles called melanosomes, which are subsequently transferred (along with their melanin) to adjacent keratinocytes in the basal epidermal layer, giving rise to skin pigmentation. The type and quantity of melanin produced and the shape, size, and distribution of the melanosomes in basal epidermal keratinocytes affect the final color of an individual’s skin. Human melanin is composed of two distinct polymers: the dark brown/black eumelanin, and the yellow/ red pheomelanin [14]. Eumelanin and pheomelanin are produced in the eumelanosomes and pheomelanosomes, respectively. Both these types of melanosomes undergo four stages of maturation, with the final stages (III and IV) resulting in synthesis and deposition of melanin. The two types of melanin differ in their composition and physical properties. Due to the incorporation of the sulfur-containing amino acid cysteine in its synthesis, pheomelanin has a higher sulfur and nitrogen content
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_53, # Springer-Verlag Berlin Heidelberg 2010
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than eumelanin. Eumelanin is made and deposited in ellipsoidal melanosomes that contain a lamellar or fibrillar internal structure, whereas pheomelanin is synthesized in spherical melanosomes and is associated with microvesicles [15, 16]. The final melanin composition, and therefore its color, is dependent on the relative amounts of pheoand eu-melanogenesis occurring in the melanocyte. Eumelanin and pheomelanin are both derived from the common substrate tyrosine. The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to Dopaquinone are both reactions catalyzed by tyrosinase, the rate-limiting enzyme in the melanin synthetic pathway. Evidence has accumulated recently for the requirement of three enzymes for initiation of melanogenesis [17]. Tyrosinase requires millimolar amounts of L-tyrosine that cannot be supplied by facilitated diffusion alone. On the other hand, 6BH4dependent phenylalanine hydroxylase mediates conversion of phenylalanine to tyrosine in amounts sufficient for tyrosinase activity. The third enzyme implicated is tyrosine hydroxylase isoform I, which converts tyrosine to L-DOPA. L-DOPA then binds tyrosinase at a site distinct from tyrosine and activates the enzyme. Other enzymes involved in melanogenesis include tyrosinaserelated protein-1 (TYRP1) and tyrosinase-related protein-2 (DCT) (> Fig. 53.1). Clinical studies on human skin have shown that in highly pigmented skin (Fitzpatrick type V and VI), that is, chronically photoexposed skin, the pheomelanin content is only slightly elevated, whereas the eumelanin content is highly elevated [18]. The reflectance or lightness (L*) of human skin, as measured by a chromameter, is correlated with the total melanin content in an exponential manner [18] (> Fig. 53.2). Lightly pigmented skin types (such as Chinese and European) have about half as much epidermal melanin as the most darkly pigmented skin types (African and Indian). In all skin types, pheomelanin is a very small part of epidermal melanin. In addition to melanin content, melanosome size, shape, and distribution play a large role in skin color. African skin has the largest melanosome size and these tend to be deposited singly, while lighter skin types (Chinese, European) have smaller melanosomes that tend to cluster together in the keratinocyte [19, 20]. Boissy et al. have focused on the distribution of melanosomes in the keratinocytes, and on the role that donor ethnicity plays in this process [19]. In these experiments, keratinocytes and melanocytes from different donors were cocultured, and it was observed that the ethnicity of the keratinocyte donor defined the distribution of the melanosomes. That is, in keratinocytes from Black
donors, the melanosomes are distributed individually, regardless of whether Black or Caucasian melanocytes were present in the cocultures. Conversely, when Caucasian keratinocytes were present, melanosomes were distributed in clusters, irrespective of the ethnicity of the melanocyte donor skin. These data point to the importance of keratinocytes in regulating melanosome distribution, and thus may have a relevant role in skin pigmentation in different ethnic skin types. Human pigmentation variation between different ethnic groups is not due to melanocyte number, as there are no differences between the groups in terms of abundance of melanocytes [21]. The levels of tyrosinase protein, the key enzyme in melanogenesis, are similar across ethnic groups as well. Interestingly, TYRP1, another important enzyme for melanin production, is more than 2.5-fold elevated in darklypigmented African and Indian skin types in comparison to more lightly pigmented Chinese and European skin types [21]. Finally, there is evidence from the literature, and confirmed recently by Alaluf et al. [21], that chronic hyperpigmentation in sun-exposed body sites (e.g., lateral forerams) is associated with increased melanocyte number. This indicates that melanocyte number (proliferation) may play a role in maintaining long-term, stable changes in skin color on chronically sun-exposed sites.
Melanosome Transfer The process of melanosome transfer to keratinocytes is poorly understood. Light and EM studies done on guinea pig skin cultures suggest that melanosome transfer may be due to direct interaction between melanocytes and keratinocytes, involving phagocytosis of bits of the melanocyte dendrite (containing the melanosomes) by the keratinocytes. Other hypotheses on transfer have been put forth, the most popular being: (i) release of melanosomes/ melanin by melanocytes, followed by their endocytosis into keratinocytes, (ii) direct inoculation (injection) of melanosomes into keratinocytes, and (iii) keratinocyte– melanocyte membrane fusion [22–24]. Whether melanin itself or the melanosomes are transferred from melanocytes to keratinocyte remains controversial. The nature of receptors involved in melanocyte– keratinocyte interactions is still speculative. Cadherins are calcium dependent cell–cell adhesion glycoproteins, of which E-cadherin is expressed by human melanocytes and is thought be the major mediator of melanocyte– keratinocyte adhesion [25]. Also, lectins and neoglycoproteins inhibit melanin transfer in melanocyte–keratinocyte
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. Figure 53.1 The melanogenesis pathway. Schematic diagram showing the enzymes involved in melanogenesis and their melanin products
cocultures [26, 27]. Other cell surface proteins proposed to be involved in the process of melanosome phagocytosis are protease activated receptor-2 (PAR-2) and keratinocyte growth factor receptor [28–32]. Work from Seiberg et al. [31–33] has shown that the transfer of melanosomes to keratinocytes is mediated by a G protein-coupled receptor – the protease-activated receptor 2 – on keratinocytes. Activation of the receptor with trypsin or a peptide agonist resulted in pigmentation in skin equivalent models, in human skin xenografted onto
SCID mice, and in Yucatan swine [33]. Serine protease inhibitors that prevent activation of the PAR-2 receptors on keratinocytes caused decreased pigmentation in the Yucatan swine model of melanogenesis, presumably by affecting melanosomal transfer to the keratinocytes [33]. Scott et al. [34] showed that UV irradiation (using a xenon arc lamp solar simulator) on human subjects (buttock skin) altered PAR-2 expression. In UV-irradiated skin, expression of the receptor was localized to the whole epidermis versus just the lower third of the epidermis on a
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. Figure 53.2 Exponential relationship between L* and melanin. Graph showing the melanin content of skin from different ethnic donors
non-irradiated control site on the same individual. Moreover, there is a delay in upregulation of PAR-2 expression in skin from phototype I subjects compared to those with type II and III skin. Several investigations point to a role for the semaphorin–plexin ligand–receptor system in melanosome transfer. Semaphorins are a class of secreted and membrane-bound proteins that are expressed widely and have been shown to play an important role in a diverse set of biological processes. Semaphorins mediate alterations in cytoskeletal elements, actin filaments, and microtubular networks. Semaphorins were originally identified for their role in neuronal development and play a crucial role in axonal guidance. Semaphorins bind two receptor families, the plexins and the neuropilins. The semaphorin 6 class of proteins are ligands for the plexin receptor family. Semaphorin 6D (Sema6D) is a member of the class 6 semaphorins, and is grouped into six isoforms through differential splicing [35]. The neural crestderived origin of melanocytes is interesting in light of semaphorins having a role in neuronal axon guidance. It is hypothesized that the semaphorin–plexin system may play a role in keratinocyte–melanocyte interactions. Using RNA-mediated interference, it has been demonstrated that knockdown of either the entire semaphorin class, specifically Sema6D, or Plexin A1 protein resulted in a significant inhibition of melanosome transfer. HaCaT keratinocytes transfected with siRNA targeted towards Sema 6D specifically or the conserved SEMA domain resulted in significant decrease in protein and a corresponding decrease in
melanosome transfer (> Fig. 53.3). Similarly, a knockdown of Plexin A1 protein in melanocytes inhibited melanosome transfer (data not shown). Independently, Scott and colleagues reported that semaphorin 7A is expressed on keratinocytes and fibroblasts, is upregulated in fibroblasts in response to UV, and binding of semaphorin 7A to plexin C1 on melanocytes inhibited dendrite formation. In contrast, binding of semaphorin 7A to b-1-integrin promoted melanocyte dendricity [36]. These results further support a role for the semaphorin–plexin receptor system in keratinocyte– melanocyte communication. Melanosome transfer is a process regulated by various environmental stimuli and physiological parameters. However, underpinning this, it is clear that it is keratinocytes that regulate this process through paracrine and autocrine factors. Evidence in literature exists for melanocyte–keratinocyte adhesion and interaction as a prelude to transfer of melanosomes to keratinocytes [25, 37]. Also, melanocyte–keratinocyte recognition is a prerequisite to melanosome transfer [38]. Some of the melanocyte-induced signaling events in keratinocytes upon melanocyte–keratinocyte interaction have been deciphered. Although melanocytes extend filopodia towards keratinocytes, it is technically not possible to monitor intracellular signaling during the actual event of cell–cell contact. Therefore, it was investigated whether isolated melanocyte cell plasma membrane fraction induced signaling in keratinocytes. When primary human melanocyte plasma membrane fraction was added to
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. Figure 53.3 Semaphorin 6D siRNA knockdown leads to a significant inhibition of melanosome transfer. Coculture of primary human melanocytes and HaCaT keratinocytes transfected with different siRNA. HaCaT keratinocytes were transfected with siRNA for Sema6D (RNAi 1099 (semaphorin 6D specific) sequence is sense-GCCACACUUUCUUCAUGCCAUAGAA- and antisenseUUCUAUGGCAUGAAGAAAGUGUGGC-) (a) or scrambled controls (b). Arrows point to melanosomes within the keratinocytes. 400¥ magnification
HaCaT keratinocytes, a transient increase in [Ca2þ]i was observed in keratinocytes (> Fig. 53.4a). Further, the calcium signal induced by melanocyte plasma membrane in HaCaT keratinocytes was found to be caused by release of Ca2þ from intracellular stores (> Fig. 53.4a). It was also noted that calcium may be necessary for pigment transfer, as transfer in melanocyte–keratinocyte cocultures was inhibited when intracellular calcium in keratinocytes was chelated [38]. This led to the hypothesis that a ‘‘ligand-receptor’’ type interaction exists between melanocytes and keratinocytes, which mediate recognition and, eventually, melanin transfer. A technical point in melanosome transfer research is whether melanosomes retain their proteins once transferred to keratinocytes, or whether keratinocytes degrade the melanosomal proteins of melanosomes. The question was indirectly answered in a study conducted by Virador et al. [39] A series of melanocyte-specific peptide antibodies to human melanosomal proteins was produced in order to study the distribution of these proteins in skin from normal individuals and people with pigmentation disorders. Polyclonal antibodies were synthesized against the peptide sequences of the following human melanosomal proteins: TYR, TYRP-1, DCT, and Pmel17 (gp100). Immunological staining of normal skin (paraffin sections) revealed that TYR, DCT, and, to a lesser extent, gp100 was not only detected in the melanocytes, but was also present in the neighboring keratinocytes as well as in some keratinocytes higher in the epidermis.
These findings suggest that it is possible to detect melanosomal proteins in keratinocytes after transfer; however, the life of these proteins in keratinocytes may still be limited.
Mutations Affecting Melanosome Transfer Evidence from three mouse coat-color pigmentation mutants revealed that melanosome transport may be a key factor in determining pigmentation, both in the hair and most likely in the skin as well. These three mutations called ashen, dilute, and leaden lead to differences in coat color of the mice by affecting melanosomal transport of melanin and not melanin synthesis itself, as this latter process is not altered in these mouse mutations [40–46]. In fact, the produced melanin is distributed in the perinuclear region of these mutant melanocytes. The genes for these mutants have been discovered, and examination of their function and biochemistry has helped to better understand the role of melanosome transport in pigmentation. The ashen gene encodes a Rab GTPase, Rab27a [46], the dilute gene encodes for myosin Va [41], and the leaden gene encodes for melanophilin [45]. Alterations at the dilute locus in mice cause a decrease in coat color intensity due to changes in melanosome movement and transfer to neighboring keratinocytes. Dilute mutant melanocytes were shown to have a
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. Figure 53.4 Primary melanocyte plasma membrane induced calcium signal in HaCaT keratinocytes (a). The data shown are representative traces of four cells in a single experiment. Primary melanocyte membrane induced rise in calcium in HaCaT cells in absence or presence (2 mM) of extracellular calcium (b). The data are mean þ SEM from three separate experiments, p > 0.1 (Joshi et al. [38])
perinuclear melanosome distribution and possessed dendrites [47]. However, melanosomes in these mutant melanocytes were less abundant in the dendrites compared to dendrites from wild type mice. Apparently, the dilute locus encodes the actin-associated motor protein, myosin Va [41]. This protein has been found to be involved in both melansome transport and dendrite formation [40]. The products of the ashen, dilute, and leaden loci all have been implicated in movement of melanosomes to dendrite tips. Rab proteins control movement and transport of intracellular vesicles, including melanosomes, by regulating the interactions between the vesicles and cytoskeletal elements and molecular motors. C-terminal prenylation mediates association of Rab proteins
with the cytoplasmic side of the cell membrane, an event that controls Rab activity [48]. Melanosomal movement to the cell periphery involves many different proteins, including the Rab protein, Rab27a, and myosin Va. This transfer of melanosomes from the perinuclear region of melanocytes to the peripheral areas and concentration at the dendrite tips [49] requires a combination of ‘‘long-range’’ bidirectional movements of the melanosomes along microtubules (along the dendrite) and local myosin Va-dependent capture and local movement of the melanosomes along actin-rich dendrite areas [50, 51]. A closely related family member of Rab27a is Rab27b, which shares more than 70% amino acid sequence identity with Rab27a [52]. The function of this protein has not been as well studied as Rab27a, although its GTPase activity has been shown [53]. In addition, transient expression of dominant-negative forms of Rab27b revealed that the native form of the protein may be involved in the number and length of dendrites as well as the movement of melanosomes to the cell periphery in melan-a murine melanocytes [53]. Myosin Va is an actin-based dimeric molecular motor protein that is involved in melanosomal movement along with Rab27a [49, 54–57]. This protein is composed of three different domains, including a motor (or head) domain containing the actin and ATP binding sites; a regulatory (neck) domain possessing several myosin light chain- and calmodulin-binding sites (also called IQ motifs); and the tail domain, which consists of an a-helix coil–coil region and a globular region [58]. Alternative splicing in the tail domain can create different tissuespecific forms of myosin Va, including a melanocyte form (specifically found in melanocytes) and the brain form (which is present in neural tissues) [59]. Several groups have recently elucidated the interactions of Rab27a, myosin Va, and melanophilin in melanosome transport [40, 44, 60–62]. In fact, these proteins interact with each other in a tripartite complex, with the melanophilin protein being the crucial link between the Rab27a and myosin Va [60]. Wu et al. [63] established the sequence of interaction of the three proteins: Rab27a binds to the melanosome, followed by binding of melanophilin to Rab27a, and then myosin Va is bound to melanophilin. The alternatively spliced exon-F in myosin Va is required for binding of myosin Va to melanophilin [63]. In human and mouse diseases such as Griscelli syndrome [64, 65], mutation of key proteins involved in the movement of melanosomes in preparation for transfer to the keratinocytes compromises/dilutes the color of the skin. The mutations that cause pigmentation issues in humans are classified into three types: Griscelli syndrome
The New Face of Pigmentation and Aging
types I, II, and III. Each of these has a mutation in the machinery involved in movement of the melanosomes from the perinuclear region to the tips of the dendrites. For Griscelli syndrome type I, patients present with albinism but also have severe neurological defects. The mutation in type I is found on the myosin 5A gene (MYO5A), which encodes for myosin Va [66]. Albinism in Griscelli syndrome type II is also associated with immunodeficiency, and can lead to fatal hemophagocytic syndrome [67]. The defect is caused by mutations in the RAB27A gene that encodes Rab27a (a GTPase). This protein is required for T cell cytotoxic granule release, and its defect may explain the immune defects observed [67, 68]. The albinism is a result of the Rab27a defect in transport of melanosomes in the transfer process. For Griscelli syndrome type III, the patient displays the characteristic hypopigmentation, but there are no associated neurological or immunological defects. The gene involved is the MLPH gene, encoding for melanophilin [69]. In addition, there have been type III patients who have a deletion in the F-exon of the MYO5A gene [69]. The mutations in both mice and humans implicate melanosome transfer as crucial for normal expression of skin and coat color, even in the absence of defects in melanin synthesis.
Regulation of Melanogenesis Human skin color is a heritable, polygenic trait, and varies widely across individuals and populations. However, environmental factors such as UVR also have a strong influence on skin color. There is a high correlation between the intensity of incident UV light at the point of anthropological origin and skin color, suggesting intense evolutionary selective pressure [70, 71]. UV radiation can degrade folic acid, a critical nutrient for reproductive health. On the other hand, UVB is required for synthesis of vitamin D. It has been speculated that skin color has evolved to maximize the production of these important nutrients while protecting the skin from UV-induced damage. Much of the knowledge on genetic regulation of skin color has come from human and animal mutations that result in drastic changes in pigmentation, such as albinism. The OMIM lists 18 genes and >100 loci involved in human albinism [72]. However, aside from these rare and extreme changes in color, only a handful of genes are known to influence the normal variations in skin color. Among these, the melanocortin receptor 1 (MCR1) has been strongly correlated with red hair, light skin, and freckling, as well as a predisposition to skin cancer [73].
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The cloning of the human genome and the development of extensive ‘‘hap-maps’’ of single nucleotide polymorphisms [74] has enabled the study of skin color via genome-wide scans, and has facilitated the identification of genes involved in normal skin color variation. Among those identified are SLC24A5, SLC45A2 (MATP), TYR, ASIP, and OCA2 [75, 76]. The role of SLC24A5 in skin color variation was identified in a South Asian population using high-density whole genome array technology [68]. In that population, polymorphisms in three genes, MATP, TYR, and SLC24A5, accounted for a large fraction of the natural variation in skin color, while SLC24A5 alone accounted for >30% of the variation in skin color. Ginger et al. [77] then directly demonstrated the role of SLC24A5 in melanogenesis. In either B16 or dedifferentiated normal human melanocytes, knockdown of SLC24A5 expression via RNA-mediated interference resulted in a decrease in melanin synthesis. The hormonal regulation of pigmentation has been reviewed extensively elsewhere [78], and will be only discussed briefly here. It is clear that keratinocytes regulate melanogenesis in a paracrine fashion, and the concept of the epidermal unit comprising 1 melanocyte and 36 keratinocytes is well accepted. However, melanocytes receive paracrine signals from and are regulated by dermal fibroblasts as well. The importance of the melanocortins in control of pigmentation has been well established. ACTH, a-MSH, and b-MSH stimulate both melanogenesis and the switch from pheo- to eumelanogensis in mammalian systems via the melanocortin receptor (MCR). Injection of MSH peptides in humans stimulates skin pigmentation, especially in sun-exposed areas [79]. MCR1, a G-protein coupled receptor, has the highest binding affinity for a-MSH and has received most of the attention. Recently however, it was observed that b-MSH binding to MCR4 may also stimulate melanogenesis. Moreover, some have proposed a receptor-independent mechanism for the stimulation of tyrosinase activity by a- and b-MSH within the melanosome [17]. The activity of the MCR is antagonized by the Agouti Signaling Protein (ASIP), which binds MCR, stimulating a switch to pheomelanogenesis. Ligation of MSH to MCR1 and the resultant increase in cAMP induces the micropthalmia transcription factor (MITF), which exerts transcriptional regulation on TYR and TYRP1. Recently, it has been suggested that hepatocyte nuclear factor-1alpha (HNF) also promotes tyrosinase transcription, and that p53 is a transcriptional regulator of HNF. Thus UV, which stimulates p53, may directly affect tyrosinase transcription via HNF in the absence of MITF.
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Endothelin 1 and 3, ligands of the G-protein coupled receptor endothelin receptor B, have been implicated in the development of melanocytes and the induction of pigmentation [80]. ET-1 is upregulated in keratinocytes exposed to UVB, and stimulates tyrosinase expression. GPCRs signal via cAMP and it is evident that a number of cAMP receptors may be involved in melanogenesis, including histamine, eicosanoids, b2-adrenoceptor, and muscarinic receptors. The nuclear hormone receptor for estrogen is expressed on human melanocytes, but its role in stimulating pigmentation is the subject of debate. While it is widely believed that circulating sex hormones play a role in the development of melasma, data suggest that melasma actually increases postmenopausally (see below). The c-kit ligand Stem Cell Factor (SCF, Steel Factor) is produced by keratinocytes, and influences the proliferation and activity of melanocytes. SCF is a key hormone in embryonic development, as it influences migration of melanoblasts from the neural crest. [81] Mutations in either the receptor tyrosine kinase, c-kit, or its ligand results in loss of pigmentation. Human mutations result in the piebaldism phenotype with patchy leukoderma [82]. Basic fibroblast growth factor (bFGF) is another tyrosinase kinase receptor ligand induced by UVB exposure to keratinocytes. In vitro bFGF stimulates melanogenesis, although its in vivo role has yet to be established [83].
Age-Related Changes Age-related changes in pigmentation were observed in several Asian populations. These studies have identified solar lentigo (SL) lesions to be one of the top pigmented lesions affecting a younger-than-expected population in Asia (see below). Solar lentigo, also known as senile lentigo or age spots, is a hyperpigmented macule that most often develops on the sun-damaged skin of the face, the back of the hands, lateral forearms, the back, and the chest. They are highly symbolic of senescence, as suggested by their common French name ‘‘cemetery flowers,’’ and are aesthetically unpleasant. SL legions range from 2 to 10þ mm, and are histologically characterized by a hyperpigmented basal layer, elongated rete ridges and increased numbers of melanocytes. Aside from the apparent activated state, the melanocytes appear otherwise normal, with melanosomes present in all stages of maturation in the cytoplasm and in dendrites. However, the basal keratinocytes have accumulation of numerous melanosome complexes or polymelansomes [84].
A look at the status of melanogenesis-specific genes reveals increases in POMC, TYR, TYRP-1, DCT, PMEL-17, OCA2, and MITF protein expression, which confirms upregulation of melanogenesis in lesional melanocytes and also proposes their involvement in legion formation [85]. However, gene expression involved in cornified envelope formation, such as profillagrin and involucrin, give weight to the possibility of a malfunctioning cornification process. Increased layers of cells in the stratum corneum point to not only a decrease in cornification, but also to a slowed desquamation process in the epidermis of SL lesions [86]. SL lesions have fewer Ki67 positive cells when compared to peri-lesional skin, an observation previously made by Unver et al. [87], suggesting that there are fewer dividing keratinocytes in the SL lesions [71]. Endothelin receptor B (ETBR) and SCF have been confirmed to be up-regulated [88, 89]. As these same markers, ETBR and SCF, have been shown to be up-regulated in other pigmented lesions such as seborrheic keratosis [90, 91], they are not unique to SL lesions. While concerns about uneven, patchy, and discrete facial hyperpigmentation are present in different ethnicities, these conditions are of particular concern in Asians, and appear to be key drivers associated with aging [92, 93]. To further investigate the importance of pigmentation to aging in Asian populations, female subjects, aged 18–65 years, were enrolled in an Institutional Review Board-approved multi-site, facial hyperpigmentation characterization study, with approximately 100 subjects per cell (more than 500 total subjects) from China (Shanghai), India (Mumbai), Thailand (Bangkok), Indonesia (Jakarta), and Japan (Tokyo). Subjects were screened for having self-perceived facial hyperpigmentation and a slight to moderate concern for facial hyperpigmentation in general. Hence, the study does not represent a random sampling of these populations, but serves as a clinical baseline under these conditions. Clinical evaluation was performed by a dermatologist of similar ethnic origin from each country to diagnose facial hyperpigmentation across multiple attributes (summarized in > Table 53.1). Additional measures included color measurements with spectrophotometry, and digital facial photographs to document clinical facial skin condition. Color measurements on non-lesional, even-toned areas of the face revealed an age-dependent increase in cumulative pigmentation, resulting in increasing values of several L* units between women aged 20–60 years (data not shown). This age-dependent increase in facial pigmentation is presumed to result from chronic, lifelong UV exposure, as upper inner arm measurements showed no age-dependence on these photo-protected
The New Face of Pigmentation and Aging
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. Table 53.1 Dermatological characterization of facial hyperpigmentation in various Asian populations. Dermatologists from each respective region diagnosed facial hyperpigmentation in females in Shanghai, China; Tokyo, Japan; Mumbai, India; Bangkok, Thailand; and Jakarta, Indonesia. Only the most prevalent conditions are contained in the table, and relatively rare disorders ( Hyperpigmentation > Pigmentation
in Aging Skin in Ethnic Groups
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48. Pereira-Leal JB, Hume AN, Seabra MC. Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease. FEBS Lett. 2001;498:197–200. 49. Wu X, Rao K, Bowers MB, Copeland NG, Jenkins NA, Hammer JA, III. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. J Cell Sci. 2001; 114:1091–1100. 50. Hammer JA, III, Wu XS. Rabs grab motors: defining the connections between Rab GTPases and motor proteins. Curr Opin Cell Biol. 2002;14:69–75. 51. Wu X, Bowers B, Rao K, Wei Q, Hammer JA, III. Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function In vivo. J Cell Biol. 1998;143:1899–1918. 52. Chen D, Guo J, Miki T, Tachibana M, Gahl WA. Molecular cloning and characterization of rab27a and rab27b, novel human rab proteins shared by melanocytes and platelets. Biochem Mol Med. 1997;60:27–37. 53. Chen Y, Samaraweera P, Sun TT, Kreibich G, Orlow SJ. Rab27b association with melanosomes: dominant negative mutants disrupt melanosomal movement. J Invest Dermatol. 2002;118:933–940. 54. Lambert J, Onderwater J, Vander HY, Vancoillie G, Koerten HK, Mommaas AM, Naeyaert JM. Myosin V colocalizes with melanosomes and subcortical actin bundles not associated with stress fibers in human epidermal melanocytes. J Invest Dermatol. 1998;111: 835–840. 55. Nascimento AA, Amaral RG, Bizario JC, Larson RE, Espreafico EM. Subcellular localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute gene. Mol Biol Cell. 1997; 8:1971–1988. 56. Wei Q, Wu X, Hammer JA, III. The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport. J Muscle Res Cell Motil. 1997;18:517–527. 57. Wu X, Bowers B, Wei Q, Kocher B, Hammer JA, III. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor. J Cell Sci. 1997;110(Pt 7):847–859. 58. Reck-Peterson SL, Provance DW Jr., Mooseker MS, Mercer JA. Class V myosins. Biochim Biophys Acta. 2000;1496:36–51. 59. Seperack PK, Mercer JA, Strobel MC, Copeland NG, Jenkins NA. Retroviral sequences located within an intron of the dilute gene alter dilute expression in a tissue-specific manner. EMBO J. 1995;14: 2326–2332. 60. Fukuda M, Kuroda TS, Mikoshiba K. Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J Biol Chem. 2002;277:12432–12436. 61. Kuroda TS, Fukuda M, Ariga H, Mikoshiba K. The Slp homology domain of synaptotagmin-like proteins 1–4 and Slac2 functions as a novel Rab27A binding domain. J Biol Chem. 2002;277: 9212–9218. 62. Strom M, Hume AN, Tarafder AK, Barkagianni E, Seabra MC. A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. J Biol Chem. 2002;277:25423–25430. 63. Wu XS, Rao K, Zhang H, Wang F, Sellers JR, Matesic LE, Copeland NG, Jenkins NA, Hammer JA, III. Identification of an organelle receptor for myosin-Va. Nat Cell Biol. 2002;4:271–278. 64. Griscelli C, Prunieras M. Pigment dilution and immunodeficiency: a new syndrome. Int J Dermatol. 1978;17:788–791.
The New Face of Pigmentation and Aging 65. Griscelli C, Durandy A, Guy-Grand D, Daguillard F, Herzog C, Prunieras M. A syndrome associating partial albinism and immunodeficiency. Am J Med. 1978;65:691–702. 66. Pastural E, Barrat FJ, Dufourcq-Lagelouse R, Certain S, Sanal O, Jabado N, Seger R, Griscelli C, Fischer A, de Saint BG. Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene. Nat Genet. 1997;16:289–292. 67. Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, Wulffraat N, Bianchi D, Fischer A, Le DF, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet. 2000;25:173–176. 68. Stinchcombe JC, Barral DC, Mules EH, Booth S, Hume AN, Machesky LM, Seabra MC, Griffiths GM. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J Cell Biol. 2001;152:825–834. 69. Menasche G, Ho CH, Sanal O, Feldmann J, Tezcan I, Ersoy F, Houdusse A, Fischer A, de Saint BG. Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). J Clin Invest. 2003;112:450–456. 70. Jablonski NG, Chaplin G. The evolution of human skin coloration. J Hum Evol. 2000;39:57–106. 71. Chaplin G, Jablonski NG. Hemispheric difference in human skin color. Am J Phys Anthropol. 1998;107:221–223. 72. Online Mendelian Inheritance in Man (OMIM), 2009. 73. Schaffer JV, Bolognia JL. The melanocortin-1 receptor: red hair and beyond. Arch Dermatol. 2001;137:1477–1485. 74. The International HapMap Consortium. A haplotype map for the human genome. Nature. 2005;437:1299–1320. 75. Stokowski RP, Pant PV, Dadd T, Fereday A, Hinds DA, Jarman C, Filsell W, Ginger RS, Green MR, van der Ouderaa FJ, et al. A genomewide association study of skin pigmentation in a South Asian population. Am J Hum Genet. 2007;81:1119–1132. 76. Han J, Kraft P, Nan H, Guo Q, Chen C, Qureshi A, Hankinson SE, Hu FB, Duffy DL, Zhao ZZ, et al. A genome-wide association study identifies novel alleles associated with hair color and skin pigmentation. PLoS Genet. 2008;4:e1000074. 77. Ginger RS, Askew SE, Ogborne RM, Wilson S, Ferdinando D, Dadd T, Smith AM, Kazi S, Szerencsei RT, Winkfein RJ, et al. SLC24A5 encodes a trans-Golgi network protein with potassiumdependent sodium-calcium exchange activity that regulates human epidermal melanogenesis. J Biol Chem. 2008;283:5486–5495. 78. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev. 2004;84:1155–1228. 79. Lerner AB, McGuire JS. Effect of alpha- and betamelanocyte stimulating hormones on the skin colour of man. Nature. 1961; 189:176–179. 80. Garcia RJ, Ittah A, Mirabal S, Figueroa J, Lopez L, Glick AB, Kos L. Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model. J Invest Dermatol. 2008;128:131–142.
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81. Reid K, Nishikawa S, Bartlett PF, Murphy M. Steel factor directs melanocyte development in vitro through selective regulation of the number of c-kitþ progenitors. Dev Biol. 1995;169:568–579. 82. Spritz RA. Piebaldism, Waardenburg syndrome, and related disorders of melanocyte development. Semin Cutan Med Surg. 1997;16:15–23. 83. Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res. 2004;17:96–110. 84. Cario-Andre M, Lepreux S, Pain C, Nizard C, Noblesse E, Taieb A. Perilesional vs. lesional skin changes in senile lentigo. J Cutan Pathol. 2004;31:441–447. 85. Motokawa T, Kato T, Katagiri T, Matsunaga J, Takeuchi I, Tomita Y, Suzuki I. Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions. J Dermatol Sci. 2005;37:120–123. 86. Aoki H, Moro O, Tagami H, Kishimoto J. Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics. Br J Dermatol. 2007;156:1214–1223. 87. Unver N, Freyschmidt-Paul P, Horster S, Wenck H, Stab F, Blatt T, Elsasser HP. Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin. Br J Dermatol. 2006;155:119–128. 88. Kadono S, Manaka I, Kawashima M, Kobayashi T, Imokawa G. The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol. 2001;116:571–577. 89. Maeda K, Ono T, Matsunaga J. The mechanism of hyperpigmentation in senile lentigo and the efficacy of skin lightening agents. Frag J. 2006;5:21–29. 90. Manaka L, Kadono S, Kawashima M, Kobayashi T, Imokawa G. The mechanism of hyperpigmentation in seborrhoeic keratosis involves the high expression of endothelin-converting enzyme-1alpha and TNF-alpha, which stimulate secretion of endothelin 1. Br J Dermatol. 2001;145:895–903. 91. Teraki E, Tajima S, Manaka I, Kawashima M, Miyagishi M, Imokawa G. Role of endothelin-1 in hyperpigmentation in seborrhoeic keratosis. Br J Dermatol. 1996;135:918–923. 92. Nouveau-Richard S, Yang Z, Mac-Mary S, Li L, Bastien P, Tardy I, Bouillon C, Humbert P, de LO. Skin ageing: a comparison between Chinese and European populations. A pilot study. J Dermatol Sci. 2005;40:187–193. 93. Tsukahara K, Fujimura T, Yoshida Y, Kitahara T, Hotta M, Moriwaki S, Witt PS, Simion FA, Takema Y. Comparison of agerelated changes in wrinkling and sagging of the skin in Caucasian females and in Japanese females. J Cosmet Sci. 2004;55:351–371.
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7 The Stratum Corneum and Aging Anthony V. Rawlings
Introduction The father of corneobiology, Albert Kligman [1], wrote in 1979: ‘‘No one dies of old skin! No matter how decrepit the integument becomes after a lifetime of assaults, it continues to perform its primary protective role. . . . But skin problems abound in the aged!’’ It is now known that dry itchy senile xerotic skin is a problem of faulty epidermal and stratum corneum (SC) maturation together with desquamation. The understanding of the changes in the chemistry and function of important stratum corneum (SC) components in aging and dry skin is a result of the tenacity of a plethora of academic and industrial scientists spanning several decades. These include studies on corneocyte size [2–4]; SC lipid levels, especially ceramides [5–9]; lipid ultrastructure and biophysics [10–12]; natural moisturizing factors (NMF) [13, 14]; SC proteases [15, 16]; corneodesmosomal proteins [11, 17, 18]; and finally corneocyte quality [19, 20]. Ultimately, changes in SC cohesion and desquamatory properties were studied by Ronald Marks [21]. Some of these changes, of course, were predicted in 1964 [22]. Key in SC function and maturation, however, is its hydration [23]. It was not until 1994 that the understanding of the perturbation of water gradients in the SC of subjects with xerotic skin was developed [24] and only in 1995 [25] it was shown that water itself was essential for corneodesmosomal degradation and ultimately desquamation. This chapter gives an overview of the latest understanding of the stratum corneum and aging (> Fig. 7.1).
Stratum Corneum Structure Corneodesmosomes obviously have a big role to play in the cohesion between corneocytes in the stratum compactum and the ‘‘apparent’’ lack of cohesion in the stratum disjunctum (SD) [26]. However, it has been learned in recent years that the interaction between corneocytes in the disjunctum layers is not as ‘‘loose’’ as originally anticipated. Using new high-pressure freezing and freeze substitution techniques for electron microscopy [27] the SC appeared to be more compact than expected with
smaller intercellular spaces and hence tighter intercorneocyte interactions. Equally using novel cryotransmission electron microscopy techniques to image vitreous sections of skin without the use of cryoprotectants [28] a more densely packed stratum corneum was again apparent. The decreased cohesive forces toward the surface layers of the SC, close to the intercorneocyte interaction, must be due to the degradation of corneodesmosomes toward the surface layers, although strong hydrophobic interaction between corneocytes was still observed due to the intercellular and covalently bound lipids. Thus, the basket weave appearance of the SC by histology and the presence of the more widely spaced SD are artifacts from the older methods. However, it is preferable to use the stratum compactum–disjunctum as the term to define their interface, which is probably one of the most biologically active parts of this important barrier tissue. In addition to the close intercorneocyte interaction, differences in the swellability of corneocytes in the different layers of the SC have also been observed [29, 30]. A lower non-swelling region (LNSR), a swelling region (SR) and an upper non-swelling region (UNSR) have been observed. The differences in the swelling regions of the SC are probably due to a combination of the loss of NMF in the outer layers of the SC, hydrolysis of filaggrin to NMF in the mid layers and lysis of non-peripheral corneodesmosomes, allowing greater intercorneocyte freedom above the stratum compactum, and transglutaminase-mediated maturation and rigidification of corneocytes toward the surface layers of the SC. As will be discussed, many of these events become aberrant in senile dry skin, but these methods need to be more rigorously applied to the study of senile SC.
Stratum Corneum Lipids All SC lipids are important for barrier function of the skin, but due to their unique structures, properties, and on a weight basis as they constitute approximately 50% of the SC lipids, ceramides have been the area of most interest in recent years [31]. In fact, 11 classes of ceramides have now been identified. The ceramide head groups are small, but can form extensive hydrogen
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_7, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 7.1 Schematic of the epidermis and the stratum corneum
. Figure 7.2 Nomenclature system of stratum corneum ceramides
bonds. They differ by head-group architecture and fatty acid chain length. Ceramides are classified in general as CER FB, where F indicates the type of fatty acid and B indicates the type of base [32] (> Fig. 7.2 and > Table 7.1). When an ester-linked fatty acid is present, a prefix E is used. Normal fatty acids (saturated or unsaturated), alphahydroxy fatty acids, and omega-hydroxy fatty acids are indicated by N, A, O, respectively, whereas sphingosines, phytosphingosines, and 6-hydroxysphingosine are indicated by S, P, and H. Sphinganine (not previously classified) is proposed to be SP in this nomenclature system. A novel long-chain ceramide containing branched chain fatty acids is also found in vernix caseosa [33]. Typical structures of human ceramides are given in > Table 7.1. Ceramides have also been found attached to the corneocyte envelope. In addition to ceramide A (sphingosine; CEROS) and ceramide B (6-hydroxysphingosine; CEROH), the presence of covalently bound omega
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. Table 7.1 Eleven classes of human Stratum Corneum ceramides Fatty acid α-hydroxy fatty acid [A]
Esterified ω-hydroxy fatty acid [EO]
CER [NDS]
CER[ADS]
CER[EODS] (not yet identified in SC)
CER[NS]
CER[AS]
CER[EOS]
CER[NP]
CER[AP]
CER[EOP]
CER[NH]
CER[AH]
CER[EOH]
Non-hydroxy fatty acid [N] Sphingoid Dihydrosphingosine [DS]
Sphingosine [S]
Phytosphingosine[P]
6-hydroxy sphingosine [H]
hydroxy fatty acid containing sphinganine and phytosphingosine ceramides has been identified [34]. These covalently bound ceramides should now be named CER OSP and CER OP. However, Hill et al. [35] could only find the presence of CER EOS (72.4%), CER EOH (19.5%), and CER OP (8.2%). Ceramides are produced as precursors in the epidermis in the form of glucosylceramides, epidermosides, or sphingomyelin (> Fig. 7.3). Epidermosides are glycated precursors of CER EOS, EOH, and EOP together with the ceramides covalently bound to the corneocyte envelope. Sphingomyelin provides a proportion of CER NS and CER AS, whereas the glucosylceramides are precursors to the other classes of ceramides [36]. The chain length distribution of the omega-hydroxy fatty acid portion of CER EOS has recently become a topic of interest. When first identified the omega-hydroxy fatty acid portion of CER EOS was reported to be mainly composed of C30 (63.6%) to C32 (14.9%) chain lengths with minor shorter chain length species [37]. However, longer chain variants
have been recently identified [38]. Rawlings et al. [39] recently reported that no chain length species were identified below C30 chain length and the bulk was either C32 or C34. Surprisingly more than 27% of the fatty acids were odd chain length species compared with previous findings of only 16%. This further heterogeneity in CER EOS composition needs to be understood for its effects on lipid lamellar packing at the molecular level and subsequently on SC function, and especially the impact of aging on these lipid species. Nevertheless, Imokawa et al. [5] first recorded the age-related decline in SC ceramides which will naturally impact on SC functioning. Reductions of all SC lipids with aging were reported slightly later [9] (> Fig. 7.4). It is the lipid-packing states, however, that influence SC barrier function. Lipids in vivo in the SC appear to exist as a balance between orthorhombic packing (a solid crystalline state) and hexagonal packing (gel). The orthorhombically packed lipids are the most tightly packed conformation and have optimal barrier properties, but a
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. Figure 7.3 Summary of main steps in epidermal biosynthesis of ceramides
. Figure 7.4 Age-related reductions in the levels of stratum corneum ceramides, fatty acids, and cholesterol from tape-strippings of the human face
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greater proportion of hexagonally packed lipid conformations are known to occur in the outer layers of the SC due to the increased presence of sebum [40]. This is consistent with a weakening of the barrier toward the outer layers of the stratum corneum. It is known that ceramides and cholesterol only form hexagonally packed lipids and it is the long-chain fatty acids that induce the orthorhombic packing states. However, it is possible that an excess of short-chain fatty acids and other lipids derived from sebum contribute to the crystalline to gel transition in the upper stratum corneum layers [41]. Naturally, during aging sebum declines and this transition will be reduced. If sebum influences desquamation in any way, then the age-related reduction in sebum lipids may contribute to the expression of xerotic skin. A sandwich model for the lamellar lipids consisting of two broad lipid layers with a crystalline structure separated by a narrow central lipid layer with fluid domains has been proposed [42] It seems that cholesterol and ceramides are important for the formation of the lamellar phase, whereas fatty acids play a greater role in the lateral packing of the lipids. Cholesterol is proposed to be located with the fatty acid tail of CER EOS in the fluid phase. CER EOS, EOH, and EOP play an essential role in the formation of the additional lamellar arrangements. The repeated distances were found to be 13 nm in dimension, composed of two units measuring approximately 5 nm each and one unit measuring approximately 3 nm in thickness. These repeat lamellar patterns were also observed by X-ray diffraction studies and were named the ‘‘long periodicity’’ (LPP) and ‘‘short periodicity’’ (SPP) phases, respectively. In the presence of long-chain fatty acids besides an orthorhombic phase the lipids also form a liquid phase. Another important ceramide, ceramide-1 as it used to be called but now known as CER EOS, has a dramatic effect on SC lipid phase behavior. For total lipid mixtures in the absence of CER EOS mostly hexagonal phases are also observed and equally no LPP phase or liquid phase is formed. Moreover, the importance of ceramide 1 or CER EOS linoleate in facilitating the formation of the LPP has been further elaborated by understanding the influence of the type of fatty acid esterified to the omega hydroxy fatty acid [43]. As a consequence, greater amounts of the LPP, orthorhombic, and liquid phases are observed mainly with linoleate-containing CER EOS, less with oleatecontaining CER EOS, and are totally absent if only stearate-containing CER EOS is present in the lipid mixtures. It seems that the presence of an unsaturated acyl chain is crucial for the formation of these phases and packing states. These studies indicate that for formation of the
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LPP, a certain fraction of the lipids has to form a liquid phase. If the liquid phase is too high (as with the oleatecontaining CER EOS) or too low (as with stearatecontaining CER EOS), the levels of the SPP increase at the expense of the LPP. It is important to remember in vivo that the fatty acid composition of CER EOS is highly complex, but it contains a large proportion of linoleic acid. Conti et al. [44] observed decreases in C18:2 and C24 fatty acids with increases in C18:1 and C16:0 in fatty acids of CER EOS in the winter months of the year. Similarly, Rogers et al. [9] demonstrated an agerelated decline in the reductions of the linoleate content of SC CER EOS with a trend of increasing oleate levels (> Fig. 7.5). Reductions in the levels of CER EOS linoleate could lead to reductions in both the LPP. It is interesting in this respect that CER EOS directly improves SC flexibility [45] and that it is vital for barrier function [46, 47]. One must not forget the influence of the environment on SC maturation and epidermal differentiation. In the same paper on the aging reductions in CER EOS linoleate levels, Rogers et al. [9] demonstrated that there was a significant reduction in the levels of SC ceramides and fatty acids, together with linoleate-containing CER EOS in subjects in winter compared with summer. Similar differences in scalp lipid levels have been observed between the wet and dry seasons in Thailand [48]. This appears to be related to changes in external humidity. Transepidermal water loss (TEWL) was reduced by approximately 30% in animals exposed to a dry (80 years) [19]. Sheu et al. reported that this may not be an aging problem in itself as these changes may be induced by an excess of sebum[54]. However, degradation of CER EOS to its corresponding acyl acid as reported by Wertz and Downing [55] may be the first step in causing a change in the lipid structure for preparing the process of desquamation. Equally, Long et al. [56] reported that the degradation of cholesterol sulfate may be involved in this process. This may be related to the reported distorted orthorhombic phase at the site of cohesive failure [57]. Thus, the relative amounts of SC lipids, sebum, and extent of washing will likely influence the presence of these structures, but they may be more easily visible as one ages. Nevertheless, this phase, that is, no organized lamellar structure, would appear consistently in subjects older than 40 [52].
Desquamation Over 4 decades ago the father of corneobiology [22] suggested that cell cohesion in the SC was dependent upon an
‘‘intercellular cement’’ that was predicted to become less stable near the surface of the skin or to be degraded by enzymes. It was not until 1979 that scientists in Professor Marks group in Cardiff [58, 59] clearly demonstrated reduced inter-corneocyte cohesion toward the outer layers of the SC as judged by mechanical cohesography or by using a standardized skin scrub stimulus. While reduced intracorneal cohesion occurs with aging a trend of reduced desquamation also occurs with increasing age, which seems to be at odds with the reported increased serine protease activity and elevated pH with aging [60]. Nevertheless, changes in desqumation and thereby SC cohesion are likely to be the result of changes in SC lipid organization, corneocyte interdigitation together with corneodesmosome numbers, and their state of degradation. Corneodesmosomes consist of the cadherin family of transmembrane glycoproteins desmoglein 1 (Dsg 1) and desmocollin 1 (Dsc 1), which bind each other on adjacent corneocytes. Inside the corneocytes, Dsg 1 and Dsc 1 are also linked to keratin microfilaments via corneodesmosomal plaque proteins such as plakoglobin, desmoplakins, and plakophilins. These provide stability and extracohesion to the SC ‘‘brick wall.’’ Otherwise the only intercorneocyte cohesive force would be that provided by intercellular as well as covalently bound lipids. Another corneodesmosomal protein, corneodesmosin (Cdsn), after secretion within the lamellar bodies with the intercellular lipids and certain proteases (but not all), becomes
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. Figure 7.6 Organization of stratum corneum lipids in tape-strippings of individuals with clinically normal skin. Transmission electron micrographs of tape-strippings. Ultrastructural changes in lipid organization toward the surface of the stratum corneum. (a) First strip: absence of bilayers and presence of amorphous lipidic material. (b) Second strip: disruption of lipid lamellae. (c) Third strip: normal lipid lamellae. (¥ 200,000)
associated with the desmosomal proteins just before transformation of desmosomes into corneodesmosomes. As all these proteins are cross-linked into the complex by transglutaminase, their controlled disruption must occur by proteolysis to reduce the intermolecular forces between the corneocytes to allow desquamation to proceed. Cdsn is thought to protect Dsg1 and Dsc1 against premature proteolysis. Cdsn undergoes several proteolytic steps. Cleavage of the N terminal glycine loop domain occurs first at the stratum compactum–disjunctum interface (48–46 to 36–30 kDa transition), followed by cleavage of the C terminal glycine loop domain in exfoliated corneocytes (36–30 to 15 kDa transition) [61]. The last step appears to be inhibited by calcium resulting in residual
intercorneocyte cohesion. Nevertheless, the presence of oligosaccharides did not protect Cdsn against proteolysis by KLK7 [62, 63]. Duhieu et al. [64] has demonstrated that the extracellular ‘‘cores’’ of epidermal desmosomes contain a highly glycosylated antigen, different from desmosomal cadherins. This protein, recognized by KM48 monoclonal antibody, is likely to be involved in the processes of keratinocyte differentiation, desmosome turnover, and epidermal cohesion. The process of corneodesmosomal degradation in tape-strippings of SC from human skin was shown by Rawlings et al. [11] together with the loss of the non-peripheral corneodesmosomes before the peripheral corneodesmosomes was also shown by the same group [17].
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Corneodesmolysis, and ultimately desquamation, is facilitated by the action of specific hydrolytic enzymes in the stratum corneum that degrade the corneodesmosomal linkages. Currently, several cysteine and aspartic enzymes are believed to be involved in this process, namely stratum corneum thiol protease (SCTP, now known as Cathepsin L-2), the aspartic proteases cathepsin E and cathepsin D, and the skin aspartic protease [65–70]. But the ones most researched are the kallikreins and several have now been immunologically identified within the SC (KLK5, 6, 7, 8, 10, 11, 13, and KLK14, where KLK5 = stratum corneum tryptic-like enzyme (SCTE) and KLK7 = stratum corneum chymotryptic-like enzyme (SCCE) [71]). Only KLK5, 8, and 14, however, are capable of degrading Dsg1 [72], but it is known that KLK8 also contributes to desquamation [73]. A depth activity profile of these enzymes are reported [74, 66], but Voegeli et al. [75] recently demonstrated that premature activation of, and elevated activity levels of KLK5, but not KLK7, on barriercompromised body sites such as the face but not the forearm (> Fig. 7.7). Elevated activity levels of plasmin, urokinase, and a newly identified SC tryptase-like enzyme were also found. An endoglycosidase, heparanase 1, has been identified within the stratum corneum, which is thought to play a role in the pre-proteolytic processing of the protecting sugar moieties on corneodesmosomal proteins [76]. Some of the desquamatory enzymes are secreted with the lamellar bodies and have been immunolocalized to the intercorneocyte lipid lamellae. Sondell et al. [77] used antibodies that immunoreact precisely with pro-KLK7
to confirm that this enzyme is transported to the stratum corneum extracellular space via lamellar bodies. In later studies, using antibodies to both pro-KLK7 and KLK7, Watkinson et al. [78] demonstrated that the processed enzyme was more associated with the corneodesmosomal plaque. More recently, Igarashi et al. [79] have immunolocalized cathepsin D to the intercellular space, whereas cathepsin E was localized within the corneocytes. Finally, KLK8 has also been reported to be localized to the intercellular spaces of the SC [80]. These enzymes, however, are transported in different lamellar granules to their inhibitor proteins. Naturally, as some of the desquamatory enzymes are present in the intercellular space, the physical properties of the stratum corneum lipids, together with the water activity in this microenvironment, will influence the activity of these enzymes. In this respect, KLK7 appears to have a greater tolerance to water deprivation than other proteolytic enzymes and this may be an adaptation to maintain enzyme activity even within the water-depleted stratum corneum intercellular space [81]. Of all the antiproteases found within the SC it is now thought that only the domains of the lymphoepithelial-Kazal-type 5 inhibitor (LEKTI) of which there are at least 15 inhibitory domains that are spliced from the mature proteins via subtilisin-like enzymes and furin [82] are responsible for attenuating the activity of the kallikreins. Interestingly, the association–disassociation constants between the desquamatory proteins and these inhibitors are influenced by pH [83]. There is a strong association of the
. Figure 7.7 Protease activity (mean SEM) on tape-stripping pools of forearm and of cheek in function of depth of stratum corneum
The Stratum Corneum and Aging
inhibitor–enzyme complexes at neutral pH, that is, as the SC is formed, and dissociation of the complexes occurs at low pH, within the outermost surface layers of the SC, where higher activities of the kallikreins are actually observed [75]. As at least pro-KLK7 is processed early in the formation of the SC and that LEKTI is secreted before the enzymes [78], presumably the inhibitors diffuse to the activated enzymes to control the later stages of desquamation. However, KLK5 is also capable of degrading LEKTI and its fragments and may in fact make them pro-inflammatory [84]. Thus, theoretically, at higher skin pHs there should be reduced desquamation as LEKTI–protease complexes occur. This may account for the apparent reduced cohesion, but not yet relating to increased desquamation. Like the levels of lipids the levels of enzymes also change according to the external environment. Declercq et al. [85] have reported an adaptive response in human barrier function, where subjects living in a dry climate such as Arizona (compared with a humid climate in New York) had much stronger barrier function and less dry skin due to increased ceramide levels and desquamatory enzyme levels (KLK5 and 7). Reduced kallikrein activity is also reported for aged SC especially for trypsin-like enzyme activity, but not chymotrypsin-like activity (> Fig. 7.8) [15] with KLK8 showing the greatest decline in mass levels [71].
. Figure 7.8 Age-related reductions in the levels of stratum corneum trypsin-like activity (KLK5-like) from tape-strippings of human leg (Chymotrypsin-like activity showed no aging changes)
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The role of the newly identified skin aspartic protease [86] and caspase 14 [87] in this process is still awaiting clarification and also their relationship to the activities of cathepsins, kallikreins, and intracorneocytic proteases [88]. Nevertheless, it would appear that the reduced levels of trypsin-like enzymes are contributing to senile xerotic skin (photodamaged skin will be considered later).
Corneocyte Envelope Maturation The change in intercorneocyte cohesion from the inner to the outer layers of the SC is also paralleled with changes in corneocyte morphology. Examining changes in corneocytes with increasing depth [89] it was reported that there was an increased folding of the cell surface leading to a more swollen appearance in the deeper depths of the SC. The cells appeared ‘‘curled’’ up, fatter, and less regularly shaped with many microvillous projections. This appeared to be close or within the stratum compactum as ‘‘corneocytes were clumped together and desmosomal attachments were seen apparently undisturbed.’’ These results suggested that there was a continuing maturation of these cells during their journey to the surface layers of the SC. Apart from corneodesmolysis and filaggrinolysis (discussed in the next section) changes to the corneocyte envelope can also occur. The CE is an extremely insoluble cross-linked proteinaceous layered structure. Disulfide, glutamyl-lysine isodipeptide bonds, or glutamyl polyamine cross-linking of glutamine residues of several corneocyte envelope proteins occurs by the action of transglutaminases (TGase) [90]. TGase 1, 3, and 5 isoforms are thought to be involved in this process. Equally, TGase esterifies lipids to involucrin on the CE [91–94]. Serge Michel et al. [19] at CIRD further investigated the morphology and composition of CEs. When viewed by Normarski microscopy, CEs were shown to have a crumpled surface when isolated from the lower layers of the stratum corneum, named fragile envelopes (CEf) and a smoother, more flattened surface when isolated from the upper stratum corneum rigid envelopes (CEr). This did not appear to be related to any change in the protein composition of the CE, but the authors did speculate on the effect of adding a hydrophobic layer of lipid. Mils et al. [95] reported that about 80% of corneocytes from volar forearm skin were smooth and rigid, whereas 90% from foot sole were rough or fragile cells. More recent work by Kashibuchi et al. [96] using atomic force microscopy confirmed these structural changes in corneocytes from the deeper layers of the stratum corneum. However, they reported that there was
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. Figure 7.9 Positive correlation between corneocyte flatness index and aging
addition, recovery of barrier function after scarring is also very much dependent on changes in corneocyte size and maturation and appears to be less so for lipid changes but more characterization is needed [99]. As will be seen later retention of immature fragile envelopes occurs in barrier-compromised conditions such as dry skin.
Stratum Corneum Natural Moisturizing Factors
an age-related reduction in corneocyte thickness and an increase in corneocyte flatness index, that is, corneocytes appear to be getting thinner but longer with age (> Fig. 7.9). This may be to countereffect the known reductions in epidermal lipid synthesis as a balancing mechanism to increase SC tortuosity and maintain ‘‘barrier’’ function, that is, TEWL. Nevertheless, SC function is severely compromised due to the reduced epidermal lamellar granule secretion of lipids and especially desquamatory proteases. CEs can also be further differentiated by their binding of tetramethylrhodamine isothiocyanate (TRITC), where the rigid envelopes stain to a greater extent [97] or based upon their hydrophobicity (staining with Nile red) and antigenicity (to anti-involucrin) [20]. It is clear from these studies that immature envelopes (CEf) occur in the deeper layers of the stratum corneum (involucrinpositive and weak staining to Nile red or TRITC) and that mature envelopes occur in the surface layers of healthy skin (apparent involucrin staining lessened and increased staining with Nile red or TRITC). The classification of fragile and rigid envelopes has subsequently been found to be a pertinent classification system as, mechanically, they have fragile and rigid characteristics under compressional force. The detection of cumulative TGase-induced cross-links in the CE is an evidence for the role of these enzymes in the maturation of these cells [97] and its activation appears to coincide with filaggrinolysis, that is, above the stratum compactum. It is also essential to consider the importance of corneocyte size in controlling skin barrier function, which seems to be underestimated. As shown by Marks, the smaller the corneocyte the greater the TEWL [98]. In
Generation of natural moisturizing factors (NMF) is summarized in > Fig. 7.10, which also highlights the importance of peptidylarginine deminases involved in the processing of filaggrin and thereby allows its hydrolysis to NMF [100]. Under most circumstances filaggrin is degraded to amino acids in the SC. Nevertheless, Harding and Rawlings [101] recently reported that there was a minor perturbation in filaggrin processing leading to the persistence of a high-molecular weight filaggrin in the superficial SC. It appears that in some individuals an imbalance in the activity of peptidyl deiminase (PAD) to general filaggrinase activity may lead to the formation of a form of filaggrin in which complete deimination (through PAD activity) by effectively depleting trypsin-sensitive protease sites (through arginine to citrulline conversion on the filaggrin protein) renders the protein refractive to filaggrinase activity. It has previously been shown that it is the general filaggrinase activity rather than PAD activity that is sensitive to changes in external RH. Changes in filaggrin and NMF levels are expected from changes in the relative humidity the skin is exposed to. Similarly, findings were reported for the water-holding capacity and free amino acid (FAA) content of the SC. Katagiri et al. [102] demonstrated that exposure of mice to a humid environment, and subsequent transfer to a dry one, reduced skin conductance and amino acid levels even after 7 days following transfer; after transfer from a normal environment, however, decreased amino acid levels recovered within 3 days. It is thought that NMF allows the outermost layers of the SC to retain moisture against the desiccating action of the environment. Traditionally, it was believed that this water plasticized the SC. However, it might be more complicated than this. The general mechanisms by which these NMF components influence SC functionality have been studied extensively [103]. The specific ionic interaction between keratin and NMF, accompanied by a decreased mobility of water, leads to a reduction of intermolecular forces between the keratin fibers and increased elastic behavior and recent studies have emphasized that it is the
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. Figure 7.10 Schematic representation of profilaggrin catabolism and filaggrin hydrolysis to natural moisturizing factors and activation of peptidylarginine deiminase
neutral and basic free amino acids, in particular, which are important for the plasticization properties of the SC. A general decline in NMF occurring with age was reported by Rawlings et al. [104] (> Fig. 7.11) and Horii et al. [13]. The latter also examined the effects of xerosis. However, Jacobsen et al. [14] reported that the levels of Ser, Glu, and Gly were increased in aged subjects, whereas the levels of Leu, Phe, Lys, Trp, and Orn were decreased. Gly, Leu, Tyr, Phe, and Lys were elevated in xerotic elderly SC. There were no changes in the total levels of amino acids, but only superficial SC was examined. As shown by the studies of Horii et al. [13] and Rawlings et al. [104] greater differences between young and aged skin are found in the deeper layers of the SC. However, a recent publication from Takashashi and Tezuka [105] have suggested that the content of FAA in the SC is actually increased in both senile xerosis and in ‘‘normal’’ aged skin compared to young. Indeed, these observations are consistent with earlier observations on the age-related increase in the levels of certain FAA
primarily found in filaggrin (serine, glutamic acid, glycine) made by Jacobsen [14]. However, in both studies they were only examining superficial SC and relatively for the same volume of space there are more cells in old skin compared with young skin. Hyaluronic acid and glycerol have recently been shown to be present naturally in the SC and are now considered to be part of the skins NMF [106]. Glycerol can also be derived from sebaceous triglyceride breakdown and again, to emphasize the importance of this molecule, studies by Fluhr et al. [107] have indicated that topically applied glycerol can completely restore the poor quality of SC observed in asebic mice (that are lacking sebaceous secretions) to normal. The importance of glycerol as a natural skin moisturizing molecule has also been shown by Choi et al. [108]. An acid pH within the SC, the ‘‘acid mantle,’’ is critical to the correct functioning of this tissue. Studies point to the essential role of free fatty acids generated through phospholipase activity as being vital for SC acidification [109],
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. Figure 7.11 Depth and age-related reduction in stratum corneum PCA levels
while Krein and Kermici [110] have recently proposed that urocanic acid (UCA) plays a vital role in the regulation of SC pH. Although this is in dispute, it is likely that all NMF components contribute significantly to the overall maintenance of pH. Other components of NMF are also not derived from filaggrin and urea, like lactate, may also be derived in part from sweat. A further understanding of the effects of lactate on SC properties has been recently described. Lactate and potassium were found to be the only components of the NMF analyzed that correlated significantly with the state of hydration, stiffness, and pH, in the SC [111]. However, the presence of sugars in the SC primarily represents the activity of the enzyme beta-D-glucocerebrosidase, as it catalyzes the removal of glucose from glucosylceramides to initiate lipid lamellae organization in the deep stratum corneum.
Dry Skin In dry, flaky skin conditions, corneodesmosomes are not degraded efficiently and corneocytes accumulate on the skin’s surface layer leading to scaling and flaking. Increased levels of corneodesmosomes in soap-induced dry skin were first reported by Rawlings et al. [11] but have been confirmed more recently by others [18] (> Fig. 7.12a, b). This is consistent with the reported increase in the number of cell layers in the SC, reduced turnover time, and larger corneocytes in senile xerosis [7]. Many corneodesmosomal proteins are now also reported to be increased in the surface layers of xerotic skin [23, 71–73]. Originally,
Dsg-1 [11] and Dsc-1 were reported [17]. Interestingly, however, in winter xerosis, the accumulation of the corneodemosomal proteins, Dsg 1 and plakoglobin, correlate with each other [18]. Cdsn protein levels, which were also increased, do not, however, have such an association suggesting that different proteolytic mechanisms occur for the different corneodesmosomal components during desquamation. Simon et al. [18] suggested that, as plakoglobin is a cytoplasmic protein, this would indicate that at least the cytoplasmic domain of Dsg 1 may be cleaved. Perhaps the intracellular portions of Dsg 1 are also degraded within the corneocyte (e.g., by the trypsin-like activity [88] or cathepsin E activity reported within the corneocyte matrix [79]). Conversely, Cdsn and cadherins might be degraded by kallikreins or cathepsin D in the lamellar matrix. This is consistent with the early electron microscope images of Rawlings et al. [11] which show that corneodesmosomes become internally vacuolated, followed by detachment of the protein structures from the corneocyte envelope before their complete degradation. The lamellar lipid matrix is also perturbed dramatically in dry skin (> Fig. 7.13) [11]. As the main desquamatory enzymes are found within this lipid matrix, the physical properties of the lamellar lipids will, therefore, influence enzyme activity. Reduced levels of stratum corneum KLK7 (SCCE) were originally reported by Harding et al. [16] in the outer layers of xerotic stratum corneum compared with normal skin (> Fig. 7.14). This has been confirmed recently by others [114] who also found that the equally important stratum corneum KLK5 (SCTE) activities were also reduced.
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. Figure 7.12 (a) Conventional transmission electron microscopy confirms the persistence of corneodesmosomes in the outer SC of winter xerosis skin. Varnish-strippings of normal (a, c) and winter xerosis (b, d) skin were analyzed by conventional transmission electron microscopy. Note that, when combined, the micrographs of normal SC correspond to the whole sample. Only part of the total height of xerosis SC, which is much thicker, is shown, however. In the outer SC of normal skin (a), corneocytes are loose and corneodesmosomes (arrows) are scarcely observed. In the outer SC of xerotic skin (b), corneocytes are more cohesive and corneodesmosomes are numerous. In the inner SC of both normal (c) and xerotic (d) skin, corneodesmosomes are numerous. Arrowheads indicate the outer surface of the samples. Scale bar: 0.5 mm. (b) Quantification of corneodesmosome density at the surface of corneocytes confirms the reduced degradation of the structures in the outer SC of winter xerosis skin. The corneodesmosome density (area occupied by corneodesmosomes divided by total area of the micrograph) was measured on electron micrographs of the inner SC (two micrographs per individual) and the outer SC (four micrographs per individual) of normal (NS, n = 2) and winter xerosis (XS, n = 3) skin samples. Histograms representing the mean values show similar densities of corneodesmosomes in the outer SC of xerotic and normal skin. The density of corneodesmosomes in the upper SC of xerotic skin, however, was significantly increased compared with normal skin (p < 0.001). Bars, standard deviations
Conversely, in SLS-induced dry skin, increased activities of these enzymes are reported [66, 75]. More recently, the overactivation of the plasminogen cascade has been associated with dry skin. Normally, being observed only in the epidermal basal layers, skin plasmin is widely distributed through the epidermis in dry skin. A urokinasetype plasminogen activator also exists in the stratum corneum [112]. These inflammatory enzymes as well as KLK5 are also increased on barrier-compromised sites such as the face and in fact show a positive correlation with TEWL (> Fig. 7.15) [75]. Clearly, these and other enzymes are
potentially involved in the inflammatory and hyperproliferative aspects of dry skin. It is now well established that, in hyperproliferative disorders such as aged and dry skin (even in dry skin of the aged) an increase in epidermopoiesis is found relative to a younger control group and there is a change in stratum corneum lipid levels and composition. Imokawa et al. [5] first recorded the age-related decline in SC ceramides, in 1992 Rawlings et al. [113] first reported an approximately 50% decline of ceramides in winter xerosis mainly due to their extraction from the outer layers of the
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. Figure 7.13 Organization of stratum corneum lipids in tape-stripping of subjects with winter xerosis. Transmission electron micrographs of tape-strippings of individuals with severe xerosis. Perturbation in lipid organization toward the surface of the stratum corneum. (a) First strip: disorganized lipid lamellae. (b) Second strip: disorganized lipid lamellae. (c) Third strip: normal lipid lamellae (¥ 200,000)
SC, and in 1993 Hara et al. [7] then published the reduced levels of SC ceramides in senile xerosis. Changes in the composition of the ceramide subtypes are reported to occur with a predominance of sphingosine-containing ceramides (at the expense of the phytosphingosinecontaining ceramides) observed in the SC of subjects with dry skin [114]. However, Saint-Leger et al. [115] could not find any changes in ceramide levels in dry skin, but found increased fatty acid levels. Likewise, Rawlings et al. [11, 113] demonstrated a positive correlation between xerosis and fatty acids. Conversely, Fulmer and Kramer [8] also showed that longer chain fatty acid species were lost from the SC samples in their SLS-induced
xerosis model versus short-chain fatty acids. Others also observed a shortening and lengthening of the acyl sphingoid bases sphingosine and 6-hydroxysphingosine, respectively and reduced phytosphingosine-containing ceramides [116]. Imokawa et al. [5] did not find reduced ceramide levels in xerotic skin (but only total SC levels, rather than superficial levels, were measured). These changes in lipid composition will, of course, influence the lamellar packing of the lipids and a reduction of CER EOS and EOH with increased concentrations of sphingosine-containing ceramides (CER NS and CER AS) and crystalline cholesterol in association with a loss of the LPP in dry skin [117]. However, although the lipid
The Stratum Corneum and Aging
. Figure 7.14 Reduction in Stratum Corneum Chymotryptic-like Enzyme (SCCE) activity in tape-strippings of stratum corneum from xerosis
ultrastructure is clearly aberrant in the outer layers of dry skin [11], more work is needed to ascribe a particular lipid phase in senile dry skin. The proportions of the different corneocyte envelope phenotypes also change in subjects with dry skin [97]. Soap washing leads to a dramatic increase in the levels of the fragile envelope phenotype at the expense of the rigid phenotype (> Fig. 7.16). It is known that stratum corneum transglutaminase activities increase toward the surface of the stratum corneum, particularly the detergent-soluble and particulate fractions. Although the same trend of the relative increase in TGase between the inner and outer corneum is true of dry skin, TGase activities are dramatically lowered in dry skin compared with healthy skin, particularly the detergent-soluble fraction, which contains mainly TGase 1 [118]. Reduced NMF levels are also implicated in dry skin conditions [13]. However, Jacobsen et al. [14] reported relative increases in Gly, Leu, Tyr, Phe, and Lys in xerotic elderly SC. Finally, Ginger et al. [119] reported that the allelic polymorphism recognized in the profilaggrin gene may be linked to a predisposition to dry skin. The profilaggrin gene codes for a 10, 11, or 12 filaggrin-repeat, and therefore an individual can be 10:10, 10:11, 11:11, 10:12, 11:11, or 12:12. Using a PCR-based approach individual
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profilaggrin allelotypes were determined and an inverse association between the 12 repeat allele and the frequency of self-perceived dry skin was identified (n = 89, p = 0.0237). This novel observation could not be explained by a simple reduction in NMF production, and provides further circumstantial evidence for profilaggrin itself (rather than filaggrin or NMF) playing a critical role in epidermal differentiation. Engelke et al. [120] also reported age-independent decrease of keratins K1 and K10 with an associated increase in the basal keratins K5 and K14. There was also premature expression of involucrin. In 1987, Leveque et al. [121] reported the ‘‘Biophysical characterization of dry facial skin’’ and pointed out that dry skin was related to a slight increase in epidermopoiesis, leading in turn to a less-stretchable stratum corneum, a physical property linked to both stratum corneum water content and thickness, but it was less well correlated with impaired barrier function. Nevertheless, the differences in the SC at different body sites should also be considered. For example, Bhwan et al. [122] reported the histopathologic differences in the photoaging process in facial versus arm skin of Caucasians between the ages of 30 and 50 years. The facial skin has a greater number of granular cell layers and a higher degree of keratinocyte atypia. Equally, at the histogical level there was a greater compact SC appearance versus basket weave on the face compared to the forearm skin, which would be consistent with the reduced amount of protein that can be removed from the face by tape-stripping [75] and the reduced SC thickness. Although, a reduced SCTE/KLK5 trypsin-like enzyme activity has been reported in the SC derived from calf tissue [15], due to the effects of UV on the face increased proteases may be found. This may be due to the intrinsically inferior barrier function [123] on the face where increased protease activity correlates with TEWL (except KLK7/SCCE chymotrypsin-like enzyme activity) or the direct effects of UV increasing the expression of KLK5/7, while decreasing the expression of LEKTI. Thus, the face may retain a thinner SC throughout life with the appropriate levels of desquamatory proteases, whereas senile xerosis might only occur on the body SC due to reduced proteases. This would be consistent with the work of Egawa et al. [124] showing an aged-related increase in SC thickness, as measured using in vivo confocal Raman microspectroscopy on the forearm but not on the face (> Fig. 7.17a, b). Nevertheless, increased proteases on the face and especially inflammatory proteases may contribute to somatosensory problems. Minimally, however, this would mean that facial skin always has a reduced barrier reserve, as coined by Cork et al. [125] throughout life, that
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. Figure 7.15 (a) Correlation of kallikrein (KLK) activities and transepidermal water loss (TEWL): SC trypsin-like KLKs, SC chymotrypsin-like KLKs (b) correlation of inflammatory serine protease activities and TEWL: plasmin and urokinase and (c) correlation of SC tryptase-like activity and TEWL
. Figure 7.16 Changes in the proportion of fragile (CEf) and rigid (CEr) corneocyte envelopes in normal and dry skin
is, the SC is always thinner and the corneocytes are always smaller on the face compared to other body sites although there are age-related changes in facial corneocyte size (> Fig. 7.18a), TEWL hardly changes on the face with age unlike other body sites (> Fig. 7.18b). Nevertheless, dry body skin is the highest unmet cosmetic body skincare need across the world [126].
Conclusion The knowledge of SC biology especially in relation to aging has increased tremendously over the last few decades. Decreases in lipid levels especially ceramides are consistently reported even on the face. In this respect decreased CER EOS linoleate levels occur. Decreased NMF levels occur
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. Figure 7.17 (a) Age-dependent variations in the depth profiles of water content in the stratum corneum of the forearm (a) and cheek (b) skin. (b) Age-dependent changes in the stratum corneum apparent thickness (SCAT) in the forearm and cheek skin
. Figure 7.18 (a) Positive correlation between corneocyte size and aging on the cheek. (b) Slight negative correlation with TEWL and aging on the cheek (even TEWL on older subjects, however, is significantly elevated compared to other body sites)
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although some report increases, which may be related to increased corneocyte size and SC thickness. Corneocytes get bigger and flatter and on non-facial body sites the SC gets thicker. Decreased KLK5/SCTE also occurs with aging on non-facial body sites, which probably contributes to the expression of senile xerosis and reduced desquamation. The increased skin surface pH and protease activity lead to reduced SC cohesion with age, but appear not to be affecting superficial desquamation positively. The increased activities of desquamatory enzymes as well as inflammatory proteases probably contributes to the maintenance of a relatively thin facial SC with elevated TEWL and reduced barrier reserve throughout all ages compared with other body sites.
Cross-references > Corneocyte
Size and Cell Renewal: Effects of Aging and Sex Hormones > Physiological Variations During Aging > Stratum Corneum Cell Layers
References 1. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–46. 2. Plewig G, Marples RR. Regional differences of cell sizes in the human stratum corneum. I. J Invest Dermatol. 1970;54(1):13–18. 3. Grove GL. Exfoliative cytological procedures as a nonintrusive method for dermatogerontological studies. J Invest Dermatol. 1979;73 (1):67–69. 4. Marks R, Dawber RP. Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Dermatol. 1971;84 (2):117–123. 5. Imokawa G, Abe A, Jin Y, et al. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol. 1991;96(4):523–526. 6. Denda M, Hori J, Koyama J. Stratum corneum sphingolipids and free amino acids in experimentally induced scaly skin. Arch Dermatol Res. 1992;284(6):363–367. 7. Hara M, Kikuchi K, Watanabe M, et al. Senile xerosis: functional, morphological and biochemical studies. J Geriatr Dermatol. 1993;1:111–120. 8. Fulmer AW, Kramer GJ. Stratum corneum lipid abnormalities in surfactant induced dry scaly skin. J Invest Dermatol. 1986;86 (5):598–602. 9. Rogers J, Harding CR, Mayo A, et al. Stratum corneums lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288: 765–770. 10. Ghadially R, Brown BE, Sequeira-Martin SM, et al. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95(5):2281–2290.
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49. Denda M, Sato J, Masuda Y, et al. Exposure to a dry environment enhances epidermal permeability barrier function. J Invest Dermatol. 1998;111:858–863. 50. Sato J, Denda M, Chang S, et al. Abrupt decreases in environmental humidity induce abnormalities in permeability barrier homeostasis. J Invest Dermatol. 2002;119:900–904. 51. Chou TC, Lin KH, Wang SM, et al. Transepidermal water loss and skin capacitance alterations among workers in an ultra-low humidity environment. Arch Dermatol Res. 2005;296:489–495. 52. Warner RR, Boissy YL. Effect of moisturizing products on the structure of lipids in the outer stratum corneum of humans. In: Loden M, Maibach HI (eds) Dry Skin and Moisturisers. Boca Raton: CRC Press, 2000, pp. 349–372. 53. Berry N, Charmeil C, Gouion C, et al. A clinical, biometrological and ultrastructural study of xerotic skin. Int J Cosmet Sci. 1999;21: 241–249. 54. Sheu HM, Chao SC, Wong TW, et al. Human skin surface lipid film: an ultrastructural study and interaction with corneocytes and intercellular lipid lamellae of the stratum corneum. Br J Dermatol. 1999;140(3):385–391. 55. Wertz PW, Downing DT. Hydroxyacid derivatives in human epidermis. Lipids. 1988;23(5):415–418. 56. Long SA, Wertz PW, Strauss JS, et al. Human stratum corneum polar lipids and desquamation. Arch Dermatol Res. 1985;277(4):284–287. 57. Chen YL, Wiedmann TS. Human stratum corneum lipids have a distorted orthorhombic packing at the surface of cohesive failure. J Invest Dermatol. 1996;107(1):15–19. 58. King CS, Barton SP, et al. The change in properties of the stratum corneum as a function of depth. Br J Dermatol. 1979;100(2):165–172. 59. Marks R, Lawson A, Nicholls S. Age-related changes in stratum corneum structure and function. In: MarksR (ed) The Stratum Corneum. Cardiff: Stratum Corneum Group, 1986, pp. 10–15. 60. Choi EH, Man MQ, Xu P, et al. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol. 2007;127:2847–2856. 61. Serre G, Mils V, Haftek M, et al. Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope. J Invest Dermatol. 1991;97:1061–1072. 62. Simon M, Jonca N, Guerrin M, et al. Refined characterization of corneodesmosin proteolysis during terminal differentiation of human epidermis and its relationship to desquamation. J Biol Chem. 2001;276:20292–20299. 63. Caubet C, et al. Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family. J Invest Dermatol. 2004;122:1235–1244. 64. Duhieu S, Laperdrix C, Hashimoto T, et al. Desmosome-binding antibody KM48 recognises an extracellular antigen different from desmosomal cadherins Dsg 1–3 and Dsc 1–3. Eur J Dermatol. 2006;15(2):80–84. 65. Lundsto¨rm A, Egelud T. Cell shedding from human plantar skin in vitro: evidence that two different types of protein structures are degraded by a chymotrypsin-like enzyme. Arch Dermatol Res. 1990;282:234–237. 66. Suzuki Y, Nomura J, Koyama J, et al. The role of proteases in stratum corneum: involvement in stratum corneum desquamation. Arch Dermatol Res. 1994;286:249–253. 67. Horikoshi T, Igarashi S, Uchiwa H, et al. Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation. Br J Dermatol. 1999;141:453–459.
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68. Horikoshi T, Arany I, Rajaraman S, et al. Isoforms of cathepsin D human epidermal differentiation. Biochimie. 1998;80:605–612. 69. Watkinson A. Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation. Arch Dermatol Res. 1999;291:260–268. 70. Bernard D, et al. Analysis of proteins with caseinolytic activity in a human SC extract revealed a yet unidentified cysteine protease and identified the so called ‘‘SC thiol protease’’ as Cathepsin L2. J Invest Dermatol. 2003;120:592–600. 71. Komatsu N, Saijoh K, Sidiropoulos M, et al. Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender. J Invest Dermatol. 2005;125:1182–1189. 72. Borgon˜o CA, Michael IP, Komatsu N, et al. A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation. J Biol Chem. 2007;282(6):640–652. 73. Kishibe M, Bando Y, Terayama R, et al. Kallikrein 8 is involved in skin desquamation in cooperation with other kallikreins. J Biol Chem. 2007;282(8):5834–5841. 74. Suzuki Y, Nomura J, Hori J, et al. Detection and characterization of endogenous protease associated with desquamation of stratum corneum. Arch Dermatol Res. 1994;285:372–377. 75. Voegeli R, Rawlings AV, Doppler S, et al. Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme. Int J Cosmet Sci. 2007;29:191–200. 76. Bernard D, Mehul B, Delattre C, et al. Purification and characterization of the endoglycosidase heparanase 1 from human plantar stratum corneum: a key enzyme in epidermal physiology. J Invest Dermatol. 2001;117:1266–1273. 77. Sondell B, Thornell LE, Stigbrand T, et al. Immunolocalization of SCCE in human skin. Histo Cyto. 1994;42:459–465. 78. Watkinson A, Smith C, Coan P, et al. The role of Pro-SCCE and SCCE in desquamation. 21st IFSCC Congress, Berlin, 2000, pp. 16–25. 79. Igarashi S, Takizawa T, Yasuda Y, et al. Cathepsin D, and not cathepsin E, degrades desmosomes during epidermal desquamation. Br J Dermatol. 2004;151:355–361. 80. Ishida-Yamamoto I, et al. Epidermal lamellar granules transport different cargoes as distinct aggregates. J Invest Dermatol. 2004; 122:1145–1153. 81. Watkinson A, Harding C, Moore A, et al. Water modulation of stratum corneum chymotryptic enzyme activity and desquamation. Arch Dermatol Res. 2001;293:470–476. 82. Komatsu N, Takata M, Otsuki N, et al. Elevated stratum corneum hydrolytic activity in Netherton syndrome suggests an inhibitory regulation of desquamation by SPINK5-derived peptides. J Invest Dermatol. 2002;118:436–443. 83. Deraison C, Bonnart C, Lopez F, et al. LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction. Mol Biol Cell. 2007;18(9):3607–3619. 84. Yamasaki K, Di Nardo A, Bardan A, et al. Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nat Med. 2007;13(8):975–980. 85. Declercq L, Muizzuddin N, Hellemans L, et al. Adaptation response in human skin barrier to a hot and dry environment. J Invest Dermatol. 2002;119:716. 86. Bernard D, Me´hul B, Thomas-Collignon A, et al. Identification and characterization of a novel retroviral-like aspartic protease specifically expressed in human epidermis. J Invest Dermatol. 2005;125(2): 278–287.
87. Fischer H, Stichenwirth M, Dockal M, et al. Stratum corneumderived caspase-14 is catalytically active. FEBS Lett. 2004;577 (3):446–450. 88. Watkinson A, Smith C, Rawlings AV. The identification and localization of tryptic and chymotryptic-like enzymes in human stratum corneum. J Invest Dermatol. 1994;102:637. 89. King CS, Nicholls S, Barton S, et al. Is the stratum corneum of uninvolved psoriatic skin abnormal? Acta Dermatol Venereol Suppl (Stockh). 1979;59(85):95–100. 90. Watkinson A, Harding CR, Rawlings AV. The cornified envelope: its role on stratum corneum structure and maturation. In: Leyden JJ, Rawlings AV (eds) Skin Moisturization. New York: Marcel Dekker, 2002, pp. 95–117. 91. Candi E, et al. Transglutaminase cross linking properties of the small proline rich 1 family of cornified envelope proteins. J Biol Chem. 1999;274:7226–7237. 92. Kim IG, Gorman JJ, et al. The deduced sequence of the novel protransglutaminase-E (TGase 3) of human and Mouse. J Biol Chem. 1993;268:12682–12690. 93. Nemes Z, Marekov LN, Steinert PM. Involucrin cross linking by transglutaminase 1. J Biol Chem. 1999;274:11013–11021. 94. Cabral A, Voskamp P, Cleton-Jansen M, et al. Structural organisation and regulation of the small proline rich family of cornified envelope precursors suggest a role in adaptive barrier function. J Biol Chem. 2001;26:19231–19237. 95. Mils A, Vincent C, Croute F, et al. The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia. J Histochem Cytochem. 1992;40:1329–1337. 96. Kashibuchi N, Hirai Y, O’Goshi K, et al. Three-dimensional analyses of individual corneocytes with atomic force microscope: morphological changes related to age, location and to the pathologic skin conditions. Skin Res Technol. 2002;8:203–211. 97. Harding CR, Long S, Richardson J, et al. The cornified cell envelope: an important marker of stratum corneum maturation in healthy and dry skin. Int J Cosmet Sci. 2003;25:1–11. 98. Marks R, Nicolls S, King CS. Studies on isolated corneocytes. Int J Cosmet Sci. 1981;3:251–258. 99. Kunii T, Hirao H, Kikuchi K, et al. Stratum corneum lipid profile and maturation pattern of corneocytes in the outermost layer of fresh scars: the presence of immature corneocytes plays a much more important role in the barrier dysfunction than do changes in intercellular lipids. Br J Dermatol. 2003;149(4):749–756. 100. Mechin MC, Enji M, Nachat R, et al. The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations. CMLS DO1 2005;1–12. 101. Harding CR, Rawlings AV. Natural moisturising factor. In: Loden M, Maibach H (eds) Dry Skin and Moisturizers. Boca Raton: CRC Press, 2006, Chapter 18, pp. 187–209. 102. Katagiri C, Sato J, Nomura J, et al. Changes in environmental humidity affect the water-holding property of the stratum corneum and its free amino acid content, and the expression of filaggrin in the epidermis of hairless mice. J Dermatol Sci. 2003;31 (1):29–35. 103. Jokura Y, et al. Molecular analysis of elastic properties of the stratum corneum by solid-state C-13-nuclear magnetic resonance spectroscopy. J Invest Dermatol. 1995;104:806. 104. Rawlings AV, Scott IR, Harding CR, et al. Stratum corneum moisturization at the molecular level. J Invest Dermatol. 1994;103: 731–740.
The Stratum Corneum and Aging 105. Takahashi M, Tezuka T. The content of free amino acids in the stratum corneum is increased in senile xerosis. Arch Dermatol Res. 2004;295(10):448–452. 106. Sakai S, et al. Hyaluronan exists in the normal stratum corneum. J Invest Dermatol. 2000;114:1184. 107. Fluhr JW, et al. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice. J Invest Dermatol. 2003;120:728. 108. Choi EH, Man MQ, Wang F, et al. Is endogenous glycerol a determinant of stratum corneum hydration in humans. J Invest Dermatol. 2005;125:288–293. 109. Fluhr JW, et al. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol. 2001;117:44 110. Krein PM, Kermici M. Evidence for the existence of a self-regulated enzymatic process within the human stratum corneum- an unexpected role for urocanic acid. J Invest Dermatol. 2000;115:414. 111. Nakagawa N, et al. Relationship between NMF (potassium and lactate) content and the physical properties of the stratum corneum in healthy subjects. J Invest Dermatol. 2004;122:755. 112. Katsura Y, Yoshida Y, Kawai E, et al. Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption. J Dermatol Sci. 2003;32:55–57. 113. Rawlings AV, Hope J, Rogers J, et al. Mechanisms of desquamation: new insights into dry flaky skin conditions. Proceedings of the 17th IFSCC, 1992, vol. 2, pp. 865–880. 114. Van Overloop L, Declercq L, Maes D. Visual scaling of human skin correlates to decreased ceramide levels and decreased stratum corneum protease activity. J Invest Dermatol. 2001;117:811. 115. Saint-Leger D, Francois AM, Leveque JL, et al. Stratum corneum lipids in skin xerosis. Dermatologica. 1989;178:151–155. 116. Chopart M, Castiel-Higounenc C, Arbey E, et al. Quantitative analysis of ceramides in stratum corneum of normal and dry skin. Stratum Corneum III. 2001.
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117. Schreiner V, Gooris GS, Pfeiffer S, et al. Barrier characteristics of different human skin types investigated with x-ray diffraction, lipid analysis and electron microscopy imaging. J Invest Dermatol. 2000;114:654–660. 118. Harding CR, Richardson J, Ginger R, et al. Role of transglutaminase in the continued cross linking of cornified envelope protein during stratum corneum maturation. 22nd IFSCC Congress P139, 2002. 119. Ginger RS, Blachford S, Rowland J, et al. Filaggrin repeat number polymorphism is associated with a dry skin phenotype. Arch Dermatol Res. 2005;297(6):235–241. 120. Engelke M, Jensen JM, Ekanayake-Mudiyanselage S, et al. Effects of xerosis and ageing on epidermal proliferation and differentiation. Br J Dermatol. 1997;137(2):219–225. 121. Leveque JL, Grove G, de Rigal J, et al. Biophysical characterization of dry facial skin. J Soc Cosmet Chem. 1987;82:171–177. 122. Bhawan J, Oh CH, Lew R, et al. Histopathologic differences in the photoaging process in facial versus arm skin. Am J Dermatopathol. 1992;14(3):224–230. 123. Egawa M, Tagami H. Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration. Br J Dermatol. 2008;158(2):251–260. 124. Voegeli R, Rawlings AV, Doppler S, et al. Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases. Int J Cosmet Sci. 2008;30(6):435–442. 125. Cork MJ, Robinson DA, Vasilopoulos Y, et al. New perspectives on epidermal barrier dysfunction in atopic dermatitis:gene-environment interactions. J Allergy Clin Immunol. 2006;118(1):3–21, quiz 22–23. 126. Matts PJ, Gray J, Rawlings AV. The ‘‘Dry Skin Cycle’’ – a new model of dry skin and mechanisms for intervention. International Congress and Symposium Series. London: The Royal Society of Medicine Press Ltd, 2005, pp. 1–38.
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48 The Use of Reconstructed Skin to Create New In Vitro Models of Skin Aging with Special Emphasis on the Flexibility of Reconstructed Skin Daniel Asselineau . Sylvie Ricois . Herve´ Pageon . He´le`ne Zucchi . Sole`ne Mine . Sarah Girardeau . Flore Nallet . Se´verine Teluob . Gae¨lle Claviez-Homberg
Introduction Aging has gained a lot more attention because of the fact that human life expectancy has increased considerably during recent decades. Aging is a complex phenomenon in which several mechanisms operate together. These include the accumulation of mutations in the genetic material, the accumulation of toxic metabolites, the formation of free radicals to produce oxidative damage, chemical modifications, and the cross-linking of macromolecules by glycation (> Table 48.1). Skin is a unique model for aging studies because it is submitted both to extrinsic influences from the environment, mostly sun exposure, and intrinsic factors, mostly of genetic origin. Intrinsic aging, also termed chronological aging, is a time-dependent process, which leads to gradual changes that affect the structure and function of all organs and tissues, which compose the organism. Skin represents a useful model to study aging in humans not only because it is affected by this process, but also because it is easily accessible. Modifications due to aging include wrinkling, laxity, dryness, and heterogeneity in pigmentation, which appear as a function of age [1, 2]. Histologically, the dermal–epidermal junction becomes flat, atrophy associated with reduction in epidermal thickness develops, and major changes take place in the dermis characterized by modification and disorganization of extracellular matrix components like collagen, elastic tissue, and proteoglycans. An attractive characteristic of skin and skin cells is that it is possible to isolate, cultivate, and use these cells to reproduce in vitro the three-dimensional architecture of skin by reconstructing serially the dermal and epidermal compartments. Those constructs are named organotypic
cultures, skin equivalents, or reconstructed skin. In this chapter, the term ‘‘reconstructed skin’’ is used to designate them.
Reconstructed Skin Reconstructed skin represents, increasingly, a very useful alternative to in vivo studies. Many different ways have been proposed to make it, [3] but because of the extensive use of membranes or other dermal substitutes, only a few of them take into account the full thickness of skin including both the dermal compartment containing living cells and the epidermal compartment composed of keratinocytes. Based on the pioneering work of E. Bell and coworkers [4], it is possible to reproduce a dermal equivalent by mixing collagen and fibroblasts in conditions leading to the formation of a dermal tissue [5]. This critical step is then followed by the reconstruction of skin by growing keratinocytes on this substrate, [6] provided the culture is raised at the air–liquid interphase (> Fig. 48.1a). As also shown in > Fig. 48.1b, it is not necessary to grow keratinocytes on top of dermal equivalents to observe complete epidermal differentiation. Complete epidermal differentiation can also be obtained when keratinocytes are embedded in the collagen fibroblast-contracted dermal equivalent. This possibility is of interest to investigate the interactions between cells in a simplified system. Reconstructed skin has been successfully used for many studies, especially for the effect of soluble factors that can be added to the tissue culture medium. For instance, the effect of retinoic acid, the acidic and active form of vitamin A or retinol, and more recently, the
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_48, # Springer-Verlag Berlin Heidelberg 2010
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. Table 48.1 Aging: theories and mechanisms Classical theories
● Oxidative stress ● Alteration of the genome /mutations ● ‘‘Error catastrophe’’ (L.E Orgel 1963) ● Programmed aging-genetic clock: Limited replicative protential (Hayflick 1966) ● Auto immune responses, deterioration of the immune system ● Accumulation of toxic metabolites ● Formation of cross links
Proposed interpretation of ‘‘old theories’’
● Oxidative stress: DNA, proteins, lipids, mitochondria ● DNA – Repair (mutations) – Function (expression) – Replication (telomere length) ● Proteins: structure and function – posttranslational modifications – Glycation (AGE, cross-links) – Transglutaminase (cross-links) – Farnesylation – Methylation, etc
. Figure 48.1 Histology of reconstructed skin grown at the air–liquid interphase. Complete differentiation was observed when keratinocytes were classically grown on the surface of the dermal equivalent in order to form the epidermis (a), as well as in a simplified system in which keratinocytes were embedded like fibroblasts in the collagen lattice to grow and form spheres (b). Bar, 25 mm
effect of vitamin C [8], has been extensively investigated in this system [7]. This 3D system has the advantage of allowing for a very precise description of the effect of such compounds both on the epidermis and the dermis, as well as the dermal–epidermal junction. Moreover, the possibility of using samples in which dermal fibroblasts have been eliminated, allows for studies of the relative roles of these two tissues. Therefore, it seemed that working at making new reconstructed skin models would be
a valuable approach for making in vitro models of skin aging.
Strategies for Skin Aging Studies Using Reconstructed Skin As previously mentioned aging is a complex phenomenon. Despite much progress in the field of reconstructed skin
The Use of Reconstructed Skin to Create New In Vitro Models
during the two previous decades, producing models of skin aging using reconstructed skin remains a difficult challenge. In fact, there were two main ways to approach this question. First reconstructed skin can be made ‘‘as usual’’ and then submitted to a mechanism presumably leading to skin aging or involved in skin aging. The advantage of this strategy is to provide information about early events of aging. The disadvantage is that it is unlikely that aging actually occurs because aging is a long-term phenomenon due to chronic exposure to adverse effects, while in vitro cultures are strongly limited in time. However, a successful follow-up of this strategy has been made in the context of UV light studies aiming at approaching photoaging. Secondly and alternatively, it is also possible to fully reproduce a given mechanism of skin aging in order to mimic aging in reconstructed skin. The advantage of this approach is that the effect of a given mechanism of skin aging will be actually reproduced and its consequences evaluated in vitro, while the disadvantage of this approach is that a single mechanism of aging will be used although aging is a complex multifactorial phenomenon. Thus, it seems restricting. This strategy has been followed in order to mimic chronological aging in vitro using the glycation reaction and interesting results were obtained. These two well-defined strategies and their respective advantages are summarized in > Table 48.2. However, there is a different way to look at aging, which was recently developed consisting of considering cellular populations and their changes as a function of aging in vivo in order to reproduce these changes in the reconstructed skin system. A study on fibroblasts subpopulations was done, which is discussed later in this chapter.
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Current Models of Skin Aging UV Exposure as an Approach of Photoaging Photoaging is due to solar exposure. The effects of photoaging have been well described [9]. The UV domains seem to be the key domains of solar light involved in skin aging especially UVB and UVA, which correspond to the two wavelength domains that reach human beings on the earth. It is also well known that UVB radiations are more energetic, but less penetrating than UVA radiations. Therefore, it is likely that in real life UVB will affect preferentially the surface of the skin while UVA will affect the skin in its depth. To approach these questions, reconstructed skin was made and submitted to both UVB and UVA single exposures performing dose–response and kinetic experiments. It was found that UVB preferentially affects the epidermal compartment producing complex alterations involving both DNA lesions and modifications of the epidermal differentiation pathway [10]. The most obvious effect of UVB exposure was the production of apoptotic keratinocytes easily histologically recognizable, also called sun burn cells, which constitute obvious markers of the effect of UVB and are also representative of their effect in real life. It was also found that UVA preferentially affects the dermal compartment by inducing apoptosis of the most superficial fibroblasts at doses at which keratinocytes seem to be unaffected suggesting differential resistance of these two cell types to UVA radiations [11].
. Table 48.2 Strategies used in the reconstruction of skin to create in vitro models of skin aging Method 1 Initiation
Method 2 Reproduction
Photoaging: UV exposure
Description of the effects of UV light on the Collagen is pre-exposed to UV radiations for the reconstructed skin system at equilibrium (= epidermal induction of dermal modifications potentially differentiation completed) relevant to photoaging CHOSEN NOT CHOSEN
Chronological aging: glycation
Induction of glycation by culturing the dermal equivalent in the presence of sugar (in excess) TESTED BUT NOT CHOSEN (toxic effects)
Benefit obtained This strategy allows: in the selected ● A description of precocious effects of UV strategy radiations but not established photoaging ● The complete reconstructed skin is UV exposed Application for anti-aging studies
Collagen is pre-incubated with sugar (ribose or glucose) prior use for preparing reconstructed skin CHOSEN This strategy allows: An approach of established chronological aging but only one mechanism is involved
In vitro photoprotection studies: solar filter studies by Anti-glycation molecules can be tested as an topical application on reconstructed skin approach to select new anti-aging agents
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Interestingly, the kinetic experiments showed that these effects were reversible since epidermal differentiation was rapidly normalized, while the dermal compartment also recovered its fibroblast population after a few days. However, as opposed to cellular recovery a slight irreversible reduction of the dermal thickness was noticed after UVA exposure due to collagen degradation by collagenase production. This observation may constitute the basis of an approach of the mechanisms of photoaging using the reconstructed skin system. These findings prompted the definition of markers of the specific effects of both UVB and UVA, that is, sunburn cells or fibroblast disappearance, which became very useful for studies in vitro, topically applied solar filters, which led to the conclusion that relevant in vitro photoprotection studies were possible [12].
Glycation of the Collagen as an Approach of Chronological Aging Glycation is a nonenzymatically driven reaction between free amine groups like those of amino acids like lysine and arginine and circulating reducing sugars like glucose. This reaction, also known as the Maillard reaction, leads to socalled advanced glycation end products (AGEs), which are eventually involved in the formation of cross-links between macromolecules. This reaction, which is a chemical reaction preferentially affects tissues characterized by poor renewal and is therefore thought to play an important role in aging [13]. In skin, glycation is thought to affect dermal macromolecules, especially those known for having a very slow turnover like collagen and elastin and because the formation of cross-links may play a role in the alteration of
. Figure 48.2 Scanning electron microscopy (a, b), bar 1 mm and transmission electron microscopy (c, d), bar 1.14 mm showing the collagen fibers in the dermal equivalents of reconstructed skin when collagen was not pre-glycated (a, c) or glycated (b, d). Note that the collagen fibers seem to be more densely packed when collagen was pre-glycated
The Use of Reconstructed Skin to Create New In Vitro Models
mechanical properties of skin like stiffness appearing as a function of age. Therefore, an investigation was done on the effect of glycation in the reconstructed skin model. Pre-glycation of the collagen was used to avoid possible toxic effects on cultured skin cells due to prolonged exposure to high concentrations of sugar. The morphology of reconstructed skin was found to be altered (> Fig. 48.2). In addition, several skin markers of interest were found to be modified in a way recalling aging in vivo. For instance, Matrix Metallo Proteinase 1 or MMP1, as well as the distribution of certain integrins like the b1 integrin were found to be modified in epidermis in a way similar to aged skin in vivo (> Fig. 48.3). At first sight it was surprising to find modifications in epidermis, but it was demonstrated that diffusible factors were involved suggesting that dermal–epidermal interactions were modified when fibroblasts were in contact with glycated collagen. This also suggests that communication between the two tissues
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can be affected during aging. The results in detail [14] and the findings are schematically summarized in > Fig. 48.4. The main conclusions were that glycation of collagen is sufficient to reproduce some manifestations of skin aging, which shows that glycation is probably an important mechanism involved in skin aging, and that the modified reconstructed skin obtained by glycation represents a model of skin aging. It is also of interest to note that this system allows studying the anti-glycation effect of various molecules or extracts, which therefore represent anti-aging candidates [15].
Specific Role of Papillary Fibroblasts in Aging The dermal part of skin is histologically heterogeneous. The superficial or papillary dermis, which is close to
. Figure 48.3 Immunolabeling of carboxy methyl lysine or CML (a–c) and b1 integrin (d–f) in reconstructed skin in the absence of glycation (a, d) or when collagen was pre-glycated either in the presence of glucose (b, e) or ribose (c, f). Note the presence of CML only when collagen was pre-glycated and the corresponding extension of the b1 labeling in most of the suprabasal layers of the epidermis. Bar, 25 mm
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. Figure 48.4 Schematic representation of reconstructed skin produced either in absence of pre-glycated collagen (left) or when preglycated collagen was used (right). Note that basement membrane components (in brown and dark green) are more abundant when pre-glycated collagen was used together with other extracellular matrix molecules of the dermis (as mentioned). As indicated, also note the increased distribution of both b1 (in green) and a6 (in yellow) integrins in the epidermis
epidermis is very thin as opposed to the deep dermis or reticular dermis, which constitutes the vast majority of this tissue. The reticular dermis is characterized by the accumulation of thick fibers, which are thought to be responsible for the mechanical properties of the dermis. It is now known that the fibroblast populations of these two regions are different, but only a small number of laboratories have investigated their relative properties including growth potential [16]. So far their fate during aging remains totally unknown. Both populations were isolated and then characterized, and their properties investigated as a function of aging. This was made possible by numerous isolations of site-matched pairs from donors of increasing age to raise age-dependant collections of these cells; their growth characteristics, cytokine, and other diffusible factor production were studied by
cell sorting and by performing cloning experiments. Reconstructed skins with dermal compartments containing one population or the other were made and compared by means of dermal contraction and ability to participate to skin reconstruction in terms of capacity to promote epidermal morphogenesis. To summarize, the reticular fibroblast population does not seem to be modified by aging as opposed to the papillary fibroblast population. The papillary fibroblast population seems to disappear in conditions that comprise different possibilities like (1) apoptosis of papillary fibroblast, (2) replacement of papillary fibroblasts by upward migration of reticular fibroblasts, or (3) differentiation of papillary fibroblasts into reticular fibroblasts during aging. This later hypothesis is a very interesting and provocative one currently under investigation because it means that papillary and reticular
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. Figure 48.5 Schematic representation of both young human skin (a: left) and old human skin (a: right) emphasizing the so far not completely elucidated modification occurring in the papillary dermis and the corresponding schematic representation of both young-like skin equivalent (b: left) or old-like skin equivalent (b: right). These schematic representations were made to emphasize the idea that reconstructed skin lacking the papillary dermal component is an easy first approximation of old skin
fibroblasts represent different differentiation states of a single cell type. The findings and these possibilities were recently published in detail and schematically represented [17]. A simplified way to conclude is to consider that a
model of reconstructed skin with a dermal compartment containing only reticular fibroblasts is a reasonable approach of a reconstructed skin model of aged skin (> Fig. 48.5).
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Flexibility of Reconstructed Skin and the Possibility to Standardize the Production: Towards RealSkin Flexibility is a very attractive property of the reconstructed skin model. The shape (> Fig. 48.6), the dermal thickness (> Fig. 48.7), how to make several – at least two – dermal compartments can be varied in laboratories, and to manipulate the cell population content of this or these dermal compartment(s) including destruction of the fibroblast population by osmotic shock (> Fig. 48.8) to provide a negative control if necessary.
. Figure 48.6 Macroscopic view of collagen-contracted lattices or dermal equivalents cast in classical round petri dishes or square tissue culture dishes. Note that the final form – round versus square – is the consequence of the shape of the tissue culture vessel used. Bar, 1 cm
Glycation of the collagen represents an example of the possibility to modify the extracellular matrix. The use of different fibroblast populations is an illustration of the possibility to change the cellular content in reconstructed skin. Such flexibility was critical to create in vitro aging models. In addition, it is also possible to standardize the fabrication of the reconstructed skin system to adapt it to production. This was achieved by casting directly the fibroblast-contracted collagen gel or dermal equivalent in inserts placed in multiwell plates (> Fig. 48.9). This system, which is a full-thickness reconstructed skin system was recently named RealSkin and is very close to the ‘‘historical’’ reconstructed skin system produced classically in individual petri dishes with the help of stainless steel rings (epidermal cell seeding) and stainless steel grids (air– liquid interphase) with the exception of the fact that tension is generated in the dermis. It is however accompanied by the presence of more abundant extracellular matrix (ECM) macromolecules (> Fig. 48.10) and increased production of MMP1 (> Fig. 48.11). Interestingly, tension seems to be correlated to increased resistance to the effect of UVA light (> Fig. 48.12).
Future Models of Reconstructed Skin with Special Emphasis on Skin Aging Classical modifications of reconstructed skin include modifications of cell populations by enrichment consisting of incorporating new cell types generally in epidermis
. Figure 48.7 Histology of different types of reconstructed skin, whose dermal compartment was the result of fibroblast-contracted gels of 7 mL (a), 10 mL (b), 20 mL (c). Note that the final thickness of the dermal equivalents obtained is the product of the initial volume used. Bar, 25 mm
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. Figure 48.8 Histology of dermal equivalent containing living fibroblasts (a) and corresponding reconstructed skin (b), and dermal equivalents in which fibroblasts were destroyed by osmotic shock (c) and corresponding reconstructed skin (d). Note the presence of fibroblasts in a and b and absence of fibroblasts in c and d. Bar, 25 mm
. Figure 48.9 Macroscopic view of classical reconstructed skin (a) made in individual petri dishes and reconstructed skin made in multiwell plates (b). Bar, 1 cm
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. Figure 48.10 Classical reconstructed skin (a–g) and reconstructed skin produced in multiwell plates (h–n). Macroscopic view (a, h), Histology (b, i), Involucrin (c, j), Collagen IV (d, k), Vimentin (e, l), Procollagen I (f, m), PG4 proteoglycan epitope (dermatan/chondroitin sulfate epitope) (g, n) immunostainings. Note increased presence of extracellular matrix material in reconstructed skin produced in multiwell plates as compared to classical reconstructed skin. Also note that vimentin labeling, which shows orientation of fibroblasts in the dermal equivalent shows no tension in the dermis of classical reconstructed skin (random orientation) as opposed to tension visible in reconstructed skin produced in multiwell plates (horizontal orientation). Bar, 25 mm
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The Use of Reconstructed Skin to Create New In Vitro Models
. Figure 48.11 Matrix Metallo Proteinase 1 production in reconstructed skin. Note increased production in classical reconstructed skin (in blue) as compared to reconstructed skin produced in multiwell plates (in red)
like melanocytes to add pigmentation of the reconstructed skin. It would also be of high interest, especially in the context of creating new models of skin aging to add new dermal cells especially endothelial cells as already done by others [18]. Endothelial cells, which are the key components of blood vessels including skin blood vessels may interact preferentially with papillary fibroblasts [19]. Moreover, dermal papillae, which are the image of skin vasculature form preferentially in the presence of papillary fibroblasts after grafting onto the nude mouse [20]. Thus, it is possible to make dermal constructs with two compartments containing papillary fibroblasts and reticular fibroblasts by making serially two gels containing first reticular fibroblasts and then papillary fibroblasts so that it is quite possible to lay down endothelial cells at the interface on top of the first gel before casting the second gel. Another expected modification of reconstructed skin of interest would be to introduce changes in the content of extracellular matrix molecules in dermal equivalents. Little has been done in that area with the exception of combining collagen with glycosaminoglycans. It would be of interest of course to introduce molecules other than collagen in dermal equivalent like elastin, for instance, it would also be of high interest to introduce various types of collagens specific from the different regions of the dermis like adding collagen III to better reproduce the
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papillary dermis. Other strategies in that area would also consist of replacing collagen classically provided by animal species like bovine collagen by human collagen or by replacing the collagen classically provided as individual molecules in solution by preparations in which the organization of collagen in fibers is better preserved in order to be closer to the actual structure and organization of collagen in skin. In the context of creating new models of reconstructed skin for skin aging studies it is very interesting to investigate human genetic diseases by creating in vitro models of these diseases, incorporating skin cells isolated and amplified from small biopsies provided by patients. For instance, the successful reproduction of the Xeroderma pigmentosum (XP) phenotype by showing lack of repair of DNA lesions in XP reconstructed skin after UV exposure [21] in a way strikingly recalling the in vivo situation. Similarly, in the future, it would be of high interest to create new in vitro models of accelerated aging by incorporating human skin cells corresponding to other genetic diseases like Progeria or Werner. An interesting point also is that skin is different as a function of the anatomic site. For instance, in epidermis in certain regions of the body specific keratins are expressed, which are not seen elsewhere. It is well known that palm of foot sole epidermis contains keratin 9, which is not found elsewhere. There are certainly many regional differences, yet to be discovered that could become the subject of specific models representing skin of any region of the body like the face versus trunk or else, which may age differently. At least this question could be investigated if corresponding reconstructed skins could be made and compared and whether they age the same way. Another striking observation is that skin is obviously different as a function of ethnic origin at least by means of pigmentation. Recently, it was illustrated that the Caucasian skin type versus African skin type is different not only in terms of pigmentation, but also morphogically in its depth since the dermal–epidermal junction is more invaginated in African skin relative to Caucasian skin (> Fig. 48.13). Moreover, the production of certain cytokines like MCP1 is different in the two types of fibroblasts [22] suggesting that not only epidermis, but also dermis could be different. Such results raise the question of whether the two types of skin age the same way or not since aging affects preferentially the dermal component of the skin. To address this question reconstructed skins are made containing either Caucasian or African skin types keratinocytes and fibroblasts in order to look for differences. This is a very interesting preliminary step prior to actual comparative aging studies, which means
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. Figure 48.12 Dose–response experiment of classical reconstructed skin (a–j) and reconstructed skin produced in multiwell plates (k–t) exposed to UVA light: control (a–p), 25 J/cm2 (b–q), 35 J/cm2 (c–r), 45 J/cm2 (d–s), and 55 J/cm2 (e–t). Histology (a–e and k–o), Vimentin labeling (f–j and p–t). Bar, 25 mm
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. Figure 48.13 Histology of respectively Caucasian human skin (a) and African human skin (b). Note pronounced dermal–epidermal invaginations in African skin. Bar, 25 mm
. Figure 48.14 Schematic representation of current ‘‘ideal’’ version of both young and old reconstructed skin models as seen at this point defined as being the closest to skin in vivo. These so-called ideal models would comprise respectively the use of cells isolated from young or old donors and the fabrication of both papillary and reticular dermal compartments containing respectively papillary and reticular fibroblasts in the presence of a preglycated collagen characterized by thick fibers for the reticular dermis, while normal unglycated collagen would be used for the papillary dermal compartment
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making reconstructed skins both age and ethnic origin dependant. Finally, there are of course many other possibilities, which are not mentioned in this brief review. It is of interest to note that in the near future not only classical anti-aging studies involving topical application of formulations will take place, but also so-called aesthetic approaches of anti-aging will become more and more fashionable. However, there is a considerable lack of reliable scientific information in that area. Therefore, experiments are initiated aiming at studying the effect produced by dermal fillers in the context of reconstructed skin in order to determine specific effects (beneficial vs adverse) as a function of the type of filler used and specific of the way the filler is distributed.
Conclusion Such considerations show that it is possible to make more complex reconstructed skin systems by varying simultaneously several parameters. For instance, the knowledge of fibroblast collection has made it possible to consider simultaneously the type of fibroblast (papillary vs reticular) the age of the donor (young vs old), and the glycation status of the collagen used (preglycated vs normal). At this point, it is possible to imagine some kind of ideal normal or aged skin made in vitro in the near future. For instance, such constructs could comprise in the case of ‘‘young’’ (normal) skin a thick reticular-like dermis containing reticular fibroblasts and made with a thick fibered collagen and a thin papillary dermis containing papillary fibroblasts and made with classical collagen on which keratinocytes would be grown while the corresponding ‘‘aged’’ skin would be made the same way except that the reticular dermal compartment would be made with glycated collagen and the papillary dermal compartment would be almost absent. This is schematically represented in > Fig. 48.14. The range of these possibilities is important and very promising for the future.
Cross-references > Aging
of Skin Cells in Culture
Acknowledgments We would like to thank Anne-Marie Minondo for performing the electron microscopy, and Catherine Olivry for the artwork.
References 1. Yaar M, Eller MS, Gilchrest BA, et al. Fifty years of skin aging. J Invest Dermatol Symp Proc. 2002;7:51–58. 2. Farage MA, Miller KW, Elsner P, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 3. Boyce ST. Cultured skin substitutes: a review. Tissue Eng. 1996;2:255–266. 4. Bell E, Sher S, Hull B, et al. The reconstitution of living skin. J Invest Dermatol. 1983;81:2S–10S. 5. Asselineau D, Prunieras M. Reconstruction of ‘‘simplified’’ skin: control of fabrication. Br J Dermatol. 1984;111:219–221. 6. Asselineau D, Bernhard B, Bailly C, et al. Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface. Exp Cell Res. 1985;159: 536–539. 7. Asselineau D, Bernhard B, Bailly C, et al. Retinoic acid improves epidermal morphogenesis. Dev Biol. 1989;133:322–335. 8. Marionnet C, Vioux-Chagnoleau C, Pierrard C, et al. Morphogenesis of dermo-epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C. Exp Dermatol. 2006;15(8):625–633. 9. Oikarinen A, Karvonen J, Uitto J, et al. Connective tissue alterations in skin exposed to natural and therapeutic UV-radiation. Photodermatology. 1985;2:15–26. 10. Bernerd F, Asselineau D. Successive alteration and recovery of epidermal differentiation and morphogenesis after specific UVBdamages in skin reconstructed in vitro. Dev Biol. 1997;183:123–138. 11. Bernerd F, Asselineau D. UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: Subsequent connective tissue repair and implications in photoaging. Cell Death Differ. 1998;5:792–802. 12. Bernerd F, Asselineau D. An organotypic model of skin to study photodamage and photoprotection in vitro. J Am Acad Dermatol. 2008;58:S155–S159. 13. Frye EB, Degenhardt TP, Thorpe SR, et al. Role of the maillard reaction in aging of tissue proteins. J Biol Chem. 1998;273: 18714–18719. 14. Pageon H, Bakala H, Monnier VM, et al. Collagen glycation triggers the formation of aged skin in vitro. Eur J Dermatol. 2007;17:12–20. 15. Pageon H, Te´cher MP, Asselineau D, et al. Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules. Exp Gerontol. 2008;43:584–588. 16. Harper RA, Grove G. Human skin fibroblasts derived from papillary and reticular dermis: Differences in growth potential in vitro. Science. 1979;204:526–527. 17. Mine S, Fortunel NO, Pageon H, et al. Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and ageing. PLoS One. 2008;3: 1–9. 18. Black AF, Berthod F, L’heureux N, et al. In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent. FASEB J. 1998;12:1331–1340. 19. Sorrell JM, Baber MA, Caplan AI. Human dermal fibroblast subpopulations; differential interactions with vascular endothelial cells in coculture: nonsoluble factors in the extracellular matrix influence interactions. Wound Repair Regen. 2008;16:300–309. 20. Asselineau D, Te´cher MP, Caplan AI, et al. Complex reconstructed skin equivalents made with papillary and reticular fibroblast
The Use of Reconstructed Skin to Create New In Vitro Models populations incorporated in distinct layers: re-expression of papillary and reticular fibroblast characteristics after grafting onto nude mice. J Invest Dermatol. 2000;114:863. 21. Bernerd F, Asselineau D, Vioux C, et al. Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient
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xeroderma pigmentosum skin in vitro. Proc Natl Acad Sci USA. 2001;93:7817–7822. 22. Girardeau S, Mine S, Pageon H, et al. The Caucasian and African skin types differ morphologically and functionally in their dermal component. Exp Dermatol. 2009;118:704–711.
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46 Tobacco Smoke and Skin Aging* Akimichi Morita
Introduction As early as 1971, Daniell [1] found that tobacco smoking has a deleterious effect on the skin, and smoker’s wrinkles are the typical clinical features of smokers. A recent epidemiological study has clearly shown that tobacco smoking is one of the numerous factors contributing to premature skin aging, which is dependent on age, sex, pigmentation, sun exposure history, alcohol consumption, and other factors [2 5]. In a further cross section study, sun exposure, pack years of smoking history, and potential confounding variables were assessed by questionnaire. Facial wrinkles were quantified using the Daniell score. Logistic statistical analysis of the data revealed that age, pack year, and sun exposure independently contributed to facial wrinkle formation [6]. In this survey, age (OR = 7.5, 95% CI = 1.87 30.16), pack year (OR = 5.8, 95% CI = 1.72 19.87), and sun exposure (OR = 2.65, 95% CI = 1.0 7.0) independently contributed to the formation of facial wrinkles, as estimated by a logistic regression analysis model. Using silicone rubber replicas combined with computerized image processing, an objective measurement of skin’s topography, the association between wrinkle formation and tobacco smoking was investigated. Sixty-three volunteers were enrolled by assessing their skin replicas, in an attempt to elucidate the association between tobacco smoking and wrinkles [7]. The replica analysis showed that the depth (Rz) and variance (Rv) of furrows (Rv) in subjects with smoking history 35 pack years were significantly higher than nonsmokers (P < 0.05). The lines of furrows (Rl) in subjects with smoking history were significantly lower than in nonsmokers (P < 0.05) [7, 8]. Tobacco smoking, which is regarded as an important environmental factor, can potentially cause ‘‘tobacco wrinkles’’ [1], although chronic exposure of skin to ultraviolet (UV) radiation results in marked alterations in the structure and composition of the epidermis and dermis, i.e., photoaging [9 11]. In a recent study, tobacco
smoking per se or smoking combined with UV exposure were strong predictors of skin aging [12].
Molecular Mechanisms of TobaccoInduced Skin Aging Tobacco smoking probably exerts its deleterious effects on skin directly through its irritant components on the epidermis and indirectly on the dermis via the blood circulation [3, 13]. The decreased stratum corneum moisture of the face contributes to facial wrinkling because of the direct toxicity of the smoke. Pursing the lips during smoking with contraction of facial muscles and squinting because of the irritating of smoke may cause the formation of wrinkling around the mouth and in the crow’s foot area [14]. The changes in the dermis of macromolecular metabolism have been brought into focus as a major factor leading to skin aging [15]. Specifically, it has been demonstrated that accumulation of elastosis material is accompanied by the degradation of matrix protein, which is mediated by matrix metalloproteinases (MMPs) in skin aging. The molecular alteration in the dermis includes the decrease of collagen synthesis, induction of MMPs, abnormal accumulation of elastic fibers, and proteoglycans [16 18].
Effects of Tobacco Smoke on Skin Models In Vitro The biosynthesis of new collagen was decreased significantly by tobacco smoke extracts in cultured skin fibroblasts [18]. The studies also showed that the production of both procollagen types I and III, the precursors of collagen, were significantly decreased from the supernatant of cultured fibroblast treated with tobacco smoke extracts, using Western blot analysis [18]. This result
∗ Originally published as Tobacco Smoke and Skin Aging in Halliwell, B.B., Poulsen, H.E. (eds.), Cigarette Smoke and Oxidative Stress, Heidelberg, Springer, 2006. pp. 379–385. Reprinted with permission.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_46, # Springer-Verlag Berlin Heidelberg 2010
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indicated that the final production of collagen secreted into the medium is reduced, regardless of the rate of collagen synthesis in the cell tested in 3H-proline incorporation. Although elastic fibers account for only 2–4% of extracellular matrix, these provide elasticity and resilience to normal skin. Tobacco smoke extracts induced the significant increase in tropoelastin mRNA in cultured skin fibroblasts. Accumulation of abnormal elastic material (termed solar elastosis) is the prominent histopathologic alterations in photoaged skin [19, 20]. Boyd et al. [21] reported that tobacco smoking could facilitate smoke’s elastosis of the subjects with an average of 42 pack years of tobacco smoking. In an in vitro study using cultured skin fibroblasts, tobacco smoke extracts induced elevation of tropoelastin. This might be attributed to premature skin aging. The expressions of MMP-1 and MMP-3 mRNA, extracellular matrix (ECM)-associated members of the MMPs gene family, were induced in cultured skin fibroblast stimulated with tobacco smoke extracts in a dose-dependent manner [18]. These results support the concept that MMPs are primary mediators of connective tissue damage in skin exposed to tobacco smoke extracts and of the premature skin aging. In addition, expression of TIMP-1 and TIMP-3 remained unchanged [18]. By inducing the expressions of MMP-1 and MMP-3, but not the induction of tissue inhibitor of MMPs, tobacco smoke extracts could alter their ratio in favor of the induction of MMPs and appears to result in a more degradative environment that produces loss of cutaneous collagen [18]. In addition, MMPs comprise a family of degradative enzymes, which are responsible for the degradation of extracellular matrix components such as native collagen, elastin fibers, and various proteoglycans. MMP-3 and MMP-7 may play a key role in the degradation of elastin and proteoglycans [22]. MMP-7 was increased in fibroblasts induced by tobacco smoke extract.
Effect of Tobacco Smoke In Vivo In a clinical study, significant higher levels of MMP-1 mRNA were observed in the buttock skin of smokers, compared with nonsmokers, using quantitative real-time PCR [23]. The elevated enzyme should lead to the degradation of collagen, elastic fibers, and proteoglycans. Therefore, the observations in dermal connective tissue induced by the treatments of tobacco suggested an imbalance between the biosynthesis and degradation, with less repair capacity on the face of the ongoing degradation,
which leads to the loss of collagen and elastic fibers, manifesting clinically as aging appearance of skin. Although staining of skin specimen and biochemical analysis of photodamaged skin demonstrated increased glycosaminoglycan content of sun-damaged skin, the underlying molecular pathogenesis remains unclear. Versican, the large chondroitin sulfate (CS) proteoglycan, has been identified in the dermis in association with elastic fibers, which contain a hyaluronic acid-binding domain. The core protein has been postulated to play a role in molecular interactions and specifically, to facilitate the binding of these macromolecules to other matrix components or cytokines such as transforming growth factor (TGF) [24]. Decorin, a small CS proteoglycan, has been shown to codistribute with collagen fibers and postulated to function in cell recognition, possible by connecting extracellular matrix components and cell surface glycoproteins [25]. Targeted disruption of decorin synthesis in mice resulted in a significant reduction in the tensile strength of skin [26]. There was a decrease in the proportion of large CS proteoglycan (versican) and a concomitant increase in the proportion of small dermatan sulfate proteoglycan (decorin) as a function of age as reported by Carrino et al. [27]. Ito et al. [28] also observed that versican was stained strongly in young rats and faintly in old rats. On the other hand, decorin was faintly stained in the young rats and distinctly stained in the old rats. There were several reports concerning the changes of proteoglycans on photoaging, especially UVB irradiation [29, 30]. The analysis of new synthesized proteoglycans showed a marked increase after UVB radiation in mice [30]. Versican and decorin immunostaining increased in photoaged tissue samples, accompanied by similar alterations in gene expression [29]. Tobacco smoke extracts decreased both versican protein and mRNA levels in cultured akin fibroblasts. However, tobacco smoke extract exposure resulted in a significant increase of decorin. These results are similar to those observed in photoaging. Based on experimental evidence, a working model for UVA damage skin was proposed, in which UV irradiation gene expression was mediated via the generation of singlet oxygen through a pathway involving activation of transcription factor AP-2 [10]. In order to define whether the reactive oxygen species (ROS) were involved in upregulation of MMPs induced by tobacco, sodium azide (NaN3), l-ascorbic acid, and vitamin E, which are potent quenchers of singlet oxygen and other ROS, were employed. NaN3, l-ascorbic acid, and vitamin E abrogated the induction of MMPs after exposure of fibroblast to tobacco smoke extract. Among the antioxidant reagents, l-ascorbic acid most obviously diminished the increase in
Tobacco Smoke and Skin Aging
MMP-1 expression level on exposure of fibroblasts to tobacco smoke extracts [18]. This points to the fact that ROS were most probably responsible for the enhanced induction of MMPs by tobacco smoke extract. The TGF-b1 is a multifunctional cytokine that regulates cell proliferation and differentiation, tissue remodeling, and repair [31]. TGF-b1 is a potent growth inhibitor in the epidermis, playing an important role in the maintenance of tissue homeostasis. In the dermis, however, TGF-b1 acts as a positive growth factor, inducing the synthesis of extracellular matrix proteins. TGF-b signals through a heteromeric complex of type I/II TGF-b receptors initiate signal transduction [32, 33]. A recent report showed that UV irradiation can cause downregulation of TGF-b type II receptor mRNA and protein, and induction of Smad7 mRNA and protein in human skin [34]. Tobacco smoke extracts induced the latent form TGF-b, not the active form, assayed by enzyme-linked immunosorbent assay (ELISA), in the supernatants of cultured skin fibroblasts [35]. The induction of endogenous TGF-b1 from tobacco-exposed cells contributes to the intracellular defense capacity. Fibroblasts responses to TGF-b1 are mediated through its active form binding to the cell surface receptor. Tobacco smoke extracts blocked cellular responsiveness to TGF-b1 through the induction of nonfunctional latent form and downregulation of TGF-b1 receptor [35]. Exogenous addition of TGF-b1 might be useful to stimulate the collagen production or to protect against the deleterious effects of tobacco smoke.
Conclusion Tobacco smoke contains numerous compounds, with at least 3,800 constituents [36]. Just which constituents contributed to the damage of connective tissue is still unclear. The tobacco-induced skin aging provides a tool for studying the effects of smoking. Also, detailed knowledge may provide a motivation to stop smoking, especially among those who are more concerned about their appearances than the potential internal damage associated with smoking.
References 1. Daniell HW. Smoker’s wrinkles: A study in the epidemiology of ‘‘crow’s feet. Ann Intern Med. 1971;75:873–880. 2. Ernster VL, Grady D, Miike R, et al. Facial wrinkling in men and women, by smoking status. Am J Public Health. 1995;85:78–82. 3. Frances C. Smoker’s wrinkles: epidemiological and pathogenic considerations. Clin Dermatol. 1998;16:565–570.
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4. Grady D, Ernster V. Does cigarette smoking make you ugly and old? Am J Epidemiol. 1992;135:839–842. 5. Kadunce DP, Burr R, Gress R, et al. Cigarette smoking: risk factor for premature facial wrinkling. Ann Intern Med. 1991;114:840–844. 6. Yin L, Morita A, Tsuji T. Skin aging induced by ultraviolet exposure and tobacco smoking: evidence from epidemiological and molecular studies. Photodermatol Photoimmunol Photomed. 2001;17: 178–183. 7. Yin L, Morita A, Tsuji T. Skin premature aging induced by tobacco smoking: The objective evidence of skin replica analysis. J Dermatol Sci. 2001b;27(Suppl 1):S26–S31. 8. Yin L, Morita A, Tsuji T. Tobacco smoking: a role of premature skin aging. Nagoya Med J. 2000;43:165–171. 9. Fisher GJ, Talwar HS, Lin J, et al. Molecular mechanisms of photoaging in human skin in vivo and their prevention by all-transretinoic acid. Photochem Photobiol. 1999;69:154–157. 10. Grether-Beck S, Buettner R, Krutmann J. Ultraviolet A radiationinduced expression of human genes: Molecular and photobiological mechanisms. Biol Chem. 1997;378:1231–1236. 11. Wenk J, Brenneisen P, Meewes C, et al. UV-induced oxidative stress and photoaging. Curr Probl Dermatol. 2001;29:83–94. 12. Leung W-C, Harvey I. Is skin ageing in the elderly caused by sun exposure or smoking? Br J Dermatol. 2002;147:1187–1191. 13. Lofroth G. Environmental tobacco smoke: overview of chemical composition and genotoxic components. Mutat Res. 1989;222:73–80. 14. Smith JB, Fenske NA. Cutaneous manifestations and consequences of smoking. J Am Acad Dermatol. 1996;34:717–732. 15. Uitto J, Fazio MJ, Olsen DR. Molecular mechanisms of cutaneous aging: Age-associated connective tissue alterations in the dermis. J Am Acad Dermatol. 1989;21:614–622. 16. Fisher GJ, Voorhees JJ. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces MAP kinase signal transduction cascades that induce Ap-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Investig Dermatol Symp Proc. 1998;3:61–68. 17. Shuster S. Smoking and wrinkling of the skin. Lancet. 2001;358:330. 18. Yin L, Morita A, Tsuji T. Alterations of extracelluar matrix induced by tobacco smoke extract. Arch Dermatol Res. 2006;292: 188–194. 19. Montagna W, Kirchner S, Carlisle K. Histology of sun-damaged human skin. J Am Acad Dermatol. 1989;21:907–918. 20. Tsuji T. Ultrastucture of deep wrinkles in the elderly. J Cutan Pathol. 1987;14:158–164. 21. Boyd AS, Stasko T, King LE Jr., et al. Cigarette smoking-associated elastotic changes in the skin. J Am Acad Dermatol. 1999;41:23–26. 22. Saarialho-Kere U, Kerkela E, Jeskanen L, et al. Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage. J Invest Dermatol. 1999;113:664–672. 23. Lahmann C, Bergemann J, Harrison G, et al. Matrix metalloprotease-1 and skin ageing in smokers. Lancet. 2001;357:935–936. 24. Fisher LW, Termine JD, Young MF. Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J Biol Chem. 1989;264:4571–4576. 25. Zimmermann DR, Ruoslahti E. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 1989;8:2975–2981. 26. Danielson KG, Baribault H, Homes DF, et al. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol. 1997;136:729–743. 27. Carrino DA, Sorrell JM, Caplan AI. Age-related changes in the proteoglycans of human skin. Arch Biochem Biophys. 2000;373:91–101.
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28. Ito Y, Takeuchi J, Yamamoto K, et al. Age differences in immunohistochemical localizations of large proteoglycan, PG-M/versican, and small proteoglycan, decorin, in the dermis of rats. Exp Anim. 2001;50:159–166. 29. Bernstein EF, Fisher LW, Li K, et al. Differential expression of the versican and decorin genes in photoaged and sun-protected skin: Comparison by immunohistochemical and northern analyses. Lab Invest. 1995;72:662–669. 30. Margelin D, Fourtanier A, Thevenin T, et al. Alterations of proteoglycans in ultraviolet-irradiated skin. Photochem Photobiol. 1993; 58:211–218. 31. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791. 32. Kadin ME, Cavaille-Coll MW, Gertz R, et al. Loss of receptors for transforming growth factor beta in human T-cell malignancies. Proc Natl Acad Sci USA. 1994;91:6002–6006.
33. Piek E, Heldin CH, Ten Dijke P. Specificity, diversity, and regulation in TGF-beta superfamily signaling. FASEB J. 1999;13: 2105–2124. 34. Quan T, He T, Voorhees JJ, et al. Ultraviolet irradiation blocks cellular responses to transforming growth factor-beta by down-regulating its type-II receptor and inducing Smad. J Biol Chem. 2001;276: 26349–26356. 35. Yin L, Morita A, Tsuji T. Tobacco smoke extract induces age-related changes due to the modulation of TGF-b. Exp Dermatol. 2003;12: 51–56. 36. Bartsch H, Malaveille C, Friesen M, et al. Black (air-cured) and blond (flue-cured) tobacco cancer risk IV: molecular dosimetry studies implicate aromatic amines as bladder carcinogens. Eur J Cancer. 1993;29A:1199–1207.
25 Unique Skin Immunology of the Lower Female Genital Tract with Age Paul R. Summers
Introduction It has been long recognized that the genital tract must be able to defend against significant microbial exposures. In this area of medicine, old theories that may have even acquired some attributes of folklore must be revised to include new knowledge. Through the last century, popular ideas regarding mechanisms of microbial defenses in the genital tract have reflected the medical thinking of each era. In the time of antisepsis of the early twentieth century, lactic acid from the lactobacillus was proposed as the chief regulatory vaginal antiseptic. Subsequently, the possibility of antiseptic action from hydrogen peroxide-producing lactobacilli was considered, although little hydrogen peroxide would be expected to be produced in the naturally anaerobic environment of the vaginal lumen. With the influence of the more recent antibiotic era, research interest has focused upon bacteriocins, unique but relatively weak lactobacillus-derived antibiotics. Theories of microbial defense have evolved further in the current, more enlightened era of immunology. Rapid advances in the area of immunology have now disclosed complex immune defenses in the genital epithelium that do have a significant antimicrobial impact, moderated by estrogen. From the immune standpoint, the lower genital tract has the following competing roles: (1) to facilitate the various aspects of reproduction and (2) to simultaneously prevent the access of locally resident microbes to the upper genital tract and to the peritoneal cavity. To facilitate a primary function in reproduction, the immune responsiveness of the lower female genital tract is blunted. Ovulation, fertilization, pregnancy, labor, and delivery of the infant are all mediated by immune mechanisms that may not be optimal for microbial defense. A blunted humoral immune response may be compensated by an active innate or cellmediated response. For example, sperm may be highly immunogenic. If sperm are detected by the humoral immune system, the development of antisperm antibodies can reduce fertility [1]. It is important for the vaginal immune system to identify potential pathogens, but not
to target sperm or the fetus, or to disrupt the immune mechanisms of fertility. Microbial and immune events in the female urethra mirror the status of the vaginal vestibule [2]. The immune function and microbial flora of the vaginal vestibule and urethra change in a parallel fashion in response to the effects of aging and hormone cycles. Hormone changes alter the morphology and mucosal defenses. Menopausal decline in innate immune defenses in the vaginal mucosa allows colonization with potential uropathogens and increases the risk for bladder infection.
Humoral Immunity The humoral immune system associated with vaginal mucosa is unique. Mucosal surfaces outside the genital tract develop in conjunction with lymphoid tissue that predominantly produces IgA. At other body sites, IgA may have a significant role in mucosal defense against microbes. With the absence of associated lymphoid tissue, vaginal mucosa releases only limited quantities of any category of immunoglobulin at all stages of life. IgG is present in vaginal secretions. The relatively small amount of IgG is serum-derived as well as locally produced in the vaginal and cervical mucosa [3]. With the relative absence of a local source of IgA, more IgG than IgA is detected in vaginal secretions [4]. The converse is true for mucosal surfaces elsewhere in the body. Cervical secretions have a higher concentration of IgA than vaginal secretions [5]. This finding is consistent with the presumed protective role of cervical mucus to prevent ascent of microbes into the endometrial cavity. The concentration of IgA in vaginal secretions declines by 90% after hysterectomy so the upper genital tract may be assumed to be the primary source of the small quantity of IgA that is present in the vaginal lumen [6]. It is reasonable to assume a similar decline in lower genital tract immunoglobulins after the menopause, with the minimal production of cervical mucus and vaginal secretions at that time in life. Cervical secretion of IgG and IgA into the vaginal pool
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varies during the menstrual cycle with the highest levels prior to ovulation during the proliferative phase, but with an 80% decline at the time of ovulation [7]. The limited amount of immunoglobulin in vaginal secretions may lower the risk for the development of antisperm antibodies. It is reasonable to speculate that sperm survival may be enhanced in some fashion by the further decline in immunoglobulins around the time of ovulation. Disruption of vaginal immunoglobulin homeostasis can be harmful. Electrical loop excision of the cervical transformation zone (LEEP) may allow an unregulated humoral immune response at that site. Serum antisperm antibodies have been identified in women who are sexually active while the cervical LEEP site is healing [8].
Innate Immunity The innate immune system has major importance in preventing invasion of potentially pathogenic microbes normally found in the lower genital tract and on the perirectal skin. During the reproductive years, an active innate immune response compensates somewhat for the blunted humoral and cell-mediated immune response in the lower female genital tract (> Table 25.1). Sexually transmitted diseases develop when sexually acquired pathogens have the ability to evade these standing defenses [9]. Human beta defensins (HBD) 1, 2, 3 and 5, secretory leukocyte protease inhibitor (SLPI), elafin, and mannose binding lectin (MBL) have been demonstrated in vaginal secretions [9]. The highest concentration of SLPI is in the cervical mucus plug, although it is expressed in secretions throughout the female genital tract. SLPI blocks the action of various destructive enzymes that may be released by pathogens. Elafin is an important protein that inhibits inflammation-related tissue damage by blocking elastase, which may be released by activated neutrophils. Elafin also has antimicrobial activity. Leukocytes and vaginal epithelial cells are the main sources of
. Table 25.1 Important characteristics of the cervical transformation zone during the reproductive years High concentration of elements of cell-mediated immunity to interact with viruses and to prevent ascent of bacteria into the upper genital tract and peritoneum Macrophages are involved in cervical ripening prior to labor Macrophages and granulocytes are involved in cervical dilation during labor
defensins [10]. Defensins are antibiotic substances that are active against various bacteria and yeast. Surfactant proteins in vaginal mucosal secretions (SP-A, SP-D) protect against viral infections, including HIV-1 and herpes simplex virus (HSV) [11]. Human neutrophil peptides (HNP 1–3) also suppress HSV in vaginal secretions [12]. These secretory products of the innate immune system are considered to be estrogen dependent, since many are the result of local mucosal metabolism, and the secretory fluid that contains these substances requires estrogen stimulation. Menopause results in a decline in the mucosaldependent elements of the innate immune system. Minor congenital defects in the innate immune system, such as polymorphisms which result in deficiency of mannose binding lectin (MBL), increase the risk of symptomatic infection [13]. MBL provides a target for complement activation by binding to the cell surface of pathogenic microbes. MBL is produced mainly in the liver and most likely arrives in the vaginal secretions as a transudate from the blood stream. MBL is a significant factor in vaginal mucosal defense against pathogens, although the MBL level in vaginal secretions is much lower than the level normally found in the systemic circulation. It is not clear whether MBL is produced by vaginal mucosal cells. During the reproductive years, toll-like receptors (TLRs) 1, 2, 3, 5, and 6 are expressed in vaginal mucosal cells. TLR 1, 2, and 5 mainly target bacteria. TLR 3 is directed against virus, and TLR 6 controls fungi [9]. The expression of TLRs is estrogen-dependent. This may explain the pre-pubertal and possibly post menopausal increased mucosal susceptibility to pathogens such as streptococcus or Neisseria gonorrhea.
Cell-Mediated Immunity Langerhans cells are abundant in vaginal and cervical mucosa [14]. In the lower female genital tract, T cells and Langerhans cells are most prevalent in the normal cervical transformation zone, so the cervical transformation zone is assumed to be the major site for cell-mediated immune reactions in this area of the human body [15]. The likely immune consequences of excision of this important area by extensive LEEP or cervical cone biopsy have not been determined (> Table 25.1). If the human skin is considered to be a major immune organ, then the cervix should be considered to have special immune function in that organ. Chronic cervicitis, often detected on cervical biopsy in asymptomatic women is actually a misnomer, as the normal cervical transformation zone is a site of significant immune activity in normal health. Pathogenic
Unique Skin Immunology of the Lower Female Genital Tract with Age
microbes can activate cervical inflammation, but the presence of numerous immune cells is actually physiologic. The increased vulnerability of the relatively fragile transitional epithelium in the transformation zone may require better standing defenses to prevent ascending infection. During the reproductive years, and to a greater extent during pregnancy, estrogen down-regulates antigen presenting cells. This results in a shift toward a Th2 immune response [16, 17]. Although this has not been studied with specific reference to the female lower genital tract, a Th2 response down-regulates the defensins and other secretory products of the innate immune system [18]. This relative immune compromise is presumed to be important for normal fertility and pregnancy. However, there are consequences, such as an increased risk for allergic contact dermatitis, as well as increased susceptibility to yeast, viruses, and other pathogens. Sexually transmitted diseases typically have mechanisms to avoid cell-mediated immunity [19]. The abundant macrophages and granulocytes in the cervical transformation zone are regulated by hormone changes of pregnancy. Reflecting the immune suppression of pregnancy, the number of macrophages in the cervical transformation zone declines in early pregnancy, and then increases in preparation for labor. Macrophages are involved in cervical ripening just prior to the onset of labor, and macrophages and granulocytes have a significant role in cervical dilation [20].
Immune Changes with Age Innate immune defenses of the vaginal mucosa are compromised with aging. Estrogen influences the expression of TLRs in vaginal mucosa [21, 22] (> Tables 25.2 and > 25.3). This loss of TLR expression increases the risk for colonization with pathogens. The post menopausal lack of epithelial cell maturation results in loss of vaginal surface barrier function. Pathogens can invade the more readily traumatized fragile epithelium Estrogen deficiency leads to a decline in mucosal secretions that contain the antimicrobial constituents of the innate immune system. The neutral vaginal pH after the menopause reflects loss of the acid defense as well as a significant decline in vaginal mucosal metabolic ability. Cell-mediated immunity is estrogen and age dependent. Langerhans cells are most prevalent in vulvar skin during the reproductive years [23]. Estrogen receptors on dendritic cells moderate the maturation of functional dendritic cells from precursor cells [24]. There is a decline in Langerhans cell function with aging, as well as a decreased Langerhans cell count by approximately 50% [25, 26].
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. Table 25.2 Characteristics of the lower female genital tract under the influence of estrogen Innate immunity
TLR 1, 2, 3, 5, 6 HBD 1, 2, 3, 5 SLPI MBL SP-A SP-D etc.
Humoral immunity
Very low IgA very low IgG IgG > IgA
Cell-mediated immunity
Depressed Th1 tendency for enhanced Th2
TLR toll like receptor; HBD human beta defensin; SLPI secretory leukocyte protease inhibitor; MBL mannose binding lectin; SP surfactant proteins
. Table 25.3 Characteristics of the lower female genital tract in the absence of estrogen Innate immunity Decreased expression of TLRs decrease in all secretory products Humoral immunity
Further decline in IgA with decreased cervical secretions
Cell-mediated immunity
Decline in langerhans cell count decline in cytokine responsiveness estrogenassociated suppression of Th1 response is eliminated
TLR toll like receptor; HBD human beta defensin; SLPI secretory leukocyte protease inhibitor; MBL mannose binding lectin; SP surfactant proteins
A decreased response to cytokines is also characteristic of aging [18]. The immunologically active cervical transformation zone is gradually eliminated by the aging process of squamous metaplasia. Antigen presenting cells are still present in the vaginal mucosa after menopause [27]. Post menopausal estrogen replacement can reactivate deficient vaginal mucosal cellmediated immune function. Asthma is a good example of the estrogen effect upon cell-mediated immunity. Asthma is influenced by the estrogen-related shift of cell-mediated immunity from a Th1 to a Th2 environment. Asthma is more prevalent in males than females prior to puberty, but higher in females with the rise in estrogen after puberty [28]. Asthma may become less severe after menopause following the decline in Th1 suppression [29]. Hormone replacement therapy after menopause may make asthma worse [30]. Similarly, post menopausal estrogen replacement may restore a Th2 environment that favors vaginal colonization with yeast.
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Conclusion Lower female genital tract immune defenses are complex and are not yet completely understood. Clearly, the immune system plays a major role in regulating vaginal microflora, but unfortunately, many pathogens have mechanisms to evade the immune defenses. Estrogen promotes the innate system, but suppresses the cell-mediated response in the lower genital tract. Humoral immunity appears to play only a small role in this portion of the female body. Immune function during the reproductive years reflects a balance between the need to protect against infection and the requirements of reproduction.
14.
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18.
References 1. Hjort H. Do antisperm antibodies reduce fecundity? A mini review in historical perspective. Am J Reprod Immunol. 1998;40:215–222. 2. Kunin CM, Evans C, Bartholomew D, Bates G. The antimicrobial defense mechanism of the female urethra: a reassessment. J Urol. 2002;168:413–419. 3. Brandtzaeg P. Mucosal immunity in the female genital tract. J Reprod Immunol. 1997;36(1):23–50. 4. Quesnel A, Cu-Uvin S, Murphy D, Ashley RL, Flanigan T, Neutra MR. Comparative analysis of methods for collection and measurement of immunoglobulins in cervical and vaginal secretions of women. J Immunol Methods. 1997;202:153–161. 5. Crowley-Nowick PA, Bell MC, Brockwell R, Edwards RP, Chen S, Partridge EE, Mestecky J. Rectal immunization for induction of specific antibody in the genital tract of women. J Clin Immunol. 1997;17:370–379. 6. Jalanti R, Isliker H. Immunoglobulin in human cervicovaginal secretions. Int Arch Allergy Appl Immunol. 1977;53:402–408. 7. Nardelli-Haefliger D, Wirthner D, Schiller JT, Lowy DR, Hildesheim A, Ponci F, De Grandi P. Specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus 16 virus-like particles. J Natl Cancer Inst. 2003;95(15):1128–1137. 8. Nicholson SC, Robindson TN, Sargent IC, Hallan NF. Fertil and Steril. 1996;65(4):871–873. 9. Horne AW, Stock SJ, King AE. Innate immunity and disorders of the female reproductive tract. Reproduction. 2008;135:739–749. 10. Klotman ME, Chang TL. Defensins in innate antiviral immunity. Nat Rev Immunol. 2006;6:447–456. 11. Meschi J, Crouch EC, Skolnik P, Yahya K, Holmskov U, Leth-Larsen R, Tornoe I, Tecle T, White MR, Hartshorn KL. Surfactant protein D bibds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication. J Gen Virol. 2005;86:3097–3107. 12. John M, Keller MJ, Fam EH, Cheshenko K, Kasowitz A. Cervicovaginal secretions contribute to innate resistance to herpes simplex virus infection. J Infect Dis. 2005;192:1731–1740. 13. Babula O, Lazdane G, Kroica J, Ledger WJ, Witkin SS. Relation between recurrent vulvovaginal candidiasis, vaginal concentrations
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of mannose-binding lectin, and a mannose-binding lectin gene polymorphism in Latvian women. Clin Infect Dis. 2003; 37:733–737. Bjercke S, Scott H, Braathen LR, Thorsby E. HLA-DR-expressing langerhans’-like cells in vaginal and cervical epithelium. Acta Obstet Gynecol Scand. 1983;62:585–589. Pudney J, Quayle AJ, Anderson DL. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol Reprod. 2005;73:1253–1263. Wira CR, Rossoll RM, Kaushic C. Antigen-presenting cells in the female reproductive tract: influence of estradiol on antigen presentation by vaginal cells. Endocrinology. 2000;141(8):2877–2885. Wira CR, Rossoll RM. Antigen presenting cells in the human reproductive tract: influence of sex hormones on antigen presentation in the vagina. Immunology. 1995;84:505–508. Thivolet J, Nicolas JF. Skin aging and immune competence. Br J Immunol. 1990;122:77–81. Chang JH, Ryang YS, Morio T, Lee SK, Chang EJ. Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation. Mol Cells. 2004;18: 177–185. Sakamoto Y, Moran P, Bulmor JN, Searle RF, Robson SC. Macrophages and not granulocytes are involved in cervical ripening. J Reprod Immunol. 2005;66:161–173. Pioli PA, Amiel E, Schaefer TM, Connolly JE, Wira CR, Guyre PM. Differential expression of toll-like receptors 2 and 4 in tissues of the human female reproductive tract. Infect Immun. 2004;72: 5799–5806. Sonnex C. Influence of ovarian hormones on urogenital infection. Sex Transm Infect. 1998;74:11–19. Harper WF, McNicol EM. A histological study of normal vulval skin from infancy to old age. Br J Dermatol. 1977;96:249–253. Paharkova-Vatchkova V, Maldonado R, Kovats S. Estrogen preferentially promotes the differentiation of CD11c+ CD11bintermediate dendritic cells from bone marrow precursors. J Immunol. 2004; 172:1426–1436. Nomura I, Goleva E, Howell MD, et al. Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol. 2003;171:3262–3269. Gilchrest B, Murphy G, Soter N. Effect of chronological aging and ultraviolet irradiation on Langerhans cells in human epidermis. J Invest Dermatol. 1982;79:85–88. Fahey JV, Prabhala RH, Guyre PM, Wira CR. Antigen-presenting cells in the human female reproductive tract: analysis of antigen presentation in pre-and post-menopausal women. Am J Reprod Immunol. 1999;42:49–57. Yawn BP, Wollan P Kurland MJ, Scanlon P. A longitudinal study of asthma prevalence in a community population of school age children. J Pediatr. 2002;140(5):576–581. Balzano G, Fuschillo S, Melillo G, Bonini S. Asthma and sex hormones. Allergy. 2001;56(1):13–20. Kos-Kudla B, Ostrowska Z, Marek B, et al. Effects of hormone replacement therapy on endocrine and spirometric parameters in asthmatic postmenopausal women. Gynecol Endocrinol. 2001;15(4): 304–311.
Specialized Skin: Genital
24 Vaginal Secretions with Age Paul R. Summers
The Source of Vaginal Secretions In the developing fetus, the vaginal epithelium is transformed from columnar to squamous prior to term birth. With the exclusion of the vaginal epithelium, most mucosal surfaces in the human body that demonstrate this type of squamous metaplasia during fetal development retain specific secretory glands. In spite of an absence of secretory subdermal glands, it is significant that the vaginal epithelial cells retain a remarkable secretory ability. Vaginal mucosa contains a microscopic intercellular network of secretory pathways. Intercellular channels are found between the tight junctions in the intermediate cell layer of the mucosa. These areas of dilation start as clefts in the parabasal cell layer of the epithelium, and appear as pores that can be seen at the mucosal surface using scanning electron microscopy [1]. The entire vaginal surface is, then, a secretory structure. The mucosal secretions of the female lower genital tract fulfill several important roles in the process of reproduction, ranging from lubrication, to microbial inhibition, to sperm facilitation. In a manner similar to mucosa at other body sites, it is presumed that vaginal secretions trap potentially pathogenic bacteria. The constant daily drainage of approximately 2 cc of secretions may, in that case, contribute somewhat to the removal of these adverse microbes [2]. More important, the confluent coating of secretions may restrict pathogens from contacting the mucosal surface, to prevent the essential first step in the establishment of infection. The most widely recognized constituent of vaginal mucosal secretions is the lactobacillus. More recent nonculture-based data have shown a number of acid-producing bacteria that may be present with or instead of lactobacillus [3, 4]. Normal vaginal secretions favor the growth of the various lactobacillus and other acid-producing bacteria strains that are considered normal flora. A mildly acidic pH and the presence of glycogen are two key factors for these strains. Metabolically restricted to anaerobic glycolysis, the lactobacillus strains release significant amounts of lactic acid into the vaginal mucosal secretions. Tradition attributes a protective role for the lactobacillus
against potential pathogens, although clinical experience suggests this presumed defensive action of the lactobacillus is strikingly inadequate. In spite of the essentially ubiquitous presence of acid-producing bacteria in normal vaginal secretions, the vaginal mucosa remains susceptible to a wide range of pathogenic microbes. During the antiseptic era of the early twentieth century, it was presumed that the lactate content of vaginal secretions contributed a significant antiseptic action to prevent infection (> Fig. 24.1). Although this simplistic view of vaginal antisepsis still remains popular, modern research has disclosed other constituents of vaginal secretions that present a more plausible explanation for antimicrobial action in the vaginal secretions. Human epithelial cells are highly active in the production of a wide range of metabolic products. In this regard, the vaginal mucosa is no exception. Many of these chemical products are released into the vaginal secretions, in some cases presumably to carry out a protective role. More than 40 different organic substances have been identified in normal vaginal secretions. Lactate is the primary acid that contributes to the low vaginal pH, but other normal constituents range from 15 typical aliphatic acids (such as acetic, myriatic, linoleic) to alcohols, glycols, and various aromatic compounds [5]. There is an important role for various elements of the innate and humoral immune systems, such as defensins and small amounts of IgA and IgG, in the vaginal fluid (refer to the chapter on immunology of the female lower genital tract). Unfortunately, vaginal pathogens are often able to evade the potential immune and mechanical barriers presented by the coating of mucosal secretions. For example, the polymicrobial pathogens of bacterial vaginosis produce hydrolytic enzymes that lyse the protein base of vaginal mucosal secretions so that pathogenic bacteria can reach the mucosa [6].
Hormone Influence upon the Vaginal Mucosal Secretions Hormone production regulates the quantity and character of vaginal mucosal secretions. Under the influence of
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. Figure 24.1 Theory of microbial inhibition proposed in the early twentieth century
estrogen, the glycogen-rich intermediate cell layer of the mucosa is the area of greatest metabolic and secretory activity. Basal and superficial cell regions are less metabolically important. Under the influence of cyclic hormone changes, constituents of vaginal secretions change significantly during the menstrual cycle [7]. Lactic acid and urea content is greatest between 48 h prior to 24 h after the luteinizing hormone (LH) surge. Mid-cycle changes probably reflect increased mucosal metabolic activity at that time [8]. After menopause, the metabolically active intermediate cell layer of the mucosa is lost, with a simultaneous decline in secretory products [9]. Furthermore, vaginal subdermal blood flow is decreased after menopause, resulting in less mucosal transuadate [10].
pH and Vaginal Secretions Tradition has assigned an acid pH around 4.5 to be the main regulatory parameter for the vagina (see Chart). Lactic acid is the major source of hydrogen ions in vaginal secretions during the reproductive years [5]. Although the common literature tends to attribute vaginal lactic acid production solely to the lactobacillus, the vaginal mucosa also releases lactate as an end result of glycolysis. A significant amount of lactic acid is a by-product of normal anerobic vaginal mucosal metabolism [9]. Lactobacilli are not the only source of vaginal lactate, and actually may not be the primary source.
Lactate from Lactobacillus vs. Mucosal Glycolysis Early studies demonstrated the dual sources of vaginal lactate, and even suggested mucosal glycolysis as the primary source. There is no direct correlation between the pH of vaginal mucosal secretions and the presence of lactobacilli, nor is there a correlation between the amount of glycogen substrate for growth of lactobacilli and the actual amount of lactobacillus [11, 12]. In the absence of a significant vaginal colonization with lactobacillus, the pH may still be in the normal acid pH range. For example, a newborn has significant lactate in the initially sterile vaginal secretions, with a pH of around 5, prior to any colonization with lactobacillus [13]. With declining maternal estrogen influence, the vaginal pH of the infant rises to the neutral range as the metabolic activity of the vaginal mucosa declines by 6 weeks of life. The vagina does not become colonized with lactobacillus until puberty. The neutral vaginal pH after menopause is associated with a significant lack of lactate as a result of diminished glycolysis in the intermediate cell layer of the mucosa, as well as a lack of lactobacillus. In view of this early research, it is surprising that the idea that the lactobacillus is the single source of lactate prevails in the current common understanding. Recent research also suggests that lactate from gylcolysis in the vaginal mucosa may have the chief regulatory role for vaginal pH. For example, pH in the vaginal
Vaginal Secretions with Age
fornices has been shown to be lower than the pH in the mid vagina, in spite of a relatively uniform distribution of lactobacillus [14]. Lactate from lactobacillus would not explain the observation that mucosal secretion during sexual stimulation appears to contain the same concentration of lactate that is found in the non-stimulated state [5]. It is also unlikely that transient alterations in lactobacillus metabolism or growth, with resulting release of excess lactate, could explain the brief decline in pH at the time of ovulation [9]. Estrogen directly and indirectly regulates vaginal mucosal metabolism, mucosal secretions, and pH (> Fig. 24.2). Microbial sources of lactate are dependent upon estrogeninduced glycogen as an energy source. Similarly, vaginal mucosal anaerobic metabolism is estrogen-dependent. Possibly, a separate mechanism for vaginal mucosal pH
. Figure 24.2 Vaginal pH and other mucosal effects of estrogen
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regulation has been identified. Under the influence of estrogen, superficial vaginal mucosal cells may secrete hydrogen ions into the vaginal lumen in a manner similar to gastric chief cells [15]. A direct link with intraepithelial lactate production was not reviewed in this study, but anaerobic metabolism of glycogen remains the prime source of intracellular hydrogen ions. Decreased mucosal metabolism after menopause alters content as well as quantity of normal mucosal secretions. Either vaginal glycogen increases under the influence of estrogen [16], or it is subjected to increased metabolism to lactate [17]. In either case, estrogen contributes to increased lactate. The vaginal pH fluctuates with the menstrual cycle, with its lowest average value around the time of ovulation. Presumably, this would be evidence of maximal anaerobic skin metabolism and lactate release.
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Sperm survive best in an anaerobic environment. It is reasonable to speculate that this enhanced anaerobic environment at the time of ovulation may contribute to sperm survival [7].
Vaginal pH with Age The vaginal pH is not just important for microbe control, but a mildly acid pH is also ideal for normal vaginal skin metabolism, including the production of various proteins that are important for vaginal immune defenses. The rise in vaginal pH after menopause results in a loss of natural skin defenses, with an increased rate in the urinary tract and yeast infections [18]. Topical application of estrogen to the menopausal vagina restores a normal pH and lowers the risk for infection [19]. It is clear that skin barrier function is compromised by the typically neutral menopausal vaginal pH. This rise in pH increases the susceptibility to contact dermatitis [20]. A menopausal rise in skin pH results in defective enzyme function and vulvar skin ceramide deficiency [21]. Stratum corneum acidity is known to be essential for a normal inflammatory response and for optimal skin barrier function [22]. This concept most likely also applies to vaginal mucosa. Application of neutral pH buffers to skin in general results in decreased stratum corneum integrity and cohesion [23]. It is only reasonable to conclude that this concern also applies to the neutral vaginal pH in the menopausal state. It is of interest to note that the vaginal pH is mildly alkaline during menses. It is possible that this transient neutral or alkaline vaginal pH during menses also contributes to a risk for contact dermatitis from menstrual sanitary pads, with special concern if the bleeding episode is prolonged.
Conclusion Vaginal mucosal metabolism is uniquely estrogen-dependent (> Table 24.1). During the reproductive years, estrogen stimulates the maturation of a metabolically active intermediate cell layer within the vaginal epithelium. This glycogen-rich cell layer is the source for much of the complex content of the mucosal secretions. Constituents of the mucosal secretions, as well as support for normal microbial flora, remain almost totally estrogen-dependent. The characteristically low vaginal pH is directly linked to anaerobic vaginal mucosal metabolism, as well as to the traditionally recognized lactobacilli and other acid-producing vaginal microflora. A low estrogen level
. Table 24.1 Menopausal effects Decreased glycogen to support lactobacillus and other microbes Decreased glycogen to support mucosal metabolism in the intermediate cell layer Significant loss of the metabolically active intermediate cell layer Decline in protective mucosal secretion Decline in hydrogen ions and other secretory products in the vaginal fluid
prior to puberty and after menopause results in inactive vaginal mucosa with little production of secretions. Vaginal pH rises with the metabolic decline in mucosal and microbial glycogen-dependent anaerobic glycolysis. The resulting neutral pH after menopause most likely results in further loss of vulvovaginal skin barrier function.
References 1. Burgos MH, Roig de vargas-Linares CE. Cell junctions in the human vaginal epithelium. Am J Obstet Gynecol. 1970;108(4):565–567. 2. Wagner G, Levin RJ. Vaginal fluid. In: Hafez ESE, Evans TN (eds) The Human Vagina. New York: North-Holland Publishing, 1978, p. 123. 3. Zhou X, Bent SJ, Schneider MG, et al. Characterization of vaginal microbial communities in adult healthy women using cultivationindependent methods. Microbiology. 2004;150:2565–2573. 4. Zhou X, Brown CJ, Abdo Z, et al. Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J. 2007;1:121–133. 5. Huggins GR, Preti G. Volatile constituents of human vaginal secretions. Am J Obstet Gynecol. 1976;126(1):129–136. 6. Cauci S, Hitti J, Noonan C, Agnew K, Quadrifoglio F, Hillier SL, Eschenbach DA. Vaginal hydrolytic enzymes, immunoglobulin against Gardnerella vaginalis toxin, and early risk of preterm birth among women in preterm labor with bacterial vaginosis or intermediate flora. Am J Obstet Gynecol. 2002;187:877–881. 7. Preti G, Huggins GR. Cyclical changes in volatile acidic metabolites of human vaginal secretions and their relation to ovulation. J Chem Ecol. 1975;1:361–376. 8. Preti G, Hugins GR. Organic constituents of vaginal secretions. In: Hafez ESE, Evans TN (eds) The Human Vagina. New York: NorthHolland, 1978, pp. 162–163. 9. Gross M. Biochemical changes in the reproductive cycle. Fertil Steril 1961;12(3):245–262. 10. Society of Obstetricians and Gynecologists of Canada. The detection and management of vaginal atrophy. Int J Gynecol Obstet. 2004; 88:222–228. 11. Weinstein L, Howard JH. The effect of estrogenic hormone on the H-ion concentration and the bacterial content of the human vagina with special reference to the Doederline bacillus. Am J Obstet Gynecol. 1939;37:698–703.
Vaginal Secretions with Age 12. Weinstein L, Bogin M, Howard JH, Finkelstone BB. A survey of the vaginal flora at various ages with special reference to the Doederline bacillus. Am J Obstet Gynecol. 1936;32:211–218. 13. Raskoff AE, Feo LG, Goldstein L. The biologic characteristics of the normal vagina. Am J Obstet Gynecol. 1943;47:467–494. 14. Tsai CC, Semmens JP, Semmens EC, Lam CF, Lee FS. Vaginal physiology in postmenopausal women: pH value, transvaginal electropotential difference, and estimated blood flow. South Med J. 1987; 80:987–990. 15. Gorodeski GI, Hopfer U, Liu CC, Margles E. Estrogen acidifies vaginal pH by up-regulation of proton secretion via the apical membrane of vaginal-ectocervical epithelial cells. Endocrinology. 2005;146(2):816–824. 16. Bo WJ. The effect of progesterone and progesterone-estrogen on the glycogen deposition in the vagina of the squirrel monkey. Am J Obstet Gynecol. 1970;107:524–530. 17. Ayre WB. The glycogen-estrogen relationship in the vaginal tract. J Clin Endocrinol Metab. 1951;11:103–110.
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18. Weisberg E, Aytin R, Darling G, et al. Endometrial and vaginal effects of dose-related estradiol delivered by vaginal ring or vaginal tablet. Climacteric. 2005;8:83–92. 19. Kunin CM, Evans C, Barhholomew D, Bates G. The antimicrobial defense mechanism of the female urethra: a reassessment. J Urol. 2002;168:413–419. 20. Berg RW, Milligan MC, Sarbaugh FC. Association of skin wetness and pH with diaper dermatitis. Pediatr Dermatol. 1994;11:18–20. 21. Fluhr JW, Kao J, Jain M, et al. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol. 2001;117:44–51. 22. Mauro TM. SC pH: measurement, origins, and functions. In: Elias PM, Feingold KR (eds) Skin Barrier. New York: Taylor & Francis, 2006, p. 225. 23. Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias PM. pH directly regulates epidermal permeability barrier homeostasis and stratum corneum integrity/cohesion. J Invest Dermatol. 2003; 121:345–353.
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58 Aging-associated Non-melanoma Skin Cancer: A Role for the Dermis Davina A. Lewis . Jeffrey B. Travers . Dan F. Spandau
Introduction
Aging and Carcinogenesis
The American Cancer Society estimates that well over one million patients are diagnosed with skin cancer each year, representing over half of all invasive and in situ cancers that occur in the United States each year [1]. The magnitude of these statistics suggests that the treatment of skin cancer in the United States is a problem both for patients and for the healthcare system. Conclusive evidence has demonstrated that the main environmental risk factor for developing skin cancer is exposure to the ultraviolet components in sunlight, primarily ultraviolet B wavelengths (UVB) [2–6]. Although skin cancer can occur at any age, there is a strong correlation between the development of skin cancer and advancing age [7]. In fact, the majority of skin cancers are found in people over the age of 60; therefore, age is also a risk factor for the development of skin cancer [1, 7]. While the correlation between aged epidermis and skin cancer is obvious, the mechanism responsible for this relationship remains obscure. Recent in vitro evidence as well as epidemiological data suggest one possible mechanism may involve alterations in the insulin-like growth factor-1 receptor (IGF-1R) signaling network [8–12]. In the skin, keratinocytes express the IGF-1R but they do not synthesize IGF-1 [13, 14]. Dermal fibroblasts support the proliferation of keratinocytes in the epidermis by secreting IGF-1 [13, 14]. Interestingly, as dermal fibroblasts age, their capacity to produce IGF-1 is severely diminished; therefore, aged skin keratinocytes are provided with a reduced supply of IGF-1 [11, 12]. This drop in IGF-1 expression is critically important for nonmelanoma skin carcinogenesis because adequate levels of IGF-1 are required to prevent UVB-induced mutations in keratinocytes. In vitro and in vivo studies have shown that IGF-1R activation protects the epidermis from initiating carcinogenic events [8–12]. The chapter will discuss the relationship between aging and cancer, the critical features of non-melanoma skin cancer (NMSC), and the newly proposed role for the dermis in driving the development of aging-associated NMSC.
Evidence accumulated thus far definitively links increasing age and the onset of cancer; in fact, the greatest risk factor for developing cancer is age [15]. At least 80% of all neoplasia occur in individuals over the age of 50 [15, 16] and the incidence of cancer rises with increasing age until the age of 90 [16]. In general, cancer is a disease that primarily afflicts geriatric patients. However, the mechanisms behind the link between cancer and aging are only beginning to be understood. Many explanations defining the relationship between aging and carcinogenesis have been postulated but few have been conclusively proven. A common theory describes the long passage of time required between the creation of initiated cells containing fixed DNA mutations and the phenotypic appearance of tumors containing the descendent clones of the original initiated cells [17]. This theory suggests that the sequential changes seen in the carcinogenic process require many years if not decades to develop, ensuring that apparent diagnosable tumors arise in older patients [17]. Separate studies have shown that the ability of individuals to affectively prevent the occurrence of initiated cells through DNA repair mechanisms declines with age [17–19]. Aging cells have an increased number of somatic mutations, probably through a combination of DNA damage as a result of environmental factors and an enhanced rate of errors occurring during DNA replication [16, 17]. As the capability to repair DNA lesions diminishes, the frequency of newly initiated potentially neoplastic cells increases as well as the probability of identifiable neoplasia. More recently, studies have identified age-dependent changes in stromal tissue which provide an increasingly favorable growth environment for initiated cells [17, 20–22]. As cells in these support tissues age, they frequently begin to lose control of normal gene expression and cellular function [20–22]. Increasingly they express genes characterized by an inflammatory phenotype that can promote the growth of previously initiated cells or enhance the progression of newly initiated tumorigenic cells [20, 21].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_58, # Springer-Verlag Berlin Heidelberg 2010
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Non-melanoma Skin Cancer: The Epidermis Cancers of the skin are the most common cancers to afflict Caucasians in the United States [1, 23]. The significant morbidity and exorbitant healthcare costs associated with the management of skin cancer provide substantial evidence of the need for research in this field. The primary environmental factor influencing the development of skin cancer is exposure to ultraviolet wavelengths in sunlight [2–5, 24–26]. Because 80% of all skin cancer are found in people over the age of 60, age is also a risk factor for the development of skin cancer. While the correlation between aged epidermis and NMSC is apparent, the mechanism responsible for this relationship remains enigmatic. The historical explanation for the correlation between skin cancer and aging argues that UVB damage inflicted on skin during adolescence initiates mutations in keratinocytes that are selectively enriched over many decades until enough genetic changes have gradually accumulated in these keratinocytes that they become carcinogenic. In fact, this mechanism has been proposed to explain the long latency period observed in other types of cancers where there is also a correlation between the development of cancer and age [27, 28]. However, recent data have suggested a modification of this theory based on the altered function of aged stromal cells (i.e., fibroblasts) affecting epithelial cells [20, 21]. This hypothesis states that the selection of initiated epithelial cells is accelerated in aged tissue due to altered gene expression in senescent fibroblasts supporting epithelial cell growth [22, 29, 30]. In addition, the aged state of cells may play a greater role in the initiation of carcinogenic DNA mutations than was previously thought to occur [31]. These new ideas on the origins of cancer have led to a new paradigm to explain non-melanoma skin carcinogenesis. In order to explain the rationale for this theory of skin carcinogenesis, the following paragraphs will summarize the current understanding of the effect of ultraviolet B (UVB) irradiation on skin, aging-associated NMSC risk factors, and how cellular senescence influences NMSC carcinogenesis.
Effects of UVB Irradiation on the Skin Sunlight is composed of a variety of wavelengths of light, which can be divided into infrared, visible, and ultraviolet light, arranged from the longest wavelengths to the shortest. The ultraviolet (UV) spectrum can be further divided into three classifications, UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm; [3]).
Wavelengths of light in the UVC range have the potential to cause the most damage to living organisms; however, nearly all UVC wavelengths are absorbed in the atmosphere and never reach the surface of the earth [3]. UVA radiation is the most abundant ultraviolet light to penetrate the atmosphere, although the data are still inconclusive as to the exact role that UVA radiation plays in human skin cancer [32]. Even though UVB radiation makes up only 0.3% of the total light that reaches the surface of the earth [4], exposure of human cells to the UVB component in sunlight can directly damage DNA and lead to the development of cancer [5]. In general, UVB radiation only penetrates the epidermal layer of the skin. Therefore, the primary cells at risk for potential UVB-induced damage reside in the epidermis, where keratinocytes are the predominant cell type. UVB irradiation of the epidermis leads to UVB-induced DNA damage in keratinocytes [33–35]. Exposure to UVB produces distinctive signature mutations in keratinocyte DNA due to the direct absorption of energy. This DNA damage consists predominantly of cytosine (C) to thymidine (T) transitions at dipyrimidine sites, including CC ! TT dimerization between adjacent pyrimidines takes place on the same DNA strand. If these mutations are allowed to persist, this DNA damage may be propagated to daughter cells perhaps giving rise to proliferative diseases including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The importance of cellular proliferation with DNA damage in carcinogenesis is elegantly illustrated by organisms composed largely of post-mitotic cells which do not develop cancers, such as the nematode C. elegans and the fruit fly D. melanogaster [36–38]. In contrast, tissues from organisms containing replicatively mitotic cells do develop cancers [37, 38]. The dose and duration of UVB received determines how the epidermis responds [34, 39, 40]. Brief exposures to UVB will arrest keratinocyte proliferation to allow for the repair of DNA damage before the keratinocyte re-enters the cell cycle. However, if the exposure to UVB is prolonged a combination of several outcomes can occur: (1) DNA damage is not repaired and keratinocytes undergo apoptosis, (2) keratinocytes become senescent as a tumor evasion mechanism, or (3) damage may be mis-repaired or partially repaired and cells continue to proliferate, propagating potentially mutagenic DNA damage. The first two observations, UVB-induced apoptosis and UVB-induced senescence, are part of the normal protective response of human skin to UVB exposure that maintains the integrity of the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes are not permitted to replicate with DNA mutations (the appropriate
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UVB response). The third observation, the failure of UVB-induced senescence leading to replication in keratinocytes containing UVB-damaged DNA, represents flawed protection from UVB damage, and the consequences of failed UVB protection may include the stabilization of initiating DNA mutations that could lead to the malignant transformation of keratinocytes (an inappropriate UVB response). Acquired mutations in key tumor suppressor genes such as p53, and RB are targets of UVB exposure [33, 41, 42]. Keratinocytes harboring p53 mutations fail to undergo UVB-induced senescence, [10], become resistant to the fail-safe apoptotic response, and acquire a growth advantage [34, 41–43]. This growth advantage allows for clonal expansion of mutant p53 cells over time contributing to the development of premalignancies and malignancies in skin [34, 41–44]. Importantly, p53 mutation hot-spots are common in NMSC at dipryrimidine sites [41, 44]. In addition to causing gene mutations, UVB exposure also induces immune-suppression [45, 46]. Reports show that UVB exposure inhibits the antigen presenting capabilities of epidermal Langerhans cells and stimulates the release of keratinocyte immune-suppressive and pro-inflammatory lipids and cytokines [39, 47–49]. The importance of this UV-induced immune-suppression and inflammation in relation to skin malignancies is logical since development of cancers requires escape from immune system function and inflammatory changes [33, 39, 47, 50]. Along with UVBinduced mutations, immune-suppression and inflammation, UVB is known to cause a change in epidermal architecture and biochemistry. In skin chronically exposed to the sun, there is an increase in actinic lesions along with a disorganization of collagen bundles [51]. UVB-induced alterations in skin biochemistry include deregulation of growth factors and their receptors, for example IGF-1, erbB1, erbB2; activation of mitogenic signaling pathways such as ras, p38 and JNK MAPKs, and inappropriate activation of transcription factors such as NF-kB [9, 52, 53].
Aging-Related Risk Factors for Developing NMSC As noted previously, there is a strong correlation between the development of skin cancer and advancing age. Childhood exposure to UV is believed to be one of the most critical risk factors in the development of skin cancers in adults [54]. One explanation for this correlation is that epidermal keratinocytes acquire UV-induced tumorigenic mutations in childhood which accumulate over time in
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selected populations of cells that manifest as skin cancers later in adult life [7, 37, 55, 56]. This explanation adheres to the multi-stage theory of carcinogenesis where initiating mutations occurring in target genes require promotional events to expand and form clones of mutated cells, eventually progressing and developing into cancers. People are exposed daily to oxidative stressors which cause DNA damage that has the potential to be fixed as mutations [57–59]. Furthermore, as human beings age, there also appears to be an increase in the production of reactive oxygen species (ROS) that may in turn increase the potential for damage to DNA [58–60]. Along with this, a decline in the function of the p53 protein has been reported in the aging process that could contribute to an increase in the frequency of mutations and tumorigenesis [56]. Furthermore, with aging, the fidelity of DNA repair mechanisms decline and therefore, may accelerate the accumulation of mutations over time [60–62]. Mutations in cancer cells are so numerous that almost certainly other factors must contribute to their development. Indeed, aging is also associated with a decreased immune function [63, 64]. Investigations have demonstrated that agerelated changes in human T lymphocytes contributed to a decrease in immunity against infections and neoplasms as well as causing an increase in autoimmune diseases [64, 65]. The combination of increased ROS production, decreased immune function, decreased p53 function, and a decreased fidelity in DNA repair mechanisms with advancing age does indeed provide a provocative environment for developing cancers.
Cellular Senescence and NMSC Cellular senescence is defined as an irreversible arrest in cellular replication in otherwise metabolically active cells. First identified as a phenomenon controlling the longevity of cells cultured in vitro [66], it is now known that replicative senescence in vitro is caused by the erosion of telomeres and an ensuing DNA-damage response [67–69]. Characteristics of senescent cells in vitro include irreversible growth arrest, increased resistance to apoptotic signals, changes in cell functions such as secretion of growth factors, cytokines, degrading enzymes, an over-expression of proteins, oncogenes and chromatin reorganization. Factors leading to senescence include finite replicative capacity via telomere shortening, DNA damage, or over-expression of mitogenic signals [36, 66, 70]. Although senescent cells cannot initiate DNA replication in response to physiological mitogens, they remain viable and continue to be metabolically active. The critical
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pathways identified in cellular senescence involve p53, p16, p21 and pRB [70]. There is compelling evidence suggesting that senescent cells accumulate in aged tissues [71–73]. In order to assess the contribution of senescent cells to aging and cancer, several markers have been employed such as senescence associated b-galactosidase, high levels of HIRA (a heterochromatin protein), and damaged teleomeres. In one study examining cultured human fibroblasts and keratinocytes, the senescent phenotype was absent in terminally differentiated keratinocytes, quiescent fibroblasts, pre-senescent cells, or immortalized cell lines [71]. In this same report, an age-related increase in senescent dermal fibroblasts and epidermal keratinocytes in human skin was observed [71]. Up to 80% of the fibroblasts examined in geriatric primate skin have senescent markers such as damaged teleomeres and high levels of HIRA [72, 73]. Given this age-associated accumulation of senescent cells, it is reasonable to propose that cellular senescence may contribute to age-related cancers by altering the surrounding tissue into a neoplastic promoting environment. The paradoxical effect of cellular senescence on an organism’s well-being has been called antagonistic pleiotropy [37, 74]. On one hand, cellular senescence is a powerful tumor suppressor limiting cell lifespan and removing damaged cells from a proliferative state preventing formation of clonal tumors [22, 44, 75]. On the other hand, the accumulation of senescent cells may contribute to aging and provide a tumor promoting environment due to their altered properties such as stromal matrix reorganization and/or degradation, secretion of growth factors and inflammatory cytokines [21, 37]. Inflammation is an important factor promoting carcinogenesis. For example, lesions visually described to be solar keratosis were identified as squamous cell carcinoma histologically when inflammation was present [50]. Simply put, the accumulation of senescent cells in aging tissue may serve to maintain tissue architecture but inadvertently due to their altered function, change the surrounding tissue milieu to an environment where damaged and mutated cells can more easily become malignant. Evidence to substantiate this hypothesis comes from investigations in which human senescent fibroblasts were found to stimulate premalignant and malignant cells to proliferate in culture and form tumors in mice due in part to senescenceinduced secretion of soluble and insoluble factors [20, 21, 76]. A study using a conditional mouse oncogenic K-rasV12 model for cancer initiation showed that senescent cells only existed in pre-malignant and not malignant tumors suggesting senescence may be an indicator in the diagnosis and prognosis of cancers [77].
Non-melanoma Skin Cancer: The Dermis Historically, aging-associated NMSC is believed to be caused by the accumulation of damaged cells over decades. For example, one of the first signs of precancerous NMSC in aged individuals has been the appearance of actinic lesions. These lesions are readily apparent in the epidermis and are treated at the level of the keratinocyte. However, what if arising actinic damage could be prevented, and thereby NMSC, by detecting changes before they have reached the epidermis? The answer to this question may be underneath the epidermis in the dermis. Considering the essential role the dermis has on epidermal function, it is surprising that it was only a decade ago that attention was drawn to age-related changes in the dermis. Therefore, although the target cell of NMSC resides in the epidermis, it is necessary to re-examine the role the dermis may play in the development of NMSC.
Dermal and Epidermal Synergism The proper functioning and well-being of the skin is reliant on synergistic interactions between the dermal fibroblasts and epidermal keratinocytes. The integration of all signals received by a keratinocyte will determine the specific path that a cell takes at any given time during differentiation. The dermis contains a variety of cell types including fibroblasts, macrophages, mast cells, dendritic cells and dermal T-lymphocytes. Composed of extracellular matrix, collagen and elastin fibers, the stroma and some basement membrane components are synthesized by dermal fibroblasts, which produce soluble factors promoting survival and growth of the tissue. When it is necessary to remodel or repair the tissue, fibroblasts produce a mixture of degrading enzymes, cytokines, and growth factors. The influence of the dermis on the epidermis, and vice versa, is far reaching. In studies where site-matched papillary or reticular dermal fibroblasts were used to construct in vitro skin equivalents, the epidermal morphology, the formation of the basement membrane, and the terminal differentiation status were influenced by the type of fibroblast used [78]. When the dermis was composed of papillary fibroblasts, epidermal keratinocytes were morphologically symmetrical and all levels of terminal differentiation were expressed. Whereas skin equivalents constructed using reticular fibroblasts impeded the formation of the basement membrane and terminal differentiation in the epidermis [78]. Furthermore, the
Aging-associated Non-melanoma Skin Cancer: A Role for the Dermis
number of fibroblasts used to construct the dermal matrix of skin equivalents also appears to be essential in establishing normal epidermal growth [79].
The Dermis, the Immune System, and Inflammation What about the role of the dermis in skin immunity? The dermis has its own armory of weapons to impede the progress of any invader which has compromised the epidermis. Acting as a sentinel, the epidermis defends against environmental and pathogenic invasion. The epidermis is equipped with Langerhans cells and T-cell receptorexpressing dendritic epidermal T-cells (DETC) to cope with incoming insults. A substantial list of dermatologic diseases can cause epidermal immunity to go awry, such as psoriasis, atopic dermatitis [80], which may upset the balance between epidermis and dermis. DETC produce and respond to insulin-like growth factor I (IGF-1). In mice that are deficient for DETC, the epidermal balance is tipped from proliferation to apoptosis and levels of insulin-like growth factor receptor (IGF-1R) are decreased [81]. Addition of either DETC or IGF-1 can correct this imbalance highlighting the influence of the immune system on growth factors in the epidermal–dermal relationship, skin homeostasis and wound repair [81]. Fibroblasts are the most abundant cells of the dermis and have a key role in the structural integrity, mechanical strength and landscape of the extracellular matrix (ECM). Fibroblasts secrete both collagen and matrix metalloproteases that regulate ECM turnover. Adding to their resume, fibroblasts have also been shown to trigger and alter the inflammatory response as well as aid in wound healing [82–85]. Additionally, in some tissue suppression of the immune response may be driven in part by fibroblasts [86]. ‘‘Activated’’ or ‘‘cancer associated’’ fibroblasts are known to produce (tissue specific) pro-inflammatory cytokines, chemokines and growth factors [82, 87, 88]. Inflammation leads to further activation of fibroblasts and the production of more inflammatory mediators [87]. This persistent vicious cycle has been well established for fibroblasts and results in chronic inflammation. Chronic inflammation can be started and sustained by disease states, cancer and activated fibroblasts. Chronic inflammation is also well known to be involved in all stages of carcinogenesis. In many cases inflammation is therapeutically treated at the level of an immune cell or blockade of the offending cytokine [87]. However, the discovery that fibroblasts can indeed initiate, promote and sustain
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inflammation should make them attractive new target for anti-inflammatory and anti-cancer therapeutics.
Dermal Aging, Senescence, and Cancer In 1956, Harman et al. suggested that free radicals were involved with the deterioration of human biochemistry with age and degenerative diseases [89]. Furthermore, reactive oxygen species (ROS) were shown to cause DNA damage and indeed promote the aging process [90]. Organism aging is therefore thought to be at least in part due to accumulation of this free radical damage over time (aging) indirectly and directly causing DNA damage [91, 92]. Fortunately the skin has powerful antioxidant defense mechanisms preventing or scavenging ROS formed and repairing DNA damage. Currently, aging-associated skin cancer has been hypothesized to be a direct result of DNA damage accumulating over time until a threshold is reached overwhelming the tissue resulting in skin cancers [7, 37, 54–56]. One powerful mechanism employed in response to stress is a state of arrested growth and altered function called senescence. Senescence was observed by Hayflick in 1961 [66] as a cell that had reached the end of its proliferative capacity in culture. Even though these cells had lost their proliferative capacity they remained viable. Some of the characteristic of senescent cells are growth arrest, resistance to apoptotic signals, and altered gene expression [88, 93]. In tissues where homeostasis hinges on precise interactions between epithelial and mesenchymal cells, presence of senescent cells may disrupt the proper function of the tissue and may have far reaching effects [94]. As humans age, there are many significant changes that occur in the body, of these the most outwardly visible are the changes to the skin. Loss of tone/elasticity, increased pigmentation and transparency are all commonly visible in aging [95]. However, what are not visible are the underlying biological, chemical and molecular changes going on under those outwardly visible changes. Cellular senescence appears to play a role in aging [75]. Brought on by DNA damage, oncogene dysfunction, other forms of stress, chromatin damage and telomere shortening (accelerated by oxidative damage), senescent cells are also found to accumulate in during normal epidermal aging [71–73]. The degree to which senescent cells help or disrupt the normal functioning of skin is unknown. What effect they have on the surrounding tissue microenvironment, adjacent cells, as well as the role they have in disease processes such as skin cancer is only just beginning
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to be unearthed. Some of the changes, such as secretion of growth factors, cytokines and degrading enzymes as well as changes in gene expression, have been seen in senescent cells.
A New Role for the Dermis in Aging-Associated NMSC As discussed, the central dogma correlating the link between skin cancer and aging is that UVB-induced skin damage during childhood and early adolescence initiates mutations in keratinocytes. Subsequently, these keratinocytes containing mutations acquire a growth advantage that over many decades to become carcinogenic. However, can it be presumed that time is the sole contributor to UVB-induced skin cancers? It is reasonable then to consider that the physiology of aging may also lend a hand to carcinogenic events. Recent data from a variety of labs have led to a modification on the origin of agingassociated skin cancer based on the accumulation of senescent dermal fibroblasts in geriatric skin [11]. This new paradigm further substantiates the importance of the interaction between dermal fibroblasts and epidermal keratinocytes in preventing the initiation of carcinogenic events. These interactions are dependent on IGF-1/IGF-1R signaling which play an important role in aging and the response of skin to UVB irradiation [8–12].
Role of the IGF-1R and IGF-1 in the Skin The stroma and some basement membrane components are synthesized by stromal fibroblasts which also produce soluble factors that promote survival and growth of the tissue. When it is necessary to remodel or repair the tissue, stromal fibroblasts produce a mixture of degrading enzymes, cytokines, and growth factors. The health and proper functioning of the skin is highly dependent on the synergistic interactions between the dermal fibroblasts and epidermal keratinocyte. One factor regulating the interaction between dermal fibroblasts and epidermal keratinocytes is IGF-1 [13, 14]. In human skin, keratinocytes express the IGF-1R but do not synthesize IGF-1. Dermal fibroblasts support the proliferation of epidermal keratinocytes by secreting IGF-1. The mature IGF-1R consists of four subunits, two identical extracellular alpha and two identical transmembrane beta subunits. The two alpha and alpha-beta subunit structures are maintained by disulphide bridges. IGF-1, IGF-2 and high concentrations of insulin can activate the IGF-1R resulting in tyrosine kinase activity. Subsequently, binding or phosphorylation of
cellular substrates in close proximity via SH2 binding domain leads to downstream signaling. The importance of IGF-1R signaling in skin carcinogenesis is clearly evident from a variety of studies. Transgenic mice overexpressing IGF-1 in the basal layer of skin epidermis exhibited epidermal hyperplasia, hyperkeratosis and squamous papillomas [96–98]. Conversely, IGF-1R knockout mice demonstrate severe hyperplasia [97]. The IGF-1R has also been shown to be important in normal epidermal differentiation [99]. Therefore, the activation of the IGF-1R can influence all stages of epidermal homeostasis. The control of longevity has also demonstrated a critical role for the insulin/IGF-1 signaling pathway in invertebrate and mammalian animal models [100–102]. Furthermore, reports have identified a key role for the IGF-1R in regulating the response of cells to oxidative stress [103].
The IGF-1R-dependent UVB Response of Human Keratinocytes Experiments that assessed the role of various growth factors on the response of keratinocytes to UVB irradiation identified that the activation status of the IGF-1R was a critical component affecting UVB-induced apoptosis in vitro (> Fig. 58.1) [8]. Inhibition of the IGF-1R, via ligand withdrawal, treatment with neutralizing antibodies, or treatment with IGF-1R-specific small molecule inhibitors prior to irradiation increased the sensitivity of keratinocytes to UVB-induced apoptosis [8–12]. Studies identified that the functional activation of the IGF-1R provided protection to human keratinocytes from UVB-induced apoptosis. However, an equally important observation was that although the activation of the IGF-1R prevents cell death, the surviving keratinocytes cannot replicate and become senescent (> Fig. 58.1) [8, 10]. The induction of senescence in response to UVB irradiation is a tumor evasion mechanism that maintains the important barrier function of the epidermis while ensuring keratinocytes cannot proliferate in the presence of irreparable UVB-induced DNA damage. This appropriate response to UVB-irradiation prevents the propagation of potentially neoplastic keratinocytes. In contrast, when the IGF-1R is functionally inactive at the time of UVB-irradiation, a portion of the keratinocytes will undergo apoptosis; however, keratinocytes that survive do not become senescent, do not repair UVB-damaged DNA, and they continue to proliferate with the potential of converting the damaged DNA into initiating carcinogenic mutations [10]. This scenario would be an inappropriate response to UVB-induced DNA damage, leading to carcinogenesis [10–12].
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. Figure 58.1 Keratinocyte IGF-1R-dependent UVB response. This cartoon demonstrates the consequences of UVB exposure to normal human keratinocytes in vitro and the role of the IGF-1R. At low doses of UVB irradiation, keratinocytes sustain mild DNA damage which can be completely repaired via normal cellular processes. These keratinocytes continue to proliferate unabatedly. High doses of UVB cause extensive DNA damage that the keratinocyte cannot repair resulting in cell death via apoptosis, or even necrosis if the UVB dose is high enough. Keratinocyte responses to both low and high doses of UVB irradiation are independent of the IGF-1R activation status. However, the keratinocyte response to a wide range of intermediate doses of UVB is completely dependent on the activation status of the IGF-1R. UVB irradiation of keratinocytes with activated IGF-1Rs incurs substantial DNA damage that cannot be completely repaired. Because of the persistence of UVB-induced DNA damage, these keratinocytes become senescent, thus preventing the replication of UVB-damaged DNA. It is important to note that when the IGF-1R is activated, no keratinocytes containing UVB-induced DNA lesions will be replicating. Unfortunately, when the IGF-1R is inactive in keratinocytes, this restriction on cellular replication with DNA damage is not in effect. Keratinocytes with functionally inactive IGF-1Rs that are exposed to UVB irradiation are more likely to undergo apoptosis; however, surviving keratinocytes can continue to proliferate, thus establishing mutations from unrepaired DNA in the daughter cells
Aging and NMSC The important in vitro discovery that epidermal keratinocytes IGF-1R activation status was crucial in response to UVB, led to a hypothesis that reduced activation of the IGF-1R may be correlated to an increased susceptibility to skin cancer in vivo. In a retrospective epidemiological study, it was found that type 2 diabetic patients using
insulin to treat their disease had a 2.5-fold decreased risk of developing non-melanoma skin cancer over the control group and type 2 diabetic patients using non-insulin medicines to treat their disease [104]. This study is important because insulin and IGF-1 have very similar molecular structures and high concentrations of insulin will activate the IGF-1R. Intriguingly, the protective effect of insulin use increased with age, implying that insulin was
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somehow protecting against the age-associated increase in non-melanoma skin cancer [104]. Since high levels of insulin activate the IGF-1R, these important data suggested the clinical relevance for the involvement of the IGF-1R signaling pathway in NMSC in vivo. Recently, the age-related changes in the IGF-1/IGF-1R signal transduction pathway have been examined in vivo. The production of IGF-1 diminishes as fibroblasts become senescent [12, 105]. Given the critical role of dermal fibroblasts in supplying IGF-1 to epidermal keratinocytes, an age-related decrease in fibroblast IGF-1 may result in keratinocytes in aged epidermis having functionally deficient activation of IGF-1R and thereby respond inappropriately to UVBirradiation. Analysis of IGF-1 in samples of geriatric individuals showed a significant reduction in IGF-1 levels when compared to young adults [12]. Accordingly, keratinocyte activated IGF-1R levels were high in young adult compared to virtual absence in geriatric individuals [12]. It has been reported that the difference between UVBinduced DNA damage repair in young verses aged human skin is the rate at which DNA damaged is cleared [61]. However, the most important point is that any DNA damage existing whilst cell proliferation continues, leaves the possibility for the propagation of mutations. In young adult skin where IGF-1 levels are high, the proliferation of keratinocytes containing UVB-damaged DNA will be prevented by a combination of DNA repair, apoptosis, and stress-induced senescence. This response to UVB irradiation which prevents the creation of tumor-initiated keratinocytes is called the appropriate UVB response. The goal of the appropriate UVB response is to ensure the integrity of the epidermis while preventing the proliferation of keratinocytes that contain UVB-induced DNA damage [11]. Because geriatric skin contains reduced levels of IGF-1, the normal UVB response is altered in aged skin [11, 12]. Following UVB exposure, keratinocytes that are proliferating despite the presence of UVB-damaged DNA can be found in geriatric skin (i.e. an inappropriate response) [12]. The role of the IGF-1R in the appropriate UVB response is demonstrated by the restoration of the appropriate UVB response in geriatric skin via treatment with exogenous IGF-1 prior to UVB irradiation [12]. Therefore, the age-related decrease in IGF-1 expression, IGF-1R inactivation and proliferation with DNA damage are major components in the development of NMSC seen in geriatric patients (> Fig. 58.2). It is important to distinguish between the role that IGF-1 plays in the initiation of UVB-induced skin cancer [11] and the previously well-documented activity that IGF-1 plays in promoting a variety of epithelial tumors [106–109]. In geriatric skin, diminished expression of IGF-1 leads to uncharacteristically decreased activation
of the IGF-1R in the epidermis. When keratinocytes are exposed to UVB in the absence of IGF-1R activation, the normal protective response to UVB is altered, so that keratinocytes with DNA damage fail to undergo stressinduced senescence and are capable of replicating chromosomes containing the UVB-damaged DNA. Therefore, the lack of IGF-1R activation at the time of UVB irradiation increases the probability of a cancer-initiating event. Previous reports of IGF-1 increasing carcinogenesis were in the context of promoting the growth of previously initiated cells, a distinctly different process. [11]. In tissues where homeostasis is dependent on precise interactions between epithelial and mesenchymal cells, the accumulation of senescent cells can disrupt the proper function of the tissue. The skin is one of these tissues where the dermal and epidermal components are interdependent on each other for the proper functioning of the organ. Therefore, cellular senescence affects the UVB response of keratinocytes in the epidermis through two distinct and opposite mechanisms; one mechanism suppresses UVB-induced transformation of keratinocytes and the second mechanism promotes keratinocyte carcinogenesis. On the positive side, keratinocytes use stress-induced senescence as a tumor evasion mechanism. The advantage to cellular senescence versus UVB-induced apoptosis is that senescence maintains the cellularity of the epidermis, thus preserving the barrier function. In other words, widespread UVB-induced keratinocyte apoptosis in the epidermis will severely compromise the epidermal barrier function while UVB-induced keratinocyte senescence will not. In this manner, the induction of senescence in UVB-irradiated keratinocytes suppresses carcinogenesis. On the negative side, cellular senescence in dermal fibroblasts will promote UVB-induced carcinogenesis in aging skin. IGF-1 expression by dermal fibroblasts is critical for the appropriate response of keratinocytes to UVB irradiation. The silencing of IGF-1 expression by senescent fibroblasts contributes to an increased initiation of transformed keratinocytes by UVB exposure. Furthermore, the altered inflammatory phenotype of senescent fibroblasts may promote the expansion of clones of initiated keratinocytes.
Clinical Implications of Dermal Involvement in NMSC Given the increase in NMSC incidence with its associated morbidity and cost, the prevention of these tumors has significant importance. Present strategies for tumor prevention include avoiding excess UV exposure. Consistent with the notion that dermal aging (both intrinsic and extrinsic) results in an ‘‘abnormal’’ UVB response, several
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. Figure 58.2 Skin IGF-1/IGF-1R-dependent UVB response. This illustration compares the response of young skin and old skin to UVB irradiation. The dermis of young adults produces sufficient levels of IGF-1 to activate the IGF-1R on epidermal keratinocytes. The appropriate activation of the IGF-1R on keratinocytes leads to the induction of stress-induced senescence following sufficient UVB exposure. In young skin exposed to UVB, replicating keratinocytes will never contain UVB-damaged DNA; if UVB-irradiated keratinocyte cannot repair all of the UVB-induced DNA damage, they become senescent. UVB-induced senescence is a tumor evasion mechanism to prevent the establishment of initiated neoplastic keratinocytes. In contrast, the expression of IGF-1 is silenced in aged dermis. The consequence of diminished dermal fibroblast IGF-1 expression is a lack of IGF-1R activation in epidermal keratinocytes. Instead of undergoing stress-induced senescence, the aged keratinocytes are able to proliferate in the presence of UVB-damaged DNA. In contrast to young skin, keratinocytes possessing UVB-induced DNA lesions can replicate in geriatric skin. This decrease in IGF-1 expression with advancing age, the subsequent decrease in IGF-1R activity, and the evasion of the normal skin UVB response contribute to the increase in non-melanoma skin cancer seen in geriatric patients
studies have demonstrated that sunscreen will prevent both actinic keratoses and squamous cell carcinomas [110–112]. For patients with established actinic keratoses precursor lesions, strategies include destruction by physical
modalities as well as by topical chemotherapy with 5-fluorouracil or immune-mediated destruction with topical imiquimod [113]. Though somewhat effective in treating established pre- or low-grade cancerous lesions, these
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treatment strategies do not appear to effect the underlying process by which aged skin is more susceptible to neoplasia. If the major deciding feature of keratinocyte response to UVB resides in the senescence status of the dermal fibroblast, then this suggests novel treatments.
Conclusion
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One possible new treatment strategy would be to develop methods to rejuvenate the fibroblasts to allow production of factors such as IGF-1. Though marketed for cosmetic purposes, skin damaging agents ranging from chemical peels, laser resurfacing, heating of the skin, and other ‘‘wounding’’ procedures could have this benefit [114, 115] and should be explored. Indeed, dermal wounding which would result in up-regulation of fibroblast genes (e.g., pro-collagen) should result in upregulation of IGF-1. It should be noted that a recent study demonstrated that the topical chemotherapeutic agent 5-fluorouracil results in the induction of dermal procollagen [116]. The ability of this chemotherapeutic agent to both remove pre-cancerous keratinocytes as well as induce dermal wounding that could protect against future UV exposure could result in an improved effect. Other therapeutic strategies that could share these ‘‘dual effects’’ of removal of mutated keratinocytes and induction of dermal rejuvenation include photodynamic therapy and topical imiquimod. Future studies should examine the dermal effects of these chemotherapeutic therapies. Since there appears to be a protective effect of exogenous insulin in skin cancer development [104], systemic treatment with IGF-1 could have a use in protecting highrisk populations. Currently used for short-stature syndromes, IGF-1 has an established side-effect profile and should also be studied [117]. Thus, this new paradigm of the role of aging in the development of skin cancer could have significant clinical implications.
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89. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11:298–300. 90. Sohal RS, Orr WC. Oxidative stress may be a causal factor in senescense. Age. 1998;21:81–82. 91. Hamilton ML, Remmen HV, Drake JA, Yang H, Guo ZM, Kewitt K, Walter CA, Richardson A. Does oxidative damage to DNA increase with age? Proc Natl Acad Sci USA. 2001;98:10469–10474. 92. Lin MT, Flint BM. The oxidative theory of aging. Clin Neuroscience Res. 2003;2:305–315. 93. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD. Microarray analysis of replicative senescence. Current Bio. 1999; 9:939–945. 94. Wall IB, Moseley R, Briard DM, Kipling D, Giles P, Laffafian I, et al. Fibroblast dysfunction is a key factor in non-healing of chronic venous leg ulcers. J Invest Dermatol. 2008;128:2526–2540. 95. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic and extrinsic factors in aging: a review. Int J Cosmetic Sci. 2008; 30:87–95. 96. Bol DK, Kigucji K, Gimenez-Conti I, Rupp T, DiGiovanni J. Overexpression of the insulin-like growth factor-1 induces hyperplasia, dermal abnormalities and spontaneous tumor formation in transgenic mice. Oncogene. 1997;14:1725–1734. 97. Wilker E, Bol D, Kiguchi K, Rupp T, Beltran L, Di Giovanni J. Enhancement for susceptibility to diverse skin tumor promoters by activation of the insulin-like growth factor-1 receptor in the epidermis of transgenic mice. Mol Carcinog. 1999;25:122–131. 98. DiGiovanni J, Bol DK, Wilker E, Beltran L, Carbajal S, Moats S, et al. Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion. Cancer Res. 2000;60:1561–1570. 99. Sadagurski M, Yakar S, Weingarten G, Holzenberger M, Rhodes C, Breikreutz D, et al. Insulin-like growth factor receptor signaling regulates skin development and inhibits skin keratinocyte differentiation. Mol Cell Biol. 2006;26:2675–2687. 100. Lin K, Hsin H, Libina N, Kenyon C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF1 and germline signaling. Nat Genet. 2001;28:139–145. 101. Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003;21:182–187. 102. Kruso H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005;309:1829–1833. 103. Ikushima M, Rakugi H, Ishidawa K, Maedawa Y. YamamotoK, Ohta J, et al. Anti-apoptotic and anti-senescent effects of Klotho on vascular endothelial cells. Biochem Biophys Res Commun. 2006;339:827–832. 104. Chuang T-Y, Lewis DA, Spandau DF. Decreased incidence of nonmelanoma skin cancer in patients with type 2 diabetes mellitus using insulin: a pilot study. Br J Dermatol. 2005;153:552–557. 105. Ferber A, Chang C, Sells C, Ptasznik A, Cristofalo V, Hubbard K, et al. Failure of senescent human fibroblasts to express insulin-like growth factor-1 gene. J Biol Chem. 1993;268:17883–17888. 106. Pollak M. Insulin and insulin-like growth factor signaling in neoplasia. Nature Rev Cancer. 2008;8:915–928. 107. Lann D, LeRoith D. The role of endocrine insulin-like growth factor-1 and insulin in breast cancer. J Mammary Gland Biol Neoplasia. 2008;13:371–379. 108. Dziadziuszko R, Camidge DR, Hirsch FR. The insulin-like growth factor in lung cancer. J Thorac Oncol. 2008;3:815–818.
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114. Meshkinpour A, Ghasri P, Pope K, Lyubovitsky JG, Risteli J, Krasieva TB, Kelly KM. Treatment of hypertrophic scars and keloids with a radiofrequency device: a study of collagen effects. Lasers Surg Med. 2005;37:343–349. 115. DeHoratius DM, Dover JS. Nonablative tissue remodeling and photorejuvenation. Clin Dermatol. 2007;25:474–479. 116. Sachs DL, Kang S, Hammerberg C, Helfrich Y, Karimipour D, Orringer J, Johnson T, Hamilton TA, Fisher G, Voorhees JJ. Topical fluorouracil for actinic keratoses and photoaging: a clinical and molecular analysis. Arch Dermatol. 2009;145:659–666. 117. Collett-Solberg PF, Misra M. Drug and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society. The role of recombinant human insulin-like growth factor-I in treating children with short stature. J Clin Endocrin Metabol. 2008;93:10–18.
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62 Atopic Dermatitis in the Aged Alexandra Katsarou . Melina C. Armenaka
Introduction Direct and indirect observations indicate that the prevalence of atopic dermatitis (AD) has increased two- to three-fold over the last 30 years [1]. A possible explanation for this increase is a higher susceptibility to sensitization due to environmental factors and the ‘‘Western lifestyle.’’ AD can be a very debilitating, persistent, and costly, long-term disease [1]. According to a populationbased survey of eczema prevalence in the USA, a substantial proportion of the population has symptoms of eczematous conditions, while 17.8 million met the empirical symptom criteria for AD [2]. Progress in understanding the epidemiology of AD has been slow due to the lack of suitable, uniformly used, simple, disease diagnostic criteria that can be used in population surveys among different countries [1]. Atopic dermatitis affects mainly children. According to many epidemiological studies, in the 1-year period prevalence measure, 5–20% of children in developed countries are affected [1]. Many differences in the prevalence of AD, between countries and between urban and rural areas within the same country, are noted in the literature [1]. Studies on ethnic groups and migrants found a large increase in the prevalence of AD in immigrant children, as compared to their country of origin [1]. These results suggest that environmental factors and a Western life style are the main causes in the development of AD. Atopic dermatitis also shows an important relationship to social class in children [1]. On the contrary, in adolescents and adults, no differences in social class over time were noted. There is relatively little information concerning adult AD. Studies from the UK and Norway found that the prevalence in adults over 20 years old is approximately 2% and less than 0.2% of adults over the age of 40 years are affected [1]. The lifetime prevalence of AD is considerably lower in the elderly, compared to younger adults [3]. Adults with a longer duration of school education appear to have a higher risk for atopic diseases [3]. A study from Thailand concerning adult-onset AD concluded that the disease is not rare in adults and develops mostly during the third decade of life. The prevalence of
AD in Nigeria was 8.5%, and 24.5% of the patients had onset after age 21 years. In another report of 2,604 patients attending a contact dermatitis clinic in Australia, 9% suffered from AD which began for the first time after age 20 and the main sites were generalized involvement, hands, and face [4]. According to a study on senile type AD in this age group, AD showed various types of eczematous lesions whose onset was in the fourth decade of life, and higher serum IgE and antibody-specific IgE antibodies levels than healthy people, but lower than in younger AD patients [5]. Among 259 adult patients with AD, after careful evaluation, only 5.4% fulfilled the criteria of intrinsic (nonallergic) AD [6]. Based on these figures, the nature and relevance of the nonallergic form of AD in adults deserves further evaluation [6]. Concerning the natural history of AD, one study suggests that 90% of affected children will be clear of eczema within 10 years, but other studies noted that only 60% of the children will be clear after the age of 16 and that 10% of hospital-based patients suffer from AD in adult life [1]. In a study from Sweden, concerning the prognosis and prognostic factors in adult patients with AD, the majority of adults (59%) with AD still had the disease after 25–38 years, when they were questioned [7]. The increased prevalence of AD in children and the fact that AD in most adults continues for many years point out that more adult and senile patients with AD are expected in the future [5, 8].
Quality of Life Atopic dermatitis is one of the commonest chronic relapsing inflammatory dermatoses, with increasing worldwide prevalence and major social and financial implications for patients. The health-related quality of life (HRQL) in children with chronic skin diseases is at least equal to that caused by many other chronic disorders of childhood, with AD and psoriasis having the greatest impact on HRQL. In adults, severe chronic inflammatory skin diseases may be considered as severe as angina pectoris, chronic anxiety, rheumatoid arthritis, multiple sclerosis, or regional esophageal cancer [9].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_62, # Springer-Verlag Berlin Heidelberg 2010
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Patients with AD have a significantly lower quality of life than the general population and healthy controls. Patients’ mental health, social functioning, and emotional functioning seem to be more affected than physical functioning, and quality of life is compromised because of disturbed sleep and fatigue during the daytime [10]. Quality of life is affected in adult AD patients and relates both to disease severity and to mental components. Among a group of adult dermatology outpatients evaluated using the Dermatology Life Quality Index, those with AD were the highest scoring group compared to those with other skin diseases [11]. Concerning the degree of handicap in relation to the choice of education and occupation, 38% of the respondents abstained from a specific education or job due to AD and there was an increased number of sick-leave days and early-retirement pensions noted [12]. Therefore, there are both personal and social consequences of AD [12]. Finally, a decrease in sexual desire due to AD was noted in 57.5% of patients, while 36.5% of partners reported that the appearance of eczema had an impact on their sex life.
Disease Subtypes, Clinical Features, Diagnostic Criteria, and Outcome Measurements Subtypes of Atopic Dermatitis Atopic dermatitis is an itchy, inflammatory, cutaneous manifestation of a systemic disorder that also includes asthma, allergic rhinitis, and food allergy. AD is an atopic disease, but all symptoms are not related to allergen exposure. Two subtypes of AD are distinguished: the ‘‘extrinsic type,’’ associated with polyvalent IgE sensitization against inhalant and/or food allergens, and the ‘‘intrinsic type,’’ without elevated IgE levels and no sensitization to inhalant or food allergens. Both forms of AD have the same clinical phenotype and associated eosinophila. Discrimination between the two types is important, because sensitization correlates with more severe skin disease, and prevention and treatment are more complicated [13]. Intrinsic AD tends to have a late onset in childhood and a female predominance [13]. As previously mentioned, a recent investigation suggests that the intrinsic type of AD is a very rare entity in adults and this raises the need to clarify the relevance of the intrinsic AD in aged patients [6]. According to a study from Japan involving 16 patients with senile AD, more than 65 years old,
12.5% had intrinsic AD, based on serum IgE levels and antigen-specific IgE antibodies [5].
Clinical Features AD usually starts in early childhood. The clinical picture and the distribution of the lesions vary depending on the age of the patient, the duration, and complications of eczema. No single diagnostic criterion exists for AD, but there are a multitude of major and minor features. Dry skin (xerosis) occurs in most atopic patients and is persistent. It is caused by reduced water content capacity of the stratum corneum and frequent irritation. Pruritus is an important clinical symptom of AD and it is essential for the diagnosis of active disease. It is always present in all phases of AD and in all ages and can be very severe, often disrupting the sleep of patient. The mechanism of pruritis is not completely understood. Several mediators such as neuropeptides, proteases, cytokines, and nerve growth factor are associated with itch in AD. In most cases of AD typical, age-related, clinical features exist [14] (> Figs. 62.1–62.3). Infantile phase (0–2 years). In most cases, AD starts within the first 3–6 months of life and is characterized by dry, erythematous, scaling areas, symmetrically located, on the cheeks, chin, perioral, and paranasal region, whereas infantile eczema tends to spare the diaper area. In more severe disease, vesicular and infiltrated plaques evolve that have a tendency for oozing and crust formation and . Figure 62.1 Case 1. AD in a 62-year-old woman with a history of allergic rhinitis Inflammation and lichenification are apparent in the extensor surface of the left arm
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. Figure 62.2 Case 2. Inflammation around the eyes and face in a 34-year-old woman
. Figure 62.3 Case 2. Lichenifications on the neck and upper back with postinflammatory diffuse hypo- and hyper-pigmentation
secondary infections may complicate the condition. Involvement of the hands, limb folds, upper trunk, arms, and legs is not unusual. The course of AD is relapsing and over time, the exudative character of dermatitis is lost. Childhood phase (2–12 years). The inflammatory lesions are characteristically located on flexural areas and signs of lichenification appear. Very often, the clinical picture is polymorphous, due to the coexistence of chronic and acute types of eczema. Skin thickening, lichenification, scratching due to persistent pruritus, acute
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erythema, with erosive or infected skin lesions, may affect the same area. Adolescent and adult phase. The main findings are dry skin, flexural inflammation and lichenification, hand and foot dermatitis, and inflammation around the eyes and neck. A variety of clinical manifestations of acute phase (scaling plaques with vesicular erosive lesions) and chronic phase (lichenification), with persistent itching, coexist in most of cases. In addition, a large proportion of adults with sensitive skin and/or irritant contact dermatitis affecting the hands had atopic eczema when they were children. In fact, the hands seem to be the most common site of AD in adulthood. The prevalence of hand dermatitis is 2–10 times higher in atopics, and occupational irritants (daily exposure to water, chemicals, detergents) and domestic work favor the development of hand eczema. Senile phase. Characteristics of AD in the senile phase remain unclear and clinical features resemble the adult phase [5]. Aged patients showed a male dominance in senile AD with a man to woman ratio of 3:1 [5]. Patients suffering from AD very often present with atypical clinical manifestations, that may be site-specific (infra-auricular striae, atopic winter feet, cheilitis, hyperlinearity of palms and soles, etc.) or clinical morphologyspecific (follicular eruption, pityriasis alba, nummular eczema, keratosis pilaris, white dermographism, etc.). Several factors influence the course of AD, including climate, textiles, and sweating. Climate. In most patients, eczema is aggravated during winter, probably due to decreased humidity. Change from a subarctic to a subtropical climate improves skin symptoms and quality of life in patients with AD. Textiles. Wool and synthetic fabrics cause irritation and itching. Patients are also sensitive to irritation from detergents and many chemicals. Sweating is a cause of itching and exacerbation of eczema. Unfavorable prognostic factors for AD are persistent dry or itchy skin, widespread dermatitis, associated allergic rhinitis, family history of AD, asthma, early age of onset, and female sex [7].
Diagnostic Criteria and Outcome Measurements The diagnosis of AD is made clinically and as the clinical manifestations are numerous, many diagnostic criteria are used in order to confirm the diagnosis. The Hanifin and Rajka diagnostic criteria have been most extensively validated from 1980 and they propose 4 major and 23 minor
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diagnostic criteria for AD [15]. A diagnosis is established if three of the major and three or more of the minor criteria occur in the patient. A scoring system developed by the European Task Force on Atopic Dermatitis (SCORing Atopic DermatitisSCORAD) is one of the best outcome measurements for atopic eczema. While Hanifin’s and Rajka’s criteria are useful for differential diagnosis, SCORAD is used to evaluate the severity of disease in clinical and epidemiological studies. It takes into account the extent of skin lesions, the severity of the clinical features, and the subjective symptoms [16].
Pathogenesis Atopic eczema is a multifactorial chronic inflammatory skin disease with a complex background that is characterized by genetic influences, skin barrier dysfunction, and immune deviation with hyper-reactivity to environmental stimuli and deficient antimicrobial immunity.
Genetics of Atopic Dermatitis The evolution of atopic dermatitis is influenced by genetic and environmental factors. AD is a complex genetic disorder and the mode of inheritance and the genes involved are not clear [17, 18]. The evidence for a strong genetic influence in the course of AD comes initially from twin studies. The difference in concordance rates between monozygous and dizygous twins gives an indication of the heritability of the disorder [17]. Segregation analysis indicates that the inheritance of AD does not fit a simple Mendelian pattern and more than one gene is responsible for the evolution of the disease. The MHC complex has certainly been implicated and the cluster of interleukins on human chromosome 5 plays an important interactive role in the final expression of atopy, asthma, and atopic eczema [18]. Several genetic studies suggest a linkage between AD and the filaggrin gene, located on the epidermal differentiation complex 1q21 [19]. Recent genetic advances, with high-throughput methods for gene identification, such as DNA micro-arrays and whole-genome genotyping, will help further dissect this complex trait [17].
Skin Barrier Function in Atopic Dermatitis The most common symptoms in patients with AD are itching and dry skin, involving both lesional and
non-lesional skin. The skin barrier is known to be damaged in patients with AD. There is a four- to eightfold increase in transepidermal water loss (TEWL) in clinically active dermatitis and a two- to fivefold increase in TEWL in clinically uninvolved skin [20]. Many studies have focused on altered content of stratum corneum lipids, as the etiology of barrier permeability dysfunction. Ceramides comprise more than 50% of the lipids and serve as the major water-retaining molecules in the extracellular space of the cornified envelope. They ensure that the skin barrier is as tight as possible [21]. Reduction of ceramides has been found in lesional and non-lesional skin of patients with AD. In particular, reduction in ceramide 1 and 3 correlated with barrier dysfunction. A possible explanation for this is that sphingomyelin metabolism is altered in AD, resulting in decreased synthesis of ceramides, and/or that the skin of patients with AD is colonized by ceramidase-secreting bacteria, which may contribute to the ceramide deficiency in the stratum corneum [21]. Normal-appearing aged skin is also deficient in ceramide, as compared with that of the younger, healthy controls [22]. These findings indicate that decrease in stratum corneum lipids, especially ceramides, is a major etiologic factor for atopic dry skin and a primary event in the evolution of aged skin [21, 22]. A deficit of n-6 essential fatty acids (linoleic acid, g-linolenic acid, columbinic acid) can lead to an inflammatory skin condition [21]. In AD, concentrations of linoleic acid tend to be elevated, but concentrations of linoleic acid metabolites are reduced, due to reduced conversion of linoleic acid to g-linolenic acid. The oral administration of gamma linolenic acid seems to improve atopic eczema. Furthermore, increased skin pH in AD enhances the action of proteases that cause breakdown of the skin barrier [21]. Soaps and detergents also increase the skin pH. Recent studies have identified mutations in the stratum corneum chymotryptic enzyme (SCCE) protease gene in patients with AD [21]. The most likely consequence of this alteration in the SCCE gene is to produce higher levels of SCCE protease. Proteases cause premature breakdown of corneodesmosomes, basic for the structural integrity of the stratum corneum, leading to a thin skin barrier and increased penetration of irritants, microbes, and allergens [21]. Washing with soap, detergents, and long-term application of topical corticosteroids, additionally increase production of SCCE. Several other genetic variations that affect skin barrier function occur in AD [21]. Null mutations in the epidermal barrier protein fillagrin gene, resident in the
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epidermal differentiation complex, have recently been identified as an important predisposing factor for eczema [19]. This finding has important clinical and therapeutic significance, because it is often the earliest sign of the atopic march and confirms the importance of epidermal barrier disruption as a primary event in the evolution of the disease. Fillagrin mutations are found in more than 50% of individuals with eczema and may indicate poor prognosis in AD, predisposing to eczema that persists into adulthood and extrinsic eczema [19] The impaired skin barrier function may contribute to the increased penetration of microbes and antigens and to cutaneous hyper-reactivity with increased susceptibility to irritants [21]. Genetic abnormalities in skin barrier function are associated with protein allergy in AD patients, because Langerhans cells take up antigen easily from the damaged skin barrier. Well-documented studies prove that the degree of epidermal barrier disruption correlates with the severity of dermatitis [21]. According to a recent study, the higher the TEWL in AD, the higher is the prevalence of sensitization to environmental airborne allergens. These data suggest the major role of epidermal barrier function in the pathogenesis of AD.
Immune Responses in Atopic Dermatitis Complex immunologic responses are involved in eczematous skin inflammation, while deficient immunity against microorganisms leads to increased skin infections.
Immune Responses Leading to Skin Inflammation In atopic dermatitis, complex interactions between immune cells and their products, cytokines and chemokines, lead to a combination of immediate immune responses and delayed cellular immune responses in the inflamed skin [23]. This is reflected in the histopathology of AD. Histopathology. Acute skin lesions are characterized by epidermal intercellular edema (spongiosis) and an epidermal and dermal perivascular cell infiltrate in which activated memory CD4+ Τ cells predominate [23]. Antigen-presenting cells bearing IgE molecules are present and mast cell degranulation is evident. Chronic lichenified lesions are characterized by epidermal hyperplasia and dermal infiltration that is dominated by macrophages, IgE-bearing Langerhans cells, activated T cells, and eosinophils [23]. Collagen deposition in the dermis is induced by skin repair cytokines, such as interleukin 11.
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The Acute Phase of Inflammation
T cell deviation and altered cytokine and chemokine levels play an important role in AD [24]. The acute inflammatory phase is dominated by T helper cell type 2 (Th2) cytokine responses. Activated memory Th2 cells that express cutaneous lymphocyte antigen (CLA) are increased in skin lesions and in the peripheral blood of AD patients, and correlate with disease severity [23]. CLA is acquired in local lymph nodes after the interaction of skin tissuedraining dendritic cells and enables cells to home in to the skin from the circulation [23, 24]. Activated Th2 cells produce interleukin 4 and 13 (IL-4, IL-13) that stimulate B cells to produce specific IgE antibodies, while downregulating T helper type 1 (Th1) responses. They also produce Il-5, an important cytokine for eosinophil development, activation, and survival [23]. The IgE antibodies bind to corresponding receptors in skin immune cells, resulting in the release of histamine and other proinflammatory mediators. Dendritic cells play a pivotal role in the acute sensitization phase. Langerhans (LCs) migrate to regional lymph nodes and prime naive T cells to expand the pool of Th2 cells [23]. In addition, LCs expressing the high-affinity IgE receptor (FceRI) and bearing IgE are increased in AD lesions and must be present in order to provoke eczematous lesions by the topical application of allergen [24]. On the other hand, regulatory T cells (Tregs) that should play a role in suppressing T cell responses to allergens, are absent in the skin and have a reduced suppressive capacity in AD [24]. Despite a ‘‘regulatory phenotype,’’ activated CD4 + CD25 + Tregs promote Th2 responses in AD patients. Environmental airborne allergens, food allergens, products of infectious pathogens, and scratching can induce Th2 immune responses and the production of specific IgE antibodies in patients with AD [23, 24]. Skin damage, caused by scratching, can lead to the production of IgE antibodies against human skin proteins [23, 24]. Such autoantibodies are found in up to 80% of AD patients during early childhood, as compared to 25% of adult patients [24]. The Chronic Phase of Inflammation
In chronic AD lesions, T helper type 1 responses are more dominant than T helper type 2 responses, resulting in cellular immune responses or delayed-type hypersensitivity responses. Interferon-g (INF-g), produced by activated Th1 cells, predominates in chronic skin lesions. Levels of Il-5, Il-12, and granulocyte-macrophage stimulating factor (GM-CSF) are also increased [23]. Keratinocytes stimulated by INF-g, release high levels of
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chemokines, including RANTES, that lead to further recruitment of T cells, dendritic cells, and eosinophils to the skin [23, 24]. It has been shown that the local production of IL12 in the skin, by antigen-presenting cells and eosinophils, induces differentiation of Th1 cells and causes the phenotype ‘‘switch’’ from Th2 (acute phase) to Th1 (chronic phase) [24]. Moreover, infectious pathogens can directly promote chronic eczema by inducing Th1 immune responses [25].
Antimicrobial Immune Defense Atopic skin is characterized by decreased ability to eliminate pathogens, as a result of defective innate and acquired immunity. As part innate immunity, keratinocytes and professional antigen-presenting cells in the skin are activated by the recognition of molecular patterns derived from microorganisms [25]. Activation involves action on Toll-like receptors (TLRs) and leads to the production of antimicrobial peptides through a vitamin D-dependent mechanism. Antimicrobial peptides have broad antimicrobial activity against viruses, bacteria, and yeast [24, 25]. In AD, the production of antimicrobial peptides, such as b-defensins and the cathelicidin LL-37, is decreased in the skin [25]. This defect is probably acquired and results from the Th2 cytokine mileu in the skin. Since vitamin D is involved in this defense pathway, it is possible that low vitamin D levels, often found in older populations, might increase susceptibility to infections. Finally, it appears that acquired immunity requires robust Th1 immune responses in order to eliminate microorganisms that come in contact with the skin, whereas Th1 responses to invading microbes are decreased in AD [25].
Environmental Triggers of Atopic Dermatitis Important environmental factors that trigger disease exacerbations are food allergens, airborne allergens, microorganisms, skin irritants, contact allergens, and psychological stress.
Foods and Airborne Allergens Atopic dermatitis is associated with hypersensitivity to foods and/or common airborne allergens. Sensitization
increases after early infancy and remains high throughout adult life [24]. According to a study from Japan, allergenspecific IgE antibodies to various inhalant allergens and/ or food allergens occur in 87.5% of elderly patients with senile AD [5].
Food Allergens In early life, AD is associated with a much higher frequency of food than aeroallergen sensitization [23]. Food allergy causes skin rashes in 40% of children with eczema, but approximately one third of children outgrow their food allergy after 1–2 years of avoidance [23]. The role of food allergy declines sharply with aging [26, 27]. A large population-based study of adolescents and adults with AD, found that food allergy was not clinically important in this age group [26]. However, adult AD patients with persistent, more severe disease are more likely to be sensitized to foods [5, 27]. Sensitized individuals can react to oral food challenges with three clinical patterns: (a) immediate hypersensitivity reactions (urticaria, angioedema, and erythema) occur within a few minutes, (b) soon after ingestion, pruritus leads to exacerbation of eczema, (c) late eczematous reactions occur after 6–48 h [27]. Combined non-eczematous and eczematous reactions may also be seen. Cow’s milk, hen’s eggs, wheat, soy, peanuts, tree nuts, and shellfish account for 90% of food allergic reactions in children and young adults with AD in the USA [27]. Foods cross-reacting to birch pollen (apple, carrot, celery, hazelnut) can trigger AD in adults sensitized to birch, even in the absence of a suspicious history [27]. Studies in German adults, documented clinically relevant allergy to pollen-associated foods in a subgroup of birch sensitized AD patients [26, 27]. In adult AD patients with a positive milk provocation challenge, milk-specific IgE was found in less than half of the patients [27]. Atopy patch tests (APTs) using topically applied food, can elicit late eczematous reactions in some of these patients. A good correlation between positive patch tests and late reactions to ingested foods has been shown in some studies, although other studies show a high rate of false-positive patch test reactions [27]. Finally, certain foods (alcohol, food additives) may cause exacerbations of eczema through nonimmune mechanisms, by acting as irritants or pseudoallergens [28]. Clinical improvement was found in 63% of adult patient after 6 weeks on a low pseudoallegen diet [28].
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Sensitization to airborne allergens usually develops at about 3–4 years of age and continues to be important in atopic adults [29]. Adult patients with persisting, severe atopic dermatitis are highly sensitized to aeroallergens, as demonstrated by skin prick tests. The highest amount of serum total IgE and aeroallergen-specific IgE has been found in AD, compared with other atopic diseases, while no significant age-related decrease is observed in aging adults [29, 30]. Important airborne allergens are dust mite, animal dander, grass, birch, molds, and cockroach. Pruritic skin lesions can develop after inhalation or skin exposure to these allergens in sensitized individuals [29]. The eczematous skin reaction that results from topical application of airborne allergens using modified patch testing (atopy patch tests) is similar to atopic eczema and is characterized by skin barrier disruption, cellular infiltration, and cytokine release [29]. Aeroallergens elicit atopy patch tests (APT) in 30–50% of AD patients, whereas positive reactions are uncommon in other atopic diseases [18, 29]. In a study involving 115 adults with AD, 54% demonstrated positive APT to least one aeroallergen, compared to 6% of healthy controls [31]. Strong evidence favors a causal role of dust mites in atopic dermatitis [29]. In addition to being airborne, they parasitize the skin and release exogenous proteases that can directly damage the epidermal skin barrier [21, 29]. In senile AD, dust mite was found to be the most important allergen and elevated mite-specific serum IgE levels occurred in 86% of patients [5]. Furthermore, the positivity of mite atopy patch tests is 45% in adults with AD [31]. Finally, avoidance of dust mites has been shown to be clinically beneficial in mite sensitive AD patients [18, 29].
induces allergen-specific Th2 cells to home to the skin [25]. Furthermore, colonization of the skin by Staphylococcus aureus and the opportunistic yeast Malassezia spp. is very frequently found in AD [33]. Experimental evidence supports the fact that these colonizing skin pathogens can lead to eczematous skin inflammation and may be responsible for treatment failures [33]. Skin colonization with S. aureus is detected in more than 90% of patient with AD and only in 5% of healthy controls [33]. Colonization rates are high in adults and they are associated with the severity of eczema [33]. Of the strains isolated from skin lesions, 30–60% secrete S. aureus enterotoxins, with superantigen properties, that cause a vigorous immune response and can exacerbate AD by promoting Th2 responses and IgE production [25, 33]. The levels of superantigen-specific IgE antibodies correlate with eczema activity [33]. S. aureus superantigens and alpha toxin also promote Th1 responses that contribute to the development of chronic eczema [25, 33]. Finally, proteinases produced by S. aureus can directly damage the skin barrier [21, 33]. Skin colonizing Malassezia spp. yeasts (Pityrosporum) are found in up to 90% of patients with AD, compared to 34% in healthy controls, and they sensitize 30–80% of patients with extrinsic and intrinsic types of eczema [33]. In a study of the affects of aging, anti-Malassezia IgE antibodies was found more often in adults, compared to children with AD [34]. A high incidence of positive patch test reactions to Malassezia are seen in AD, especially in adults with head-and-neck eczema [33]. Finally, observations support that decreasing skin colonization, with antibiotics or antifungal treatments, decreases the severity of skin lesions [33]. However, this beneficial clinical effect is only short-lived and recolonization occurs.
Microorganisms
Water, Skin Irritants, and Contact Allergens
In AD, skin barrier dysfunction, either resulting from a genetic defect, or acquired by scratching and environmental influences, facilitates the increased penetration of microbes and their products [32]. Decreased antimicrobial immunity predisposes patients to develop bacterial skin infections (impetigo, paronychias), localized viral infections (herpes simplex, warts, mollusca contagiosa), or disseminated viral infections (eczema herpeticum, molluscatum, and vaccinatum) and fungal skin infections by Trichophyton rubrum and Candida albicans [24, 25]. Acute infections can cause AD flares, by several immune mechanisms, including stimulating Il-12 production that
Water hardness may be important in AD. According to an ecological study, water hardness acts on existing dermatitis by exacerbating the disease or prolonging its duration, rather than as a cause of new cases [1]. Atopic skin is known to be prone to react to irritants [18, 21]. In daily life, soaps, detergents, and excessive washing, can induce flares of AD and predispose to the development of irritant hand dermatitis [21]. Irritants can act synergistically with allergens to increase skin inflammation in patients with AD [35]. The consecutive application of the irritant sodium lauryl sulfate and aeroallergens on the skin of sensitized atopic adults, led to a
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more severe barrier disruption than the application of each component alone [35]. Due to skin barrier dysfunction, contact allergens can penetrate the skin more easily and according to a recent study of unselected adults in Norway, AD was a risk factor for allergic contact sensitization [36]. The number of positive contact allergens increases with age in atopic patients [37]. At least one positive patch test, to a standard series has been found in approximately 40% of AD patients [38]. The most common contact allergens were nickel, cobalt, fragrances, and rubber. Atopic dermatitis is the most common cause of occupational dermatitis and doubles the risk of developing irritant dermatitis in some occupations, particularly those involving wet-work, although abrasive hand cleaners and greases also contribute [23, 39]. Occupational exposure to irritants, or to contact allergens, can induce the appearance of widespread eczema in atopic patients in whom eczema was quiescent for years, or was never present. Eczema can persist even after removal from the high-risk occupation. Pre-employment counseling of adolescents and adults with atopic eczema is crucial, so that they can make correct decisions on their future occupation [39].
Psychological Factors Patients with AD often suffer from stress-related exacerbations, exhaustion, depression anxiety, and helplessness [40]. Many studies have explored the personality type of patients. Atopic dermatitis patients have been described as anxious, emotionally unstable, tense, and perfectionist; but finally it appears that there is no specific personality type unique to AD, and patients tend to suppress emotions [40]. Neuropeptides, endogenous opioids, and serotonin, released after stress challenge, have also been associated with itching [40]. Neuropeptides in the skin act directly on blood vessel walls and act indirectly, as mediators of inflammation, by inducing release of cytokines from mast cells and endothelial cells, and as immuno-modulators via corticotropine-releasing hormone. Observations that psychological stress may induce AD flares can be explained by studies showing that stress favors a shift in immunity toward a T helper type 2 cell allergic response [40]. Additionally, patients with AD appear to have an inherited hypothalamic deficiency that impairs normal hypothalamic–pituitary–adrenal axis function [40]. Gender differences in response to stress may implicate the hypophalamic–pituitary–gonadal
pathway, since female hormones generally enhance inflammation [40]. Skin barrier function is also altered by stress, by means of increased cortisol levels that cause decreased lamellar body secretion and down-regulation of epidermal expression of antimicrobial peptides [21]. Psychologic and stress-reduction interventions have shown to improve patient well-being and significantly improve skin manifestations [40].
Diagnostic Tests Elevation of eosinophil levels and total and allergen-specific serum IgE levels are common in AD and are associated with more severe dermatitis [21]. Diagnostic tests for evaluating allergy in AD are summarized in > Table 62.1. Sensitization to allergens can be demonstrated by measurement of allergen-specific IgE antibody determination in the serum (RAST, ImmunoCAP) and by skin prick tests [18]. If skin tests are used (> Fig. 62.4), it is important to have in mind that skin reactivity to histamine (a positive control) begins to decrease significantly after the age of 50 and reaches a plateau after the age of 60 [41]. The induction of a local eczematous reaction after application of the allergen using atopy patch tests (APTs) is another important tool for detecting relevant allergens in AD. APTs should be applied to intact, untreated skin of the back for 48 h, and read at 48 and 72 h [42]. They have been shown to be reproducible for dust mite and other aeroallergens (> Fig. 62.5), using petrolatum as a vehicle [42]. An aeroallergen panel is now commercially available. APTs can also be used for food allergy testing, preferably using freshly made extracts, since food allergens are often unstable [27]. Recently, Malassezia has . Table 62.1 Diagnostic tests for evaluating allergy in AD IgE-mediated (immediate) Total serum IgE Specific serum IgE (aeroallergens, foods, human proteins/ autoallergens, S. aureus enterotoxins, Malassezia) Skin prick tests (aeroallergens, foods) Oral provocation challenges (foods) T cell-mediated (delayed) Atopy patch tests (aeroallergens, foods, Malassezia) Standard patch tests (standard series, occupational series, medicaments) Modified oral provocation challenges (foods)
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. Figure 62.4 Prick tests to a panel of aeroallergens applied to the forearm. Positive reactions show a local wheal and flare (H = histamine, C = negative control)
. Figure 62.5 Patch test results read at 72 h in a patient with AD. An irritant reaction to hypo-allergenic adhesive tape is evident. The right half of the photo corresponds to the area of the back where aeroallergens (atopy patch tests) were applied and there are no significant reactions. The left half corresponds to standard patch tests. The basic European set (upper left) showed a (++) reaction to colophony 20%, while positive reactions to three allergens in the cosmetic panel were also seen (lower left)
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The diagnosis of eczematous reactions to foods in patients with AD requires oral provocation challenges to prove the clinical relevance of history, positive skin prick tests, positive food-specific IgE, or atopy patch tests [27]. Because food-induced eczema usually needs more than 6 h to develop and may require repeated ingestion, oral challenge protocols need to be modified, or else positive reactions may be missed [27]. Patients should be observed for longer than 1 day after each challenge [27]. Furthermore, a diagnostic elimination diet, lasting 4–6 weeks, is often recommended, whereas in older patients, individually tailored diet with foods that rarely cause food allergy or ‘‘pseudoallergy’’ can be used [27, 28]. Finally, patch testing with a standard series of contact allergens and occupational series depending on work-place exposure may be helpful (> Fig. 62.5). Adult patients unresponsive to topical treatment should also be tested for contact sensitization to topical medicaments [18].
Treatment AD is a chronic relapsing inflammatory skin disease of childhood, but also of adult life. The successful approach to the management of AD requires a combination of simultaneous multiple actions and treatments to prevent, identify, and eliminate trigger factors, and to improve skin barrier function.
General Measures and Basic Treatment The education of the patient and/or their families, and the communication between doctor and patient are a very important part of successful management of AD. Recommendations and instructions must be written step by step. The avoidance of specific triggering factors (aeroallergens, foods, contact allergens) and nonspecific triggers (contact irritants, soaps, prolonged hot water showers, environment with low humidity, wool and synthetic clothing, perfumes, make-up) is indicated for all patients and all types of AD. The prevention of stress, anxiety, and depression is also very important and the support of a psychologist in many patients is proposed [18].
Topical Treatment been used in investigational units [18, 23]. The demonstration of a positive APT reaction can reinforce the need for allergen avoidance and this can lead to significant clinical improvement.
Topical treatment comprises the foundation of AD treatment and is indispensable for all patients suffering from AD.
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Emollients
Topical Calcineurin Inhibitors (TCI)
They help restore and preserve the stratum corneun barrier and also decrease itching and the need for topical treatment. Emollients should be applied continuously, even if no active inflammation is evident. Ointments are the most occlusive. Urea-containing moisturizers improve skin barrier function and reduce skin susceptibility to irritants, whereas salicyl acid can be added to an emollient for the treatment of chronic hyperkeratotic lesions. Formulations containing lipids identical to those of stratum corneum, in particular, ceramide supplementation could better improve the dysfunctional barrier. Emollients must be applied several times daily and should be continued long after other topical treatments have been stopped. Folliculitis is a side-effect when the occlusive action is pronounced and in this case, the emollient must be changed.
Pimecrolimus and tacrolimus are the two available calcineurin inhibitors with steroid-free, anti-inflammatory, and immunomodulatory effects [18]. They act by inhibiting inflammatory cytokine transcription in activated T cells and other inflammatory cells via inhibition of calcineurin. Their action is more specific than corticosteroids in the inflammatory process and they are not associated with skin atrophy and thinning, striae, glaucoma, and other steroid-related side effects. They can be used on the face, eyelids, neck, and any other area with sensitive and thin skin. The most common side-effect is a burning sensation of the skin of short duration, related to the skin barrier dysfunction. According to many clinical trials, no evidence of systemic toxicity or local and systemic skin infections have been noted. However, it is recommended to minimize exposure to UVR and to use sun protective agents. The early use of topical calcineurin inhibitor can lead to better long-term disease control, with fewer flares and less need for topical corticosteroid rescue therapy. Tacrolimus ointment is more effective than pimecrolimus cream in adults with moderate to severe AD, but both agents have a similar safety profile [45]. Finally, combined topical therapy, with corticosteroids and TCI, is proposed by many practitioners, because the two classes have different and possibly complementary mechanisms of action. The recent guidelines of International Consensus Conference on AD, recommend corticosteroids for acute control of disease progression and as intermittent treatment in maintenance therapy with TCIs.
Topical Steroids Topical Steroids have been the corner stone of treatment of AD for more than 50 years and are still an important tool for the management of AD, especially for acute flares. A large number of topical corticosteroids are in use, ranging from high to low potency of action. Topical steroids should be applied no more than twice daily, as short-time therapy for the acute phase of AD. Many therapeutic schemes are used in order to obtain the optimal therapeutic effect. Intermittent use might be as effective as initial therapy with a high-potency steroid, followed by a time-dependent dose reduction, or a change over to a lower potency preparation [14, 18]. Despite their widespread use, side effects are not very frequent for low to medium potency topical steroids, although 72.5% of people worry about using topical corticosteroids on their own or their child’s skin [43]. Ultra-high and high-potency topical corticosteroids are used for short-term treatment of lichenified areas in adult patients. To prevent tachyphylaxis, side effects and rebound phenomena, it is proposed to use them once daily, in combination with frequent application of emollients for the first weeks and then alternate day use is recommended. Wet-wrap dressings, using diluted steroids and/or emollients, are very effective as a very short-term therapy for acute erythrodermic dermatitis, therapy-resistant AD, and intolerable pruritus [44].
Topical Antimicrobials To decrease the bacterial and fungal load on involved and uninvolved skin, topical antiseptics, such as triclosan, chlorexidine and antifungals, such as ketoconazole shampoo, have been shown to be effective and can be topically used, added to bath water, or to bath emollient. According to many studies, exacerbations of AD are the result of bacterial infections and super-infections, especially to S. aureus. Topical antibiotics, alone or in combination with corticosteroids, are effective in mild and localized forms. Fucidin is the most popular topical antimicrobial agent in many countries, with good skin penetration, but long-term therapy results in drug resistance [18].
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Systemic Treatment
Azathioprine
Antihistamines
Due to late onset of action and side effects (myelosuppression, hepatotoxisity immunosuppression), treatment with azathioprine in AD patients is very limited [46].
Sedating antihistamines are effective for reducing itching and combinations of antihistamines some times are needed for better itch control.
Antimicrobial Treatment Antibiotics are indicated for widespread bacterial infection, especially of S. aureus. Clarithromycin, cephalosporins, or semisynthetic penicillins used for 7–10 days are effective, but it has been shown that recolonization occurs soon after treatment [18]. The increased prevalence of antibiotic resistance of S. aureus is now recognized as a growing problem in patients with AD [25]. Antimicrobial peptides promise to play an alternative role to conventional antimicrobials. Systemic antiviral agents are very important in the cases of herpes simplex viral infections.
Phototherapy and Photochemotherapy Phototherapy is a well-established second-line treatment for adult and adolescent patient with AD [14, 18]. UVB in combination with UVA is more effective and the addition of topical corticosteroids can improve the favorable response.
Immunotherapy Allergen-specific immunotherapy for 1 year, in patients with AD and allergic sensitization to house dust mites, is able to improve eczema and to reduce the use of topical steroids [18]. Further studies are needed to this direction, in order to establish to role of immunotherapy for AD.
Systemic Corticosteroids
Probiotics
Short courses of oral steroids in cases of acute flare-ups are indicated (20–40 mg of prednizolone/adult dose) [14]. Very gradual decrease of the steroid dose may also diminish the possibility of rebound phenomenon. Long-term oral corticosteroids therapy is associated with many wellknown side effects.
Probiotic bacteria are suggested to reduce eczema symptoms in children with and without food allergy [25]. The probiotic effect is attributed to the normalization of increased intestinal permeability and the improvement of the immunological defense barrier (IgA) of the intestine. In adult patients with moderate AD, food supplementation with probiotic bacteria-rich yogurt was effective and this effect depended on the recovery of intestinal mucosal barrier function.
Cyclosporin A (CyA) CyA in vivo, inhibits calcineurin depended pathways and reduces cytokines (IL2, IFN-g). In cases of severe AD, CyA is effective, but due to possible side effects, particularly renal toxicity and high blood pressure, it should be used in limited patients. Serum creatinine and blood pressure levels must be performed regular intervals. According to a recent systemic review, concerning systemic treatment in patients with severe AD, in eleven studies CyA effectiveness was shown [46]. These studies demonstrated decreased severity of AD, a dose-related response was found and effectiveness was similar in adults and children, although tolerability was better in children. The lowest effective dose should be administered for the shortest treatment period, in order to minimize side effects.
Conclusion The increased prevalence of AD in children and the fact that AD in most adults continues for many years point out that more adult and senile patients with AD are expected in the future.
Cross-references > Cutaneous
Effects and Sensitive Skin with Incontinence in the Aged > The potential of Probiotics and Prebiotics for Skin Health
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56 Carcinogenesis: UV Radiation* Douglas E. Brash . Timothy P. Heffernan . Paul Nghiem
Introduction Skin cancer offers the best picture of how a carcinogen causes human neoplasia. The basic principles of carcinogen exposure and slow development – discovered when Sir Percivall Pott traced scrotal cancers in adults to childhood employment as a chimney sweep – also apply to sunlight-induced cancers [1, 2]. The process begins with carcinogen exposure, DNA damage, and failure to repair DNA or apoptotically eliminate the damaged cell [3–6]. A mutant gene arises in a single cell, which then expands into a mutant clone [7]. Rare cells of this clone repeat the carcinogenesis cycle to generate mutations in additional genes. Sunlight acts at each of these steps.
Epidemiologic Observations The lifetime expectation of skin cancer in Australia is 60%. In the southern US and Hawaii, non-melanoma skin cancers exceed all other cancers combined. Basal and squamous cell carcinomas (BCC and SCC), and an SCC precursor, actinic keratosis (AK) are most frequent on sun-exposed skin, in outdoor workers, and at lower latitudes. SCC increase more quickly with dose and low latitude than BCC, and occur later in life, implying that SCC require more sun-related steps. In contrast, one third of BCC occur on body sites having only intermittant sunexposure, such as the trunk and legs. Melanoma also depends on sunlight. The relation to latitude is clear, yet it is often stated that the predilection for the back and lower legs makes the relation to sunlight uncertain. This predilection reflects the large surface area of the back and legs. When expressed as lesions per unit area, melanomas are 10–20-fold more frequent on the face and male ears than on intermittently exposed sites such as the lower legs in women, shoulders, back, or neck [8]. The slow-growing lentigo maligna melanoma and its precursor, lentigo maligna (Hutchinson’s freckle), occur on
exposed body sites of light-skinned individuals. Melanomas are rare on the buttocks and soles. Melanoma appears to have two distinct origins: (1) A chronic sun damage (CSD) etiology affects the head and neck and is associated with chronic elastosis – a classic indicator of chronic sun damage – as well gene amplification of the cell cycle genes CDK4 and CCND1 [9]. (2) A non-CSD route involves intermittant sun exposure of sites such as the trunk. Non-CSD melanomas carry mutations in the BRAF or NRAS oncogene – upstream regulators of cell cycle genes – and the patients have variant alleles of the melanocortin 1 receptor [10]. Intermittently exposed body sites are the location of the melanoma increase in recent decades, melanomas in patients under age 50, and the additional melanomas seen near the equator [8]. Recreational sunburn may explain these and the two-fold higher melanoma incidence in office workers compared to outdoor workers. Sunlight is also implicated by the susceptible population: both classes of skin cancer are more frequent in light-skinned individuals with blonde or red hair who burn rather than tan. Compared to black-skinned individuals, nonmelanoma skin cancer risk rises tenfold in Asians and 100-fold in Caucasians, with a further 2–12-fold for blonde or, especially, red hair. The divergence is less for melanoma, about 1:1:15. In black skin, BCC is rare even in patients with the hereditary nevoid basal cell carcinoma syndrome (NBCCS or Gorlin syndrome). Skin tumors in black patients are often scar-related, but these may be associated with sunlight as well. These effects result from less melanin in light skin [11], less shielding by pheomelanin than eumelanin, and greater production of photosensitized reactive oxygen from pheomelanin. Molecular epidemiology has provided the most direct evidence for UV as the active component of sunlight: UVB signature mutations are present in human BCC, SCC, and
*This chapter is adapted with permission from Ch. 112 of Wolff, E, Goldsmith, L, Katz, S, Gilchrest, B, Paller, A and Leffell, D (eds.), Fitzpatrick’s Dermatology in General Medicine, 7th ed., vol. 1, pp 999–1006, Mc-Graw-Hill, 2007. M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_56, # Springer-Verlag Berlin Heidelberg 2010
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AK (see UV Signature Mutations). Mutant cells are associated with elastotic dermis, indicating chronic sun exposure. UVB’s effectiveness is due to its ability to partially penetrate the ozone layer and stratum corneum and then be absorbed by DNA [12]. The ozone layer absorbs all but one part per million of UVC (used in germicidal bulbs), which otherwise would be lethal; UVA penetrates well but is poorly absorbed by DNA. Nevertheless, chronic UVA can induce tumors in mice and malignantly transforms predisposed human cells. The cumulative dose of sunlight required to cause BCC or SCC in adults is fairly large, approximately 10,000 and 70,000 h of exposure, respectively. Psoriasis patients who had low PUVA exposure and received 300 UVB treatments. Some melanomas appear to be independent of sunlight: tumors of the mucosa, palms, soles, and nailbeds are equally frequent in whites and blacks, have remained constant while melanomas of the skin have become epidemic, and are not associated with precursor nevi. Ocular melanomas are more frequent in whites than blacks, but do not depend on latitude and have not increased in the last few decades.
The Skin Cancer Epidemic The incidence of melanoma and non-melanoma skin cancers has doubled each decade since the 1930s [8]. AK, lentigo maligna, and lentigo maligna melanoma – typically lesions of the middle-aged and elderly – are now seen in young adults. But because cancer requires several events, it is not guaranteed that this increase in skin cancer is caused by an increase in one of the sunrelated events. The best evidence for sun is that increases have been greatest for intermittently sun-exposed sites such as the trunk and limbs, with little change in melanomas of the head and neck. Increased recreational exposure is usually blamed. Another suspect has been ozone depletion; because of a steep absorption curve in the UVB region, small changes in ozone concentration greatly affect UVB penetration. (UVC is fortunately still blocked.) The Antarctic ozone hole caused a 50% ozone reduction over southern Chile and Argentina in the last 2 decades, with UVB increasing up to 40-fold. Yet skin cancers in these areas are increasing at the same rate as elsewhere. The Arctic ozone hole has been offset by screening from air pollutants, yet skin cancers in Scandinavia are rising. An iatrogenic source of increased skin cancer incidence is PUVA (psoralen + UVA) therapy for psoriasis,
which increases the risk of SCC eightfold; in some patient cohorts, it raises melanoma >14-fold; BCC is not affected. Cancer is now increasing as a result of tanning beds. Individuals whose first sunbed exposure occurred as a young adult, or who had long durations or high frequencies of tanning bed exposure, already have a 70% higher risk of melanoma [13].
Characteristics of UV-Induced Cancers and Precancers In the US, 1,000,000 BCCs are diagnosed annually, as well as 100,000 SCCs and 60,000 melanoma. Survival differs strikingly. Fewer than one in 10,000 BCCs will metastasize and threaten the patient. This number increases to 1 in 40 for SCC, with clinical experience indicating that SCCs on sun exposed skin are less likely to metastasize than those arising in scars. One in seven invasive melanomas is lethal. Merkel cell carcinoma is a sun-induced cutaneous neuroendocrine cancer that will kill one in three patients diagnosed with it. Its incidence has tripled in the past 15 years to approximately 1,000 per year in the US. The type of exposure preferentially leading to each malignancy differs. Cumulative lifetime sun exposure is strongly associated with SCC incidence. BCC and AK instead seem to depend on reaching a certain threshold of UV exposure, often attained in youth, such that sensitive individuals develop BCC at a relatively early age and the incidence does not increase with further exposure. Case-control studies link melanoma with intense exposure early in life, with one or two blistering sunburns doubling the melanoma risk. This effect may be an underestimate if, as mentioned above, melanoma has two origins, one depending on chronic sun damage and one on intermittant exposure and sunburn. Children are particularly sensitive to sunlight: moving from England to Australia before age 20 confers the higher Australian incidence of AK, SCC, BCC, and melanoma, but the risk is much less when adults immigrate. This is not simply due to children spending more time outdoors, as Fig. 56.1). The most frequent is TT, but TC and CC cyclobutane dimers are also made. A single bond between the 6 position of one pyrimidine and the exocyclic group of the other instead creates a pyrimidine-pyrimidone (6–4) photoproduct. Both photoproducts distort the DNA helix and are recognized by DNA repair enzymes. UVA is 20-fold more frequent in sunlight but requires 1,000-fold greater doses for its biological effect. UVA induces T-containing cyclobutane dimers and lesser numbers of oxidized purines and pyrimidines and singlestrand breaks [15]. UVA generates these lesions indirectly by photosensitization. UVA also efficiently photoisomerizes UVB-induced (6–4) photoproducts to their poorly repaired and highly mutagenic Dewar isomers.
Photosensitized Reactive Oxygen Species Both UVB and UVA can be absorbed by cytoplasmic ring-containing molecules such as NADH, riboflavin,
quinones, tryptophan and tyrosine, and the heme group of catalase. The resulting energetic molecule can interact with DNA to produce a T-containing cyclobutane dimer [15] or can produce reactive oxygen species. In the latter pathway, the chromophore’s energy is transferred to oxygen, resulting in singlet oxygen (1O2; an excited state of oxygen) or, if an electron is transferred, superoxide (O2●–). In the presence of water, these lead to hydrogen peroxide (H2O2) and thence, in the presence of Fe+2, to the hydroxyl radical (●OH). Hydroxyl radicals produce oxidative DNA damage resembling that after gamma radiation. Reactive oxygen species react with lipid membranes and the redox-sensitive catalytic site of phosphatases (see Cytoplasmic Signaling, below). Their production of 8-hydroxy-deoxyguanosine in DNA probably accounts for the occasional non UV-like mutations after UVB. UV also upregulates nitric oxide (NO), a more stable radical species that can participate in similar reactions after diffusing long distances and traversing lipid membranes.
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Excision Repair Nucleotide excision repair (NER) is the key protection mechanism against the lethal and mutagenic effects of UV-induced cyclobutane dimers and (6–4) photoproducts. Two NER pathways have been identified, global genome repair (GGR) and transcription coupled repair (TCR). GGR removes DNA lesions throughout the genome, whereas TCR is specialized for DNA lesions in the transcribed strand of transcriptionally active genes. In humans, excision repair requires the concerted action of six repair factors (XPA, RPA, XPC, TFIIH, XPG, and XPFERCC1) composed of nearly 20 polypeptides, some identified and named according to the seven complementation groups of xeroderma pigmentosum (XP). The enzymatic steps of NER include: recognizing damaged DNA, forming dual incisions that bracket the UV lesion, removing the damaged oligomer (24–32 nucleotides in length), gap filling by DNA synthesis, and ligating the repaired strand [16]. Global NER is considered error-free because the complementary undamaged strand is used as a template for repair synthesis. This core machinery of GGR is also used by TCR in active genes. Whereas in GGR the XPC protein recognizes distortions in the DNA double helix, the damage recognition signal for TCR is an RNA polymerase II complex stalled at a UV lesion, which attracts the core GGR machinery. RNA pol II sterically hinders the accessibility of NER factors and so is removed from the damage site by the CSA and CSB proteins. CSA and CSB are the genes mutated in Cockayne’s syndrome (CS), an autosomal recessive disorder characterized by cutaneous photosensitivity and physical and mental retardation. Induction of these repair factors is genetically regulated (see DNA damage signalling).
UV Signature Mutations UV leaves a characteristic signature when it interacts with DNA, and this signature remains in the tumor decades later. It has been used to answer many questions about the origin of cancer. A cyclobutane dimer or (6–4) photoproduct can lead to a mutation in two ways (> Fig. 56.1). When the lesion is copied during DNA replication, the DNA polymerase may read a damaged cytosine as a thymine and insert an adenine opposite it. At the next round of replication, the polymerase correctly inserts thymine across from adenine with the result being a C ! T substitution. Although the TT cylobutane pyrimidine dimer is the best known and most frequent photoproduct,
the thymines are not mutagenic because the XPV gene encodes a specialized polymerase (Pol eta) that adds adenines across from a T-containing cyclobutane dimer. Alternatively, a mutation can arise because cyclobutane dimers accelerate deamination of their cytosines to uracil (or 5-methylcytosine to thymine), leading to a C ! T substitution; no polymerase error is involved. In either case, C ! T mutations occur only where a cytosine lies next to a thymine or another cytosine, because the major UV photoproducts join adjacent pyrimidines. If two adjacent cytosines mutate, the result is CC ! TT. This distinctive pattern of mutation, C ! T where the C lies next to another pyrimidine, including CC ! TT, is unique to UV radiation and is called the UV signature [17]. UV signature mutations provide a tool for deducing backward from mutations found in tumors to the original carcinogen. Nearly all experimentally created UVB or UVC mutations are located at adjacent pyrimidines and about two thirds are signature mutations. The remaining third, typically C ! A and T ! C substitutions or 1–2 base insertions or deletions, are still caused by UV but probably arise by photosensitized production of reactive oxygen. Because this oxidative class is caused by many carcinogens, these mutations cannot identify whether their source was UVB, UVA, tobacco smoke, or intracellular oxidative phosphorylation. A set of tumors carrying the classic UV signature mutations must, however, also have some tumors with these UV-induced oxidative mutations. UVA, in contrast, only weakly induces UVB signature mutations by photosensitization but also generates oxidation-like mutations and T ! G changes. The latter are rare with UVB or other carcinogens and have been proposed to be a UVA fingerprint.
p53 UV signature mutations identified p53 as critical for preventing SCC and BCC but not melanoma. The P53 protein is a transcription factor that controls genes involved in the cell cycle, apoptosis, and DNA repair; it also acts directly on apoptosis proteins [6]. The p53 gene is mutated in about half of all human cancers and is termed a tumor suppressor gene because cancer arises from losing its normal function rather than gaining an abnormal function as oncogenes do. Over 90% of SCC in US patients contain these mutations, as well as three quarters of AK. Although nearly all BCC overexpress P53 protein, only half carry p53 mutations. Each mutation changes the amino acid, indicating that the mutation was selected for and contributed to tumor development, rather than being
Carcinogenesis: UV Radiation
simply an indicator of sun exposure. These p53 mutations are most frequent at nine mutation hotspots in important functional regions of the protein. Compared to internal cancers, some skin cancer hotspots are displaced several nucleotides to lie at a site of adjacent pyrimidines. Some sites may be hotspots because repair is slower there [18]. Other skin hotspots, like internal cancer hotspots, lie at 5-methylCG sites where body temperature slowly deaminates 5-methylcytosine to thymine; UV accelerates this process. The p53 mutations in AK indicate that these dysplasias are clonal rather than toxic reactions; patients with multiple AK’s have different mutations in each lesion [19]. The similarity of AK and SCC mutations supports the idea that AKs can progress to SCC. Aggressive skin tumors from patients exposed to both sun and chemicals contain multiple unrelated p53 mutations, as if multiple tumors arose in an abnormal field and merged. XP tumors contain very frequent CC ! TT mutations, perhaps because slow repair allows more time for cytosine deamination. Double-base mutations are also seen in conjunctival SCC, a tumor associated with HIV in sunny areas. Sunscreens reduce the level of UV signature mutations. In contrast, arsenic-induced BCC and SCC have non-UV p53 mutations; p53 mutations in BCCs from sun-shielded body sites resemble those seen with oxidative damage. Sunlight mutates p53 early. Normal sun-exposed skin carries 60,000 clones of p53-mutant keratinocytes, 3–3,000 cells in size [20]. By hematoxylin-eosin staining, the cells in these mutant clones appear completely normal. The early appearance of p53 mutations makes it possible to trace lineages in tumor development. Microdissecting lesions containing AK, carcinoma in situ, and SCC reveals that each stage contains the same p53 mutation. Although this result shows that each stage arose from the same founder lesion, it does not show that the stages derived from each other. To show a lineage, it is necessary to find additional mutations that appear in succession. In microdissected BCCs, one p53 mutation is present throughout the tumor, with various second mutations in different regions of the tumor. Once both p53 alleles are mutated, the cell is prone to aneuploidy, increasing the likelihood of a mutator phenotype.
Hedgehog Pathway Nearly all sporadic BCC’s have inactivating mutations in the PTCH tumor suppressor gene, a part of the hedgehog pathway; the remainder have activating mutations in its target, SMO. The hedgehog pathway appears to be a
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‘‘gatekeeper’’ for basal cell carcinogenesis, needing to be mutated early in BCC: minute BCCs have PTCH mutations, as do all histological subtypes; no BCCs have loss on other chromosomes without involvement of PTCH; and a congenital lesion that can progress to BCC, the sebaceous nevus, has PTCH allelic loss in 40% of cases [21]. In sporadic BCCs about three quarters of PTCH mutations are UV-like (either UV signature mutations or the expected UV-induced oxidative mutations) and a further 15% are one- or two-base insertions or deletions, often adjacent to a C ! T at a dipyrimidine site. BCCs from XP patients contain UV-like PTCH and SMO mutations, with CC ! TT mutations overrepresented. About 20% of the mutations in sporadic BCCs are not UV-like and resemble germline mutations seen in Gorlin syndrome patients – deletions or insertions larger than 2 bp. This finding may relate to the clinical observation that one third of BCCs occur on parts of the body that are not chronically sun-exposed, as well as the correlation between truncal BCC and defects in the glutathione radical-scavenging system (see Genetic Risk Factors). PTCH mutations tend to code for stop codons or frameshifts that completely inactivate the protein. In hereditary BCCs, nearly all tumors arose after losing the normal allele. This allelic loss appears related to sunlight, since NBCCS tumors are most frequent on sun-exposed skin and are rare in blacks. UVB rarely causes this type of large genetic rearrangement so, in analogy to the x-ray sensitivity of NBCCS patients, UVA-photosensitized reactive oxygen may be important.
Psoralen + UVA (PUVA) Psoralen irradiated with UVA forms adducts at TA, TG, or TT sequences, as well as crosslinks between the two DNA strands at these sites. In human PUVA-induced keratoses, SCCs, and BCCs, about one-quarter of mutations in p53 or HRAS are psoralen-like mutations at the T of TA, TG, or TT; this proportion increases as the PUVA dose increases. The majority of mutations, however, are UV signature mutations. These UV-like mutations could have arisen from the UVA in PUVA, separate UVB treatments for psoriasis, or environmental UVB.
Melanoma Mutations Despite the correlation between melanoma and sunlight, genes with UV signature mutations are not prevalent. The CDKN2A locus is frequently mutated or deleted in
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familial and sporadic melanoma. Its two distinct tumor suppressor proteins, INK4A (also known as P16) and ARF, inhibit cell cycle progression via RB and P53, respectively. INK4A inhibits CDK4/6’s inactivation of the retinoblastoma protein, RB. ARF inhibits MDM2-mediated degradation of P53. Germline mutations in CDKN2A are observed in 25–40% of familial melanomas. In sporadic melanoma, allelic loss of CDKN2A is more common than the rare INK4A inactivating mutations or inactivation of INK4A by promoter methylation. The role of sunlight in these genetic events is unknown. Oncogene mutations are found in the RAS-BRAFMEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway. This signaling cascade is normally activated upon growth factor stimulation and its sequential phosphorylation regulates cell proliferation and differentiation. Activating mutations in MAPK signaling remove the growth factor requirement. RAS mutations are present in 10–20% of melanomas and have been correlated with UV exposure. The most prevalent RAS pathway mutations in melanomas are in the BRAF gene, particularly the BRAF V600E point mutation that renders the kinase constitutively active and enhances ERK activation [22]. This mutation is present in nearly 80% of acquired melanocytic nevi, suggesting a potential early role of BRAF in melanoma development. The V600E mutation is not UV-like and is associated with intermittant sunlight exposure.
GADD45. Additionally, P53 functions as a chromatin accessibility factor, modifying the structure of damaged DNA and making it more accessible to repair factors. P53 also transactivates the cell cycle arrest protein P21, although in keratinocytes UV induces P21 even without P53. UV primarily slows down S phase (‘‘S phase delay’’) and induces a modest G2 arrest, unlike ionizing radiation which uses P53 to induce G1 arrest. It is often said that cell cycle arrest facilitates DNA repair and survival, but there is little evidence supporting this concept; deleting the P21 cell cycle arrest protein has no effect on repair after UV. These ‘‘guardian of the genome’’ roles of P53 are complemented by a ‘‘cellular proofreading’’ role in which P53 erases aberrant cells by apoptosis rather than repairing them [19]: it transcriptionally activates the death receptor Fas and proapoptotic effectors Bax, Bak, Bid, and PUMA; it directly activates Bax protein at the mitochondrion; and it inactivates E2F1, which otherwise inhibits anti-apoptotic Bcl-2 [7]. This suite of UV responses is lost when P53 is mutated by sunlight. DNA damage also activates the cytoplasmically-sequestered transcription factor NFc´B, which then activates pro-inflammatory cytokines such as IL-10, growth signals, and anti-apoptotic signals. Finally, DNA photoproducts trigger UV-induced systemic immunosuppression and suppression of dendritic cell antigenpresenting activity, but not inflammatory edema.
UV-Induced Steps in Cancer and Cancer Prevention
‘‘The UV response’’ initially referred to the P53-independent activation of JNK, its target c-JUN and, via the FOSJUN transcription factor AP-1, induction of genes for collagenase, metallothionein, and c-JUN and c-FOS themselves [23]. The signal begins when reactive oxygen species generated by UVB or UVA photosensitization inactivate phosphatases by converting a highly sensitive cysteine residue in the catalytic site to sulfenic acid [24]. Dephosphorylation of growth factor receptor dimers and death receptor trimers is slowed, leading within minutes to more phosphorylated active receptors. Activated death receptors (involved in apoptosis) such as Fas and TNFa receptor then cluster even without a ligand, recruiting the adapter proteins DAXX and FADD and activating cytoplasmic kinases and scaffold proteins. These activate ASK1 and MEKK 4,7 kinases and their target, JNK [25]. Phosphatase inhibition can activate JNK directly. In parallel, AP-1 also induces genes for the death receptor ligands, FasL and TNFa. These ligands, together with UV upregulation of FAS receptor via P53, create a delayed feedback loop that, as with ionizing radiation, appears to
Skin tumors arise on a background of sun-damaged skin. To prevent sun damage, the skin reacts to acute and chronic UV exposure by multiple stress responses.
DNA Damage Signaling A cell with damaged DNA upregulates normal P53 protein. UV signaling is initiated by cyclobutane dimers and (6–4) photoproducts specifically in the small minority of actively-transcribed genes, with stalled RNA polymerase not only recruiting excision repair proteins but also initiating signaling [5, 6]. In an unknown way, this activates ATR and CHK1 kinases, which phosphorylate P53 at sites that make it resistant to proteasomal degradation mediated by HDM2. P53 then transcriptionally activates a large repertoire of genes, including the repair proteins P48, which is required for GGR and is defective in XP group E, and
Cytoplasmic Signaling
Carcinogenesis: UV Radiation
prolong the rapid but transient response triggered by UV’s inactivation of phosphatases. Without this prolongation, UV-activated NFc´B quickly terminates the JNK response. This loop is important because transient JNK activation leads to cell proliferation, but constitutive JNK activation induces apoptosis. AP-1 also induces immunomodulatory cytokines such as IL-12, which facilitates nucleotide excision repair [26]; the AP-1-induced metalloproteinases degrade dermal extracellular matrix molecules such as collagen and may contribute to photoaging [27]. UV also blocks initiation of protein translation, via kinases that inactivate elongation factor eIF2a.
Cellular Responses Apoptosis UV signaling generates sunburn cells – basal and suprabasal keratinocytes with dense, pycnotic nuclei and intensely eosinophilic cytoplasm. This apoptotic morphology is accompanied by pathognomic DNA double-strand breaks and cleaved caspase 3. UV-induced apoptosis requires signals from both DNA damage and the cytoplasm: DNA photoproducts in active genes trigger P53 and its regulator Mdm-2, but apoptosis also requires JNK and is partially blocked by antioxidants. Although TNFa is required, injecting TNFa does not lead to sunburn cells so UV-induced cytoplasmic signaling is not sufficient. In fibroblasts, keratinocytes, or melanocytes with normal P53 and irradiated with physiologic UVB or UVC doses, apoptosis proceeds through the intrinsic mitochondrial pathway rather than the death-receptor/caspase 8 pathway. Apoptosis then prevents cancer by removing UVdamaged cells, termed ‘‘cellular proofreading’’. Mice accumulate mutations at a rapid rate if they are defective in apoptosis due to a defect in p53 or Fas ligand, or due to overexpressing the anti-apoptotic protein Survivin [7, 28]. In the case of Survivin, this increases SCC. The epidermal hyperplasia that occurs several days after UV may replace cells lost by apoptosis or may remove additional damaged or mutant cells by desquamation. The signal for hyperplasia involves both DNA photoproducts and the EGF receptor.
Stem-Cell Populations In chronically UVB-exposed human skin, p53-mutant clones are found at the two sites of epidermal stem
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cells: the hair follicle, whose bulge region contributes to follicle development and transiently to wound repair, and the interfollicular epidermis, which maintains epidermal homeostasis and can also generate follicles [29]. SCC are thought to originate in interfollicular epidermis, whereas histological evidence and the expression pattern of PTCH indicate that BCC originate in the follicles [30]. Hedgehog signaling through PTCH is crucial for maintaining skin stem cell populations, and for regulating hair follicle and sebaceous gland development. Chronically UV-irradiated human or mouse skin contains scattered basal cells with unusually high levels of DNA photoproducts. The tumor promoter TPA, which induces skin stem cells to proliferate, causes these cells to disappear and be replaced by clusters of p53-mutant keratinocytes. This behavior resembles that of stem cells that are quiescent and poorly repaired, at least on the parental DNA strand, until triggered to divide. The immortalization-promoting enzyme telomerase is normally present only in the epidermal basal layer, but is elevated in sun-exposed skin, skin precancers, and cancers.
Clonal Expansion of Mutant Cells A single mutant cell must clonally expand to reach a clinical size. Less obviously, clonal expansion facilitates the multiple genetic hit mechanism of cancer. Physiologic UV doses create mutations at a frequency of ˜104/gene per cell division (> Fig. 56.2). The specific mutations needed to activate an oncogene would be rarer. Spontaneous mutations, which reflect errors by the replication machinery or DNA damage due to body temperature, are also rare, on the order of 105. The probability of mutating five genes, such as an oncogene and both alleles of two particular tumor suppressor genes, is then at best 1020. With 106 proliferating keratinocytes per cm2 in human skin, and 1 m2 exposed, fewer than one person in 1010 would have a tumor. Accounting for the 60% lifetime expectation of skin cancer in Australia solely in terms of genetic hits in one cell is impossible. In contrast, clonal expansion increases by 1,000-fold the number of targets for the next mutation. A stem cell’s clonal expansion is normally limited to its stem cell compartment [31]. Sunlight is a key driver of clonal expansion beyond this point. The p53-mutant clones in human skin are larger in chronically sunexposed skin. In mice, clones stop growing and regress when UVB treatment ends, indicating that clone expansion is due to a UV-induced physiological event rather than an irreversible mutation. One of these physiological
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. Figure 56.2 The carcinogenesis cycle UV makes chemical changes in DNA that create mutations when DNA is copied. Some mutations alter the function of the gene in which they occur and lead to a new cell phenotype. The abnormal cell expands into a clone that becomes the target of further DNA damage
events is UV-induced apoptosis [7]. Once a p53 mutation arises, the cellular proofreading mechanism backfires. Subsequent UV exposures eliminate damaged normal cells but spare apoptosis-resistant mutants. A mutant normally restrained within its stem cell compartment then escapes to colonize the newly-vacated compartment. Repeating this process results in quantized clonal expansion. Apoptosis retards or accelerates skin cancer depending on the stage. It prevents new p53 mutations, accelerates the expansion of p53-mutant clones and papillomas, and prevents the mutations that convert a papilloma to SCC.
Cell–Cell Communication Cell– cell interactions prevent abnormal cells from proliferating inappropriately. In human autotransplant experiments, BCC’s transferred from their original site regressed – suggesting that an abnormal underlying dermis is required for the tumor to persist. Dermal fibroblasts suppress transformed keratinocytes by secreting TGFb that induces squamous differentiation. Normal keratinocytes also suppress their transformed neighbors, and UV interferes with these signals. Normal human keratinocytes eliminate adjacent transformed keratinocytes (mutated in p53 and HRAS) by inducing cell cycle arrest and differentiation. Physiologic doses of UVB cause apoptosis and differentiation in the normal cells, but not in the transformed keratinocytes, allowing the latter to clonally expand [32]. Other intercellular signals are mediated by integrins – membrane receptors for extracellular matrix proteins such as collagen (a2b1 integrin), laminin (a3b1), and fibronectin (a5b1). Keratinocytes bound to such ligands provide a ‘‘do not differentiate’’
signal through MAP kinase pathways, suppressing keratinocyte apoptosis and allowing a stem-cell pool to be maintained. Integrin receptors are often dysregulated in tumors. UVB irradiation downregulates the b1 integrin subunit; UVA downregulates the gap junction communication protein connexin 43, resembling the action of the tumor promoter TPA. Melanocyte proliferation is normally regulated by keratinocytes via cell–cell adhesion receptors such as E-cadherin, P-cadherin, and desmogleins; these receptors are lost in vertical growth phase melanomas. UV stimulates keratinocytes to secrete endothelin-1, which then downregulates melanocyte E-cadherin and upregulates their N-cadherin. This E to N-cadherin switch diverts melanocyte interactions away from keratinocytes and toward fibroblasts and melanocytes. Endothelin also downregulates the a6 integrin subunit, upregulates the avb3 and a2b1 integrins that anchor melanocytes to dermal collagen and are associated with vertical-growth stage melanoma, inhibits gap junction communication by phosphorylating connexin 43, and activates metalloproteinases associated with basement membrane invasion.
Immune Surveillance In humans, the primary evidence cited for immune surveillance in preventing UV-induced skin cancer is the 10–20-fold increase in AK and SCC on previously sunexposed skin in transplant patients receiving chronic immune suppression to prevent organ rejection. In one Australian study, 27% of deaths in a heart transplant cohort were due to skin cancer. The increase begins months to a few years after immune suppression is
Carcinogenesis: UV Radiation
initiated, and both the AK and SCC are unusually aggressive. Melanomas are also increased. Several lines of evidence mar the interpretation as immune surveillance: Cyclophosphamide is a well-known mutagen. Cyclosporine promotes tumor growth in vitro and in immune deficient mice where there is no immune system to be suppressed. Azathioprine (Imuran) is a mutagen when followed by UVA irradiation. HIV patients do have a modestly increased incidence of SCC, but these tumors at sun-shielded sites are associated with human papilloma virus. Skin cancers are often said to develop in patients who are immunodeficient due to leukemia or lymphoma. Most published reports lack controls or patient data, but an eightfold elevation in chronic lymphocytic leukemia seems valid. Most of these patients had received immunosuppressive drugs, or radiation, so the mutagenicity caveat applies. In contrast, Merkel cell carcinoma may be truly sensitive to immune function because its incidence increases 10–20-fold not only in solid-organ transplant recipients but also in HIV patients (13-fold) and in chronic lymphocytic leukemia.
Animal Models of Skin Cancer UV Radiation Wavelengths and Carcinogenesis Showing causality requires manipulating an experimental system, which usually cannot be done in humans. Early experiments generated fibrosarcomas by irradiating mouse ears. SCC are generated by irradiating back skin daily for about 4 months with doses of UVB several-fold above the minimal erythemal dose. The sequence of events resembles that in humans, with p53-mutant clones appearing early, followed by reddish lesions that resemble AKs both visibly and histologically. SCC induced by UVB contain UV signature p53 mutations. p53-mutant clones may be a precursor lesion for SCC because (a) the relationship between UVB dose and induction time for an AK equals that for a p53-mutant clone plus an additional similar event and (b) mutant clones and SCC have the same sensitivity to DNA repair knockouts. Growth of an existing SCC no longer depends on UV. The action spectrum for carcinogenesis in the hairless mouse closely approximates that for erythema in human skin and edema in murine skin, with the most effective wavelengths at 295–305 nm in the UVB region. Calculations show that this peak is the product of the 260 nm DNA absorption peak and absorption by the skin [12]. Activity decreases sharply at wavelengths above this range.
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The UVA used in tanning beds can also cause skin tumors in mice; p53 mutations are rare. UVB acts as both initiator and promoter for mouse skin papillomas and UVA acts as a promoter. Tumor promoters are agents that increase the frequency of tumors, but only when delivered after the initiator mutagen and only while the promoter is present. Initiation is considered to contribute irreversible genetic events whereas promotion stimulates reversible growth acceleration. The promotion concept developed from studies of chemically-induced papillomas, especially those now known to be mutated at Hras and at low risk for conversion to SCC, which are transiently increased after treating with a chemical such as phorbol ester. Similarly, 90% of UVB-initiated p53-mutant clones regress within 3 weeks after UVB is terminated; T or B cells are not required. AKs also often regress once irradiation stops but SCCs do not, indicating that invasive tumors no longer need a promoter. Because it is now clear that tumorigenesis consists of multiple cycles of mutation and growth, dividing cancer development into initiation and promotion phases has been largely superseded by a focus on the timing of specific genetic and cellular mechanisms. Studies in mice also revealed that: cyclobutane dimers are responsible for UVB-induced apoptosis, hyperplasia, p53-mutant clones, and SCC [33]; repairing the UVBinduced oxidative lesion 8-OH-dG reduces SCC by half; p53/ mice are highly susceptible to UV-induced AK and SCC whereas UV induces BCC in Ptch+/ mice; and basaloid budding and BCC can be induced by overexpressing the hedgehog pathway [30]. Physiological status also affects tumor development: exposure to stress (fox urine) reduces the latency of UV-induced SCC from 21 weeks to 8 weeks. Modeling melanoma has been more challenging. A melanoma-susceptible fish demonstrated the ability of UVA-induced reactive oxygen to induce melanomas. In the opossum Monodelphis domestica, UVB + UVA induces melanomas and melanocytic hyperplasia and UVA can itself induce melanocytic hyperplasia. Generating melanomas in mice requires genetic manipulation. Ocular and dermal melanomas arise without UV when the SV40 virus early region sequences are put under the control of the tyrosinase promoter. When the metallothionein promoter drives hepatocyte growth factor, melanocytes are produced in the epidermis – not their normal location in mice – and a single high dose of sunlamp UV in the neonate, but not adult, generates melanomas months later. When the tyrosinase promoter is used to drive mutant HRAS in a mouse deleted for the ARF gene, melanomas arise months later but sooner if the mice receive a
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single high neonatal UV dose. Half the UV-induced tumors carry amplified CDK6, reminiscent of chronicsun damage melanomas in humans. It is crucial to realize, however, that both metallothionein and tyrosinase promoters are UV-inducible, so the single UV dose used here is not guaranteed to model the role of UV in causing human melanoma.
Immune Function and Skin Cancer The mouse model reveals an important immunological component to tumor progression. Murine UV-induced tumors are highly immunogenic, but early in the course of chronic UV radiation, before primary tumors are evident, mice lose their ability to reject UV-induced tumors. UV therefore has a systemic immunosuppressive effect. Natural killer T lymphocytes are the suppressor T cells responsible [34].
Genetic Risk Factors for UV Carcinogenesis Pigmentation and Initial Damage Melanin has a large role in resistance to skin cancer. The Fitzpatrick skin types are determined not only by baseline pigmentation but also by a person’s response to UV (always burns, tans easily, or rarely burns). This simple UV skin response scale can account for up to a 100-fold difference in susceptibility to skin cancers. Similarly, the many molecular etiologies of oculocutaneous albinism, which result in a deficiency of normal melanin, markedly increase the risk of skin cancer. Increased skin cancer among albinos is mostly nonmelanoma skin cancer but melanomas seem elevated as well. Another hereditary risk factor, red or blonde hair, is now understood at a molecular level as commonly caused by polymorphisms in the melanocortin-1 receptor. This receptor is a G-protein coupled receptor that binds melanocyte stimulating hormone (MSH) and lies at a key point in melanogenesis. Certain mutations in this receptor render it insensitive to normal pigmentation signals, resulting in pheomelanin instead eumelanin. This is an important distinction in terms of skin cancer prevention. Red or yellow pheomelanin is a markedly less effective free radical scavenger; indeed, UV exposed pheomelanin is degraded with a net formation of superoxide [11]. Therefore, pheomelanin can act as a photosensitizer, even inducing apoptosis in nearby cells [35]. Decreased protection and increased damage from pheomelanin in the epidermis may partly
underlie the markedly increased risk of skin cancer associated with red-haired individuals. Reactive oxygen species are largely absorbed by radical scavengers such as glutathione before they cause significant membrane or DNA damage. Specific polymorphisms in the genes for glutathione S-transferase confer a two-fold risk for truncal AK, SCC, and BCC [36]. This genetic risk increases to sixfold in patients with high sun exposure and 12-fold in sun-exposed transplant patients.
DNA Repair and Apoptosis Xeroderma pigmentosum highlights the association between DNA repair capacity and skin cancer. Although defects in any of the XP genes result in some defect in NER, the severity of the XP disorder – number of skin tumors and life expectancy – correlates with DNA repair capacity [16]. The correlation between repair capacity and cancer incidence is also present in the general population. Patients with AK have 30–50% reduced excision repair in their normal fibroblasts or lymphocytes. These data suggest that relatively subtle variability in DNA repair capacity contributes to an individual’s risk of developing skin cancer.
Viral Sensitivity In the rare inherited sensitivity to HPV infection termed epidermodysplasia verruciformis, roughly one-half of patients will develop SCC. These SCC are typically on sun exposed body sites and occur decades earlier than in the general population. HPV may be important in UV carcinogenesis in immunosuppressed transplant patients, as it is detected twice as frequently in SCCs from these patients as in immunocompetent controls.
Prevention The potential of education and sunscreen to control skin cancer remains largely unproven due to the long latency between sun exposure and skin cancer appearance. Adult sunscreen use can reduce AK by two-fold. Randomized studies of school children who aggressively applied sunscreen indicate that common nevi can be reduced. A regimen of sun protection longer than the typical 2–4-year study would likely decrease nevi and subsequent melanoma risk more impressively. Encouraging data are emerging from Australia where education campaigns,
Carcinogenesis: UV Radiation
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covered sidewalks, and covered playgrounds at schools have led to a stabilization of the incidence of non-melanoma skin cancers. Clearly, an effective campaign to reduce skin cancer needs to begin with school age children. This is due to the combined factors that children under the age of 18 spend a significant portion of their time in outdoor recreation and sun damage sustained early in life has more time to contribute to UV carcinogenesis. One could ask ‘‘how much sun is too much?’’ The answer will vary immensely depending on many factors such as pigmentation, whether sun exposure occurs in modest doses or large exposures, the immune status of the individual, and subtle differences in DNA repair capacity. Although highly controversial, there may be some benefit to sun exposure. Several studies have suggested two-fold better survival following a diagnosis of invasive melanoma among patients with extensive sun exposure as compared to those with less exposure. A potential explanation is greater vitamin D synthesis induced by UV.
A recent development in the area of chemoprevention is the ability to augment normal DNA repair pathways. If one could increase DNA repair capacity, the mutations, chromosomal aberrations, and cell death known to be causal for skin tumors should be reduced. The most direct application of this concept is the use of a viral enzyme (T4 endonuclease V) capable of recognizing cyclobutane dimers and accelerating the initial incision step of the nucleotide excision repair pathway. This enzyme has been formulated into a liposomal preparation allowing penetration into the relevant layers of skin. Indeed, studies in XP patients have shown efficacy in decreasing the residual DNA damage as well as the number of actinic keratoses. There are thus diverse emerging approaches by which chemopreventive therapies may increasingly serve as an adjunct to classical UV protection with sunscreens and avoidance of UV exposure to skin.
Conclusion
> Melanoma
Despite the use of suncreens and public awareness of the effects of long-term exposure to UV, the incidence of both melanoma and non-melanoma skin cancers continues to increase. This has led to investigation of novel chemopreventive agents that interfere with the development of cancer through diverse mechanisms. Perhaps the most important recent advance in this area is the use of imiquimod (Aldara), an activator of the innate immune system through the TLR7 receptor. Imiquimod has recently been approved for treating AKs and superficial BCCs and reports of imiquimod effects also exist for atypical melanocytic proliferations such as lentigo maligna. In terms of foods, tea appears to have chemopreventive activity. Orally administered green or black tea reduces UV-induced skin cancers in mice to less than half of control levels. Polyphenols and caffeine appear to be the major chemopreventive components. Topical application of caffeine was similarly effective in mice. The protective effect of caffeine is attributed to its ability to induce apoptosis following UV irradiation. Low fat diets have been well studied as an approach to preventing skin cancer. Animal studies have shown that high fat diets shorten the time between UV exposure and tumor formation and markedly increase the number of tumors per animal. In human trials of patients with nonmelanoma skin cancer, restricting calories from fat reduced the appearance of AKs by two thirds and the development of new non-melanoma skin cancers by one half.
Cross-references and Skin Aging Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma
> Sunlight
References 1. Leigh I, Newton-Bishop JA, Kripke ML. Skin Cancer. Plainview: Cold Spring Harbor Laboratory Press, 1996, pp. 361, Cancer Surveys, vol. 26. 2. Brash DE, Ponte´n J. Skin precancer. In: Ponte´n J (ed) Precancer: Biology, Importance, and Possible Prevention. Cold Spring Harbor: Cold Spring Harbor Press, 1998, pp. 69–113. 3. Brash DE. Sunlight and the onset of skin cancer. Trends Genet. 1997; 13:410–414. 4. de Gruijl FR, Ananthaswamy HN. Biological effects of ultraviolet radiation. Mutat Res. 2005;571(special issue). 5. Hussein MR. Ultraviolet radiation and skin cancer: molecular mechanisms. J Cutan Pathol. 2005;32(3):191–205. 6. Latonen L, Laiho M. Cellular UV damage responses – functions of tumor suppressor p53. Biochim Biophys Acta. 2005;1755:71–89. 7. Raj D, Brash DE, Grossman D. Keratinocyte apoptosis in epidermal development and disease. J Invest Dermatol. 2006;126:243–257. 8. Green A, MacLennan R, Youl P, Martin N. Site distribution of cutaneous melanoma in Queensland. Int J Cancer. 1993;53:232–236. 9. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Brocker EB, LeBoit PE, Pinkel D, Bastian BC. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353(20):2135–2147. 10. Landi MT, Bauer J, Pfeiffer RM, Elder DE, Hulley B, Minghetti P, Calista D, Kanetsky PA, Pinkel D, Bastian BC. MC1R germline variants confer risk for BRAF-mutant melanoma. Science. 2006; 313(5786):521–522. 11. Kollias N, Sayre RM, Zeise L, Chedekel MR. Photoprotection by melanin. J Photochem Photobiol B. 1991;9(2):135–160.
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12. Freeman SE, Hacham H, Gange RW, Maytum DJ, Sutherland JC. Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light. Proc Natl Acad Sci USA. 1989;86:5605–5609. 13. Gallagher RP, Spinelli JJ, Lee TK. Tanning beds, sunlamps, and risk of cutaneous malignant melanoma. Cancer Epidemiol Biomarkers Prev. 2005;14(3):562–566. 14. Wang SY. Photochemistry and Photobiology of Nucleic Acids. New York: Academic, 1976, 596 pp., vol. I. 15. Douki T, Reynaud-Angelin A, Cadet J, Sage E. Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation. Biochemistry. 2003;42(30):9221–9226. 16. Cleaver JE. Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nat Rev Cancer. 2005;5(7):564–573. 17. Brash DE, Rudolph JA, Simon JA, Lin A, McKenna GJ, Baden HP, Halperin AJ, Ponte´n J. A.role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA. 1991;88:10124–10128. 18. Tornaletti S, Pfeifer GP. Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science. 1994;263:1436–1438. 19. Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, Brash DE. Sunburn and p53 in the onset of skin cancer. Nature. 1994;372:773–776. 20. Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM, Persing JA, Leffell DJ, Tarone RE, Brash DE. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci USA. 1996;93:14025–14029. 21. Gailani MR, Bale SJ, Leffell DJ, DiGiovanna JJ, Peck GL, Poliak S, Drum MA, Pastakia B, McBride OW, Kase R, Greene M, Mulvihill JJ, Bale AE. Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9. Cell. 1992;69: 111–117. 22. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, PritchardJones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949–954. 23. Bender K, Blattner C, Knebel A, Iordanov M, Herrlich P, Rahmsdorf HJ. UV-induced signal transduction. J Photochem Photobiol B. 1997;37:1–17.
24. Tonks NK. Redox redux: revisiting PTPs and the control of cell signaling. Cell. 2005;121:667–670. 25. Devary Y, Rosette C, DiDonato JA, Karin M. NF-kB activation by ultraviolet light not dependent on a nuclear signal. Science. 1993;261:1442–1445. 26. Schwarz A, Stander S, Berneburg M, Bohm M, Kulms D, van Steeg H, Grosse-Heitmeyer K, Krutmann J, Schwarz T. Interleukin-12 suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair. Nat Cell Biol. 2002;4:26–31. 27. Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, Voorhees JJ. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature. 1996;379:335–339. 28. Hill LL, Ouhtit A, Loughlin SM, Kripke ML, Ananthaswamy HN, Owen-Schaub LB. Fas ligand: a sensor for DNA damage critical in skin cancer etiology. Science. 1999;285:898–900. 29. Levy V, Lindon C, Harfe BD, Morgan BA. Distinct stem cell populations regenerate the follicle and interfollicular epidermis. Dev Cell. 2005;9(6):855–861. 30. Oro AE, Higgins KM, Hu Z, Bonifas JM, Epstein EH, Scott MP. Basal cell carcinomas in mice overexpressing sonic hedgehog. Science. 1997;276:817–821. 31. Zhang W, Remenyik E, Zelterman D, Brash DE, Wikonkal NM. Escaping the stem cell compartment: sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations. Proc Natl Acad Sci USA. 2001;98:13948–13953. 32. Mudgil AV, Segal N, Andriani F, Wang Y, Fusenig NE, Garlick JA. Ultraviolet-B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression. J Invest Dermatol. 2003;121:191–197. 33. Jans J, Schul W, Sert YG, Rijksen Y, Rebel H, Eker AP, Nakajima S, van Steeg H, de Gruijl FR, Yasui A, Hoeijmakers JH, van der Horst GT. Powerful skin cancer protection by a CPD-photolyase transgene. Curr Biol. 2005;15(2):105–115. 34. Moodycliffe AM, Nghiem D, Clydesdale G, Ullrich SE. Immune suppression and skin cancer development: regulation by NKT cells. Nat Immunol. 2000;1(6):521–525. 35. Takeuchi S, Zhang W, Wakamatsu K, Ito S, Hearing V, Kraemer KH, Brash DE. Melanin acts as a potent UVB sensitizer to cause an atypical mode of cell death in murine skin. Proc Natl Acad Sci USA. 2004;101:15076–15081. 36. Ramsay HM, Harden PN, Reece S, Smith AG, Jones PW, Strange RC, Fryer AA. Polymorphisms in glutathione S-transferases are associated with altered risk of nonmelanoma skin cancer in renal transplant recipients: a preliminary analysis. J Invest Dermatol. 2001;117: 251–255.
64 Cutaneous Effects and Sensitive Skin with Incontinence in the Aged Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Introduction Urinary and fecal incontinence affects a significant portion of the elderly population. The increase in the incidence of incontinence is not only dependent on age but also on the onset of concomitant aging issues such as infection, polypharmacy, and decreased cognitive function. If incontinence is left untreated, a host of dermatologic complications can occur, including incontinence dermatitis, dermatological infections, intertrigo, vulvar folliculitis, and pruritus ani. The presence of chronic incontinence can produce a vicious cycle of skin damage and inflammation due to the loss of cutaneous integrity. Minimizing skin damage caused by incontinence is dependent on successful control of excess hydration, maintenance of proper pH, minimization of interaction between urine and feces, and prevention of secondary infection. Even though incontinence is common in the aged, it is not an inevitable consequence of aging but a disorder that can and should be treated. Appropriate clinical management of incontinence can help seniors continue to lead vital, active lives as well as avoid the cutaneous sequelae of incontinence.
Prevalence of Incontinence As part of the aging process, the bladder becomes more irritable, holds less, and empties less efficiently [1]. These normal changes, when accompanied by concomitant illnesses or medications, obstetrical injury, dementia, or changes in nutrition or hormonal status, can produce incontinence [1, 2]. While reported prevalence rates vary widely, incontinence tends to increase with age and becomes a relatively common affliction in those over 50 [3]. A communitybased study of American women over 50 found that 48.4% experienced urinary incontinence, 15.2% suffered from fecal incontinence, and 9.4% experienced both [4].
Studies have reported the prevalence of fecal incontinence in nursing homes to be as high as 50% [5]. Risk factors include advancing age, female gender, and multiparity [5]; the numerous underlying causes of fecal incontinence are shown in > Table 64.1. Incontinence is thus an important clinical problem in the elderly today, and one that is expected to grow.
Incontinence: Cutaneous Effects Although urinary and fecal incontinence increase with age, neither are natural sequelae of aging, but disorders which could be treated [6]. After 6 months, transient incontinence is more likely to be established as chronic, with a decreased prognosis for long-term resolution [1]. Incontinence of any type creates a hostile environment for the skin [1]. The skin’s ability to provide a barrier between the internal and external environment depends on the integrity of the skin and its histological structure, the presence of intra- and extracellular lipids, and the skin’s pH [7]. Genital hygiene is of particular importance to the health and well-being of older women, as perineal dermatitis is frequently encountered in patients with urinary and/or fecal incontinence [8]. Moreover, the risk of pressure ulcers and incontinence dermatitis can be significant when older women suffer impaired mobility and urinary or fecal incontinence [3]. Occlusion of the skin, often induced by incontinence pads or other containment devices, has a profound influence on the skin surface as shown in > Table 64.2. Skin occluded by diapering is wetter, with higher pH, bacterial count, and susceptibility to erosion [9]. Barrier permeability, as well as molecular and cellular homeostasis [10], is affected as well. Aged skin is additionally compromised [11]. Skin becomes drier, with more tendency to crack [12]. It also becomes thinner, more fragile, and less resistant to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_64, # Springer-Verlag Berlin Heidelberg 2010
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infection. Loss of elasticity leaves it more susceptible to injury, while its ability to repair itself diminishes. Skin aging also results in less surface sensory perception, increasing the risk of injuries like pressure ulcers [11]. Incontinence in aged skin has the potential to produce chemical irritation, mechanical injury, and increased susceptibility to infection. Chemical irritation can be defined as an imbalance of moisture, salts, enzymes, or other chemicals [8]. The pH of normal health skin ranges from 4.0 to 6.8, with an average of approximately 5.5. With exposure to excess moisture, pH can increase to as much as 7.5; pH with exposure to urine can reach 8.0 [8]. The associated disruption of the acid mantle interferes with the production of lipids and enzymes critical for barrier integrity, as well as keratinization critical to the repair of damage caused by prolonged exposure to urine or feces [8]. The acid mantle of the skin provides significant resistance against dehydration as well as bacterial invasion [13]. Disruption of the acid mantle by incontinence allows for secondary infection [14]. Staphylococcus, indigenous to perineal skin, is the most common culprit [8]. Secondary infection by Candida albicans, a common resident of the gastrointestinal (GI) tract, is also common [8]. In those with impaired immune function, the overgrowth
. Table 64.1 Contributing factors in fecal incontinence
of cutaneous pathogens, or invasion of fecal bacteria is more likely to be a complication [3]. Exposure to excess water alone can cause damage in 48 hours, with multiple possible effects as displayed in > Table 64.3. Stool and urine contain numerous substances with the potential to further irritate skin: ammonia, which raises pH; urea, which gets converted to ammonia in the presence of bacteria-containing feces
. Table 64.2 Dermatological effects of occlusion Parameter Measured pH
Increases pH [68]
Integrity of stratum corneum
Disrupts lipid organization and metabolism [68] Prevents expected increase in epidermal lipid synthesis [69] Increases TEWL [69] Increases hydration of stratum corneum [70]
Bacterial counts
Increased [9]
Function of Stratum Corneum
Prevents recovery of elevated TEWL [69] Increases permeability, especially to nonpolar lipids [68, 71]
Intrinsic Factors (Concomitant Diseases) Anorectal dysfunction [44] Arthritis [67] Cancer [44]
Influence of Occlusion
Inhibits barrier restoration [72] Carbon dioxide emission rate
Increased [9]
Cellular Function
Decreases mitotic activity [73] Inhibits DNA synthesis [74]
Dementia [6]
Induces intercellular adhesion molecule 1 [75]
Diabetes [44] Fecal impaction [6]
Increases CD3 + epidermal lymphocytes [75]
Gastrointestinal infections [44] Inflammatory bowel disease [44] Liver failure [44]
Inhibits increase in epidermal cell proliferation [76]
Neuromuscular dysfunction [6]
Reduces epidermal pool of IL-1a [77] Increases skin surface temperature [10]
Neurosensory dysfunction [8] Stroke [67] External Factors Obstetrical injury [5] Patient restraint [67] With kind permission from Wiley-Blackwell publisher adapted from Farage, MA, KW Miller, E. Berardesca and HI Maibach. 2007. Incontinence in the Aged: Contact Dermatitis and Other Cutaneous Consequences. Contact Dermatitis: 57:211–217 [82]
Visible changes
Deepens skin furrows [70] Increases inflammation [75] Increases frequency of hydration dermatitis [78]
DNA = deoxyribonucleic acid, TEWL = transepidermal water loss With kind permission from Wiley-Blackwell publisher adapted from Farage, MA, KW Miller, E. Berardesca and HI Maibach. 2007. Incontinence in the Aged: Contact Dermatitis and Other Cutaneous Consequences. Contact Dermatitis: 57:211–217 [82]
Cutaneous Effects and Sensitive Skin with Incontinence in the Aged
. Table 64.3 Dermatological effects of water Parameter Measured Visible changes
Influence of Water Increases erythema [79, 80] Increases irritation [10]
Function of stratum corneum
Trend to increased cutaneous blood flow [79, 80] Increases TEWL [79, 80] Increases permeability to low molecular weight irritants [24] Increases frictional coefficient, causing increased susceptibility to trauma [14, 24]
Pathogenesis
Increases risk of pressure ulcers [13] Increases risk of loss of skin integrity [13, 24] Increases susceptibility to bacterial colonization [14, 24]
pH
Increase in pH [81]
TEWL = transepidermal water loss With kind permission from Wiley-Blackwell publisher adapted from Farage, MA, KW Miller, E. Berardesca and HI Maibach. 2007. Incontinence in the Aged: Contact Dermatitis and Other Cutaneous Consequences. Contact Dermatitis: 57:211–217 [82]
[15]; and digestive enzymes that are erosive to skin [8], creating increased erythema, transepidermal water loss (TEWL), and susceptibility to fungal infection [16]. A 3-week exposure to physiological concentrations of fecal enzymes and bile salts in vivo produced significant barrier disruption and erythema in one human model [16]. Excess moisture on the skin will eventually produce mechanical damage. Twice as much energy is required to produce frictional erosions on dry skin as on skin subjected to 24-hour water exposure [17], therefore, incontinence is a major risk factor in pressure ulcers [18]. Skin hydration following occlusion is significantly higher and slower to dissipate in aged skin [19]. Overhydrated skin is also more vulnerable to tearing [14, 17], particularly when immobile patients are moved [8]. In addition, overzealous scrubbing during cleansing procedures on fragile elderly skin can strip away the protective horny layer, an event usually limited to epidermis, but which may involve huge areas of skin [8]. Any breach of skin integrity in an elderly individual can become a serious injury due to the potential for infection, loss of fluid and electrolytes, decreased thermoregulatory function, and impaired metabolism and communication [3].
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Incontinence Dermatitis Perineal dermatitis, or incontinence dermatitis, is a broad term describing skin problems in patients with urinary or fecal incontinence. The condition creates much pain and discomfort in elderly sufferers, causing inflammation and tissue damage to the vulva, perineum, perianal region, and buttocks [3]. The prevalence of risk of perineal dermatitis, predictably, increases as mobility declines. Frequency of incontinence, baseline skin condition, overall health, management of excess moisture [20], cognitive impairment, and concurrent medications are also factors [21, 22], as shown in > Fig. 64.1. Sixty percent of fecal matter is comprised of microorganisms [23]. Urine and feces in contact with the dry, cracked skin characteristic of the elderly is absorbed into the crevices, providing an excellent environment for bacterial growth [18, 24, 25]. When urine and stool mix, bacteria present in the stool convert urea to ammonia [26], which raises the pH of the skin thus destroying the acid mantle and facilitating penetration of irritants. Feces also contain proteolytic and lipolytic digestive enzymes, normally deactivated in the digestive tract [13], which are reactivated in the presence of ammonia, with the potential to erode skin [16, 26]. Perineal dermatitis is thus more likely when stool and urine are simultaneously present [27], as pathogens in stool overwhelm the skin’s defenses, leading to breakdown and subsequent colonization [28]. In a study of geriatric psychiatric patients, all patients with both urine and fecal incontinence developed perineal dermatitis within 2 days [27]. Incontinence dermatitis in older people begins with mild erythema of the skin, which may worsen and develop into blistering and erosion [8]. Severe or improperly managed cases can result in full-thickness wounds [29]. Initial inflammation may be harder to detect in darker skin. With urinary incontinence, dermatitis begins between the labial folds; dermatitis associated with fecal incontinence originates in the perianal area and progresses to the posterior aspect of the upper thighs [8]. Unusual patterns may reflect occlusion of the skin by a containment device [8]. Selection of proper treatment must take into account the fragility of elderly skin [17], as additional tissue injury is an all too common sequelae of cleansing [22]. Treatment for incontinence dermatitis must be carefully considered to avoid further unintentional damage [30]. Although the use of products such a vegetable shortening, shaving cream, or veterinary supplies is common, legal liability makes this practice unsupportable [13].
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Cutaneous Effects and Sensitive Skin with Incontinence in the Aged
. Figure 64.1 Copyright ß(2006) From Farage M. and M. Bramante. Genital Hygiene: Culture, Practices, and Health Impact. In: The Vulva: Anatomy, Physiology, and Pathology. Editors: Farage MA, Maibach HI. CRC Press 2006 (3). (Reproduced with permission from Routledge/Taylor & Francis Group, LLC)
Dermal Infections Fungal Infections Fungal infections of the skin related to incontinence involve primarily one of two organisms: Tinea and Candida albicans. Tinea is a fungal infection of the vulvar skin folds, more commonly known as ringworm. Though rare, its prevalence rises in older women because of diminished cellular immune response [12, 31]. Characteristic presentation is a ring-shaped eruption with an actively advancing border but healing center. Any pruritic, scaly eruption of the vulva, however, should be scraped for microscopic examination and treated with antifungal therapy, if appropriate. Avoiding overhydration of the skin will generally prevent this condition [3]. Secondary infection with Candida albicans will result in erythematous, punctuate pustules with a central confluence; satellite lesions may be visible at the border of
the infection [32]. Lesions can take on a macular appearance from friction [8]. Yeast may extend into the groin and thigh folds [21], with curd-like plaques on mucous membranes [2]. Even mild inflammation is accompanied by significant burning, requires pain management [21], and can be accompanied by intense pruritus of affected areas [2]. Chronic infection can cause darkened or reddened skin which may be mistaken for a pressure ulcer [8]. The gastrointestinal tract is an important reservoir of Candida albicans, and fecal incontinence increases the risk of Candida colonization [33]. Routine administration of amoxicillin doubled the counts of Candida albicans observed from both the rectum and skin, with an associated increase in the risk of dermatitis [34]. Treatment for Candidiasis consists principally of topical antifungal agents [2]. Use of systemic antibiotics should be discontinued, when possible [2], while affected areas are kept dry and, as much as possible, exposed to air [2].
Cutaneous Effects and Sensitive Skin with Incontinence in the Aged
Bacterial Infections A primary skin function is the prevention of invasion by external pathogens. Compromised skin may be less effective, allowing the proliferation and invasion of microbials [24]. Staphylococcus can readily colonize skin already compromised by incontinence dermatitis [33]. About 20% of cases of necrotizing fasciitis, a particularly destructive necrosis of the skin and deeper tissues, have been associated with the presence of infected hair follicles [35]. In addition, the use of incontinence therapies such as trans-obturator and vaginal tape has a demonstrated association with both necrotizing fasciitis and cellulitis [36, 37].
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. Figure 64.2 Pressure Ulcer in the sacral area. A patient laying in the bed after spinal injury
Intertrigo and Vulvar Folliculitis Exposure to excess moisture and excretory products associated with incontinence encourages the development of both intertrigo and vulvar folliculitis in the genital area. Intertrigo, a maceration of the tissue due to heat, moisture, and friction [2], affects areas with opposing skin surfaces such as the labia, perineum, and genitocrural folds [38]. Vulvar folliculitis, a result of bacterial infection in the presence of increased moisture, warmth, and decreased hygiene, is the development of red, tender pus-filled papules surrounding the hair follicles which may be associated with general staphylococcal or streptococcal infection. Both conditions respond to fastidious hygiene and keeping the perineal area dry [3].
Pruritus Ani Anogenital pruritus, characterized by perianal itching, can be the result of minor incontinence and but can also result from overzealous cleansing with harsh soaps [2]. It is characterized by intense itching accompanied by erythema and/or excoriation. Chronic scratching may result in perianal inflammation and skin damage, particularly in the presence of impaired mental function [21].
Pressure Ulcers Pressure, or decubitus, ulcers are an area of localized cutaneous damage typically associated with pressure from bony protuberances on aged skin [39] (> Fig. 64.2). Overhydration of the skin, typically in the form of incontinence, increases the susceptibility of the skin to this type of injury [14, 18]. Skin needs to be as dry as possible and
patients repositioned frequently [11]. The frequency of pad changes for incontinent patients is directly related to the risk of Stage II pressure ulcers [40]. Adequate nutritional status should be maintained [11]. Little research, however, has been performed with regard to an effective skin care regimen for decubitus ulcer prevention. One retrospective study demonstrated that the use of a combined skin cleanser/protectant product on residents with incontinence decreased the incidence of nosocomial pressure ulcers in the sacral/ buttock area [41]. A 6-month prospective study found that the use of a body wash and skin protectant with incontinence patients reduced the risk of Stage I and II pressure ulcers from 11.3% to 4.8% [42].
Management of Incontinence Few systematic trials document the impact of specific cleansing regimens on the prevention or treatment of incontinence dermatitis. The only published prospective study of preventative care was a preliminary trial of structured intervention in 15 institutionalized patients with dementia [27]. An equal number developed dermatitis (2 in the structured care intervention group and 3 in the unstructured care group) regardless of whether cleansers, moisturizers, or moisture-barrier preparations were used. Dermatitis developed only in those with urofecal incontinence and followed more than 4 incontinent episodes in 24 hours. None of the patients were capable of informing caregivers of incontinent episodes. The small number of subjects and their poor mental health limit the conclusions that can be drawn from this study.
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Cutaneous Effects and Sensitive Skin with Incontinence in the Aged
Thus, patients who are incapable of reporting incontinent episodes should be monitored closely and promptly cleansed. Wet or soiled garments should be changed promptly and skin cleansing should follow every incontinent episode [13]; efforts should be made to prevent further moisture from reaching the skin [22]. Skin cleansing must be performed using cleansers specifically formulated for incontinent patients, which have an acidic pH and a no-rinse formulation [22] in order to avoid frictional forces during cleansing [13]. Where possible, air drying of the skin is optimal [13]. The pH of cleansing products should be verified before use, as many common products, even when supposedly appropriate for incontinent patients, have pH outside the recommended range [13]. Skin should be kept dry [13] by using superabsorbent incontinence pads [24]. Barrier ointments, which protect the skin from contact with moisture while at the same time preventing friction from diapers and bed linens, should also be used [43]. Case reports have supported their effectiveness [44]. Moisturizers can contain medicating ingredients which bind moisture, thus helping to heal damaged skin and prevent drying [13, 22]. Topical antibiotics and antimicrobials should be employed only when an infection is actually present [22]. Antifungal powders and creams can be applied underneath barrier creams or ointments where needed to control incontinence-associated fungal rashes [22]. The chronically incontinent person should be monitored closely for signs of impending loss of skin integrity, with particular attention to skin folds and creases. In women who are obese, skin folds of the lower abdomen must also be exposed and examined, particularly in women who are diabetic or immunocompromised [8].
Sensitive Skin with Incontinence Incontinence in the aged population is quite common, with a prevalence of about 50% both in community [4] and institutionalized [5] populations. In addition, the majority of women in industrialized countries (50% to 90%, depending on the population studied), as well as a rapidly increasing percentage of men in those countries, believe that they have sensitive skin [45]. As the proportion of the elderly continues to increase in industrialized nations, the numbers of individuals who suffer from both incontinence and sensitive skin are likely to increase as well. It is possible that this population may experience
more serious dermatological effects related to incontinence that those individuals without sensitive skin. The physiological changes that occur as skin ages would predict an increased susceptibility to irritants, including urine and feces [46]. Existing studies, however, are ambiguous with regard to the influence of age on skin sensitivity. Clinical assessment of the erythematous response to irritants in older people suggest that susceptibility generally decreases with age [47]. However, objective signs of irritation often show little correlation with the intensity of subjective complaints [47]. A study of sensory perceptions of sensitive skin conducted on 1,029 individuals in Ohio stratified subjects into four age groups (subjects under 30, subjects in their thirties, subjects in their forties, and those over fifty and evaluated subjective data according to age [47]. Those over 50 were more likely to claim sensitive skin than younger adults, and more likely to perceive genital skin (to the exclusion of other body sites) to be more sensitive [47]. Older adults also stated that their skin had become more sensitive over time (46%) [47]. In a large Italian study including over one hundred elderly subjects that performed lactic acid sting tests on every subject, the intensity of the stinging response was inversely proportional to the age of the patient [48]. Urinary incontinence, however, represents a substantial psychosocial burden and is associated with a variety of psychiatric disorders, including depression, anxiety, decreased self-esteem, reduced social and personal interaction, and other psychological disorders [49–52, 64]. It is possible, then, that incontinence may modulate these individual’s perceptions of skin sensitivity, particular in the genital area. The vulva, formed partially from embryonic endoderm, differs from skin at exposed body sites [57] and displays differences in irritant potential which seem to be dependent on the relative permeability of irritants in vulvar skin: vulvar skin was shown to be significantly more reactive than forearm skin to benzalkonium chloride and maleic acid [54], but less reactive than the forearm to sodium lauryl sulfate [55, 57]. The non-keratinized skin of the vulva, however, exhibits clearly increased permeability [57] related to the absence of keratin, a loosely packed, less structured lipid barrier, and a relative thinness as compared to keratinized skin [57]. Buccal tissue, similar in structure, is often employed in a surrogate model for vulvar testing; buccal skin has been demonstrated to be 10 times more permeable than keratinized skin [58]. Although the vulvar area may be particularly susceptible to cutaneous irritation [56], little objective published
Cutaneous Effects and Sensitive Skin with Incontinence in the Aged
data exists on the relationship between repeated exposure to habitual vulvar irritants and sensitive skin [53]. When tested, the vulvar area was less responsive to both venous blood and the products of menses than the upper arm [55]. The contribution to irritation by topical agents is substantial [60, 61] and often underestimated [63]. In fact, 29% of patients with chronic vulvar irritation were demonstrated to have contact hypersensitivity, and 94% of those were determined to have developed secondary sensitization to topical medications [62]. Thus, incontinence may aggravate overlying issues in an aged patient as well, with sequelae which may differ in intensity in those with sensitive skin. Recent studies have evaluated skin sensitivity in the vulvar area with regard to sensory responses to consumer products meant for the vulvar area. It was hypothesized that patients with erythema related to a previous genital infection may represent a population of sensitive subjects; however, no increase in sensory effects to exposure to feminine hygiene pads was observed [53]. In a similar population, however, in which observed erythema was evaluated against perceived sensory effects, women who perceived themselves as particularly susceptibility to facial erythema were significantly more likely to have vulvar erythema, a potential indicator of a underlying biological origin [53]. Interestingly, a separate study evaluated perceptions of sensitive skin in women with urinary incontinence, expecting to observe an increased sensitivity of genital skin [65]. Increased sensitivity specific to the genital area was not observed, but incontinent women were significantly more likely to assess themselves as having overall skin sensitivity than continent subjects (p = 0.014; 86.2% in incontinent subjects versus 68.3% in controls) [65].
Conclusion The economics of incontinence reflect its impact on older adults in the US: $1.1 billion/year is spent on incontinencerelated products, and $16.4 billion/year on incontinencerelated care [66]. Restoring normalcy to the lives of incontinent seniors and offering proper clinical care will greatly decrease the risk for serious dermatologic, social and psychological problems in the aging population [1, 64]. Dermatologists who are knowledgeable about geriatric issues can help to maintain the health and quality of life of their older patients. Additional prospective clinical trials are needed to study the long-term efficacy of preventive hygiene measures as well as therapeutic interventions [3].
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Cross-references > Aging
and Skin Sensitivity Dermatitis in the Aged > Irritant Contact Dermatitis > Perceptions of Sensitive Skin with Age > Solutions and Products for Managing Female Urinary Incontinence > Susceptibility to Irritation in the Elderly: New Techniques > Atopic
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Cutaneous Effects and Sensitive Skin with Incontinence in the Aged 61. Basketter DA, Wilhelm KP. Studies on non-immune immediate contact reactions in an unselected population. Contact Dermatitis. 1996;35:237–240. 62. Marren P, Wojnarowska F, Powell S. Allergic contact dermatitis and vulvar dermatoses. Br J Dermatol. 1992;126:52–56. 63. Farage MA. Vulvar susceptibility to contact irritants and allergens: a review. Arch Gynecol Obstet. 2005;272:167–172. 64. Farage MA, Miller KW, Berardesca E, Maibach HI. Psychosocial and Societal Burden of Incontinence in the aged population: a review. Arch Gynecol Obstet. 2008;277:285–290. 65. Farage M. Perceptions of sensitive skin: women with urinary incontinence. Arch Gynecol Obstet. 2009;280(1):49. DOI: 10.1007/ s00404-008-0870-6. 66. Agency for Health Care Policy and Research (AHCPR). Overview: urinary incontinence in adults, clinical practice guildeline update. http://www.ahrq.gov/clinic/uiovervw.htm. cited June 7, 2009. 67. Nelson RL, Furner SE. Risk factors for the development of fecal and urinary incontinence in Wisconsin nursing home residents. Maturitas. 2005;52:26–31. 68. Rippke F, Schreiner V, Doering T, et al. Stratum corneum ph in atopic dermatitis: impact on skin barrier function and colonization with Staphylococcus Aureus. Am J Clin Dermatol. 2004;5: 217–223. 69. Grubauer G, Elias PM, Feingold KR. Transepidermal water loss: the signal for recovery of barrier structure and function. J Lipid Res. 1989;30:323–333. 70. Zhai H, Maibach HI. Occlusion vs. Skin barrier function. Skin Res Technol. 2002;8:1–6. 71. Zhai H, Maibach H. Effects of occlusion: percutaneous absorption. In: Bronaugh R, Maibach H (eds) Percutaneous Absorption, DrugCosmetics-Mechanisms-Methodology, 4th ed. Boca Raton: Taylor and Francis: 2005: 235–245. 72. Taljebini M, Warren R, Mao-Oiang M, et al. Cutaneous permeability barrier repair following various types of insults: kinetics and effects of occlusion. Skin Pharmacol. 1996;9:111–119.
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63 Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes Shingo Sakai . Hachiro Tagami
Introduction Patients with diabetes often have dry scaly skin [1–3]. Moreover, diabetes mellitus induces various forms of dermopathy such as bullosis diabeticorum, necrobiosis lipoidica diabeticorum, scleredema diabeticorum, and acanthosis nigrican [4]. Non‐healing ulcers occur in approximately 15% of patients with diabetes, and therefore it is imperative to prevent ulcer formation and improve wound healing in these patients [5, 6]. In general, diabetic atrophy is thought to result from complications such as vasculopathy and neuropathy. Insulin resistance and hyperglycemia contribute to the impaired physiologic function observed in various tissues of these patients. Hyperglycemia induces cellular abnormalities via several mechanisms, including non‐enzymatic glycosylation, oxidative–reductive stress, aldose-reductase activation, and activation of diacylglycerol-phosphate kinase C (PKC), etc. [7, 8]. For example, diabetes induces advanced glycosylation end products in the collagen of the dermis [9, 10]. These end products are observed in aged skin also [11] and are postulated to produce the characteristic skin stiffness [12, 13] and delayed wound healing [14, 15] seen in older adults. Reports also indicate that reduced collagen synthesis and increased matrix metalloproteinase production occur in the skin of patients with diabetes [16, 17]. Hence, the dermis of patients with diabetes may share some of the features of the dermis of aged skin [18, 19]. Within the epidermis, insulin acts as an essential growth factor for the proliferation [20] and migration [21, 22] of keratinocytes. The inhibition of keratinocyte proliferation seen in patients with diabetes may contribute to the delayed wound healing. Insulin also regulates keratinocyte differentiation [23]. Interestingly, the surface area of corneocytes is reportedly larger in patients with diabetes than in normal individuals [24], suggesting that diabetes mellitus impairs epidermal turnover. However, it is difficult to distinguish the effects of diabetes on the functional properties of the stratum corneum (SC), because diabetes often accompanies aging. For example,
pruritus due to diabetes is difficult to be distinguished clinically from that noted in senile xerosis [4]. A new model of diabetes has been developed in hairless mice by using streptozotocin (STZ) [25], an agent that destroys insulin-secreting pancreatic beta cells [26]. STZtreated animals serve as a model of type I diabetes. The hairless mouse model exhibits many of the features seen in human patients with uncontroled diabetes mellitus, including hyperglycemia, polydipsia, and polyuria [27, 28]. The hairless mouse model of STZ-induced diabetes avoids the obstacles to functional and biochemical analysis of the skin posed by the presence and growth of fur. Using this model, the effects of experimentally induced diabetes on SC hydration and barrier function, as well as changes in the SC content of lipids and soluble amino acid were assessed [25]. These properties of the SC were also examined in patients with diabetes mellitus [29]. As will be detailed herein, the experimental and clinical study results indicate that diabetes mellitus induces epidermal changes similar to those observed in aged skin.
Biophysical Properties of the Stratum Corneum in the Hairless Mouse Model of Diabetes Mellitus Changes in the Functional Properties of the Stratum Corneum Streptozotocin (STZ) (150 mg/kg) rapidly induced diabetes symptoms in hairless mice. Blood glucose concentrations increased significantly from the second day after the injection, and the increase continued time-dependently for up to 3 weeks. After the induction of hyperglycemia, high-frequency conductance (HFC) of the skin (a parameter linked to SC hydration) decreased in a time-dependent fashion, relative to that of the untreated mice. Three weeks after the STZ injection, HFC levels in the SC of the diabetic animals were about a half of those of the control group (> Fig. 63.1). In contrast, no difference in transepidermal
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_63, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 63.1 Mice with streptozotocin-induced diabetes exhibit decreased water content of the stratum corneum without impairment of the water barrier function. CONTROL, buffer-injected group; STZ, streptozotocin-treated group. *Significant (p < 0.05); N.S., not significant. Values represent means with SEM from five animals per group
. Figure 63.2 Mice with streptozotocin-induced diabetes exhibit normal metabolism of amino acids in the stratum corneum. The amino acid content in the SC (a) and epidermal (pro) filaggrin content (b) were assayed at 3 weeks after a single injection of streptozotocin.*Values represent means with SEM from five animals per group. Significant (p < 0.05). FG shows filaggrin monomer protein
water loss (TEWL), a measure of skin barrier function, was observed. These findings suggest that experimentally induced diabetes in mice impairs water homeostasis in the SC without altering its water barrier function.
Changes of the Amino Acid and Lipid Content of the Stratum Corneum In mice with STZ-induced diabetes, the amino acid content of the SC increased slightly by 3 weeks after treatment (> Fig. 63.2). Immunoblotting analysis demonstrated that the contents of profilaggrin and filaggrin, precursors of the water-soluble amino acids, were almost unchanged in the epidermis of these animals (> Fig. 63.2). This suggests that the normal processing of profilaggrin to amino acids is functional in the epidermis of the mice with STZ-induced diabetes. The content of the total SC lipids per unit area of the skin was higher in the STZ-treated group than in the
control group (> Table 63.1); only the triglyceride content decreased significantly after the induction of diabetes. This result suggests that triglyceride metabolism in the sebaceous glands was impaired in mice with experimentally induced diabetes. The state of SC hydration is thought to be regulated principally by three factors: the water-soluble natural moisturizing factor (derived mainly from profilaggrin [30]), intercellular lipids [31], and sebum lipids [32]. In the dry skin of patients with atopic dermatitis and in the dry skin of older adults, the levels of both SC amino acids [30, 33] [34] and SC ceramides (the main components of intercellular lipids [35–37]) reportedly decrease. The dry skin of senile xerosis is also characterized by significant decreases in triglycerides [36, 38, 39]. In mice with experimentally induced diabetes, decrease in the water content of the SC was independent of decrease in the content of SC amino acids and ceramides, but paralleled a decrease in SC triglycerides. Alterations in triglycerides are similar to the changes seen in older patients with xerosis.
Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
. Table 63.1 Decreased triglyceride content in the stratum corneum of mice with streptozotocin-induced diabetes Content (mg/cm2) STZ-treated mice
Control mice
Ceramide I
1.5 0.2
1.9 0.1*
Ceramide II–V
9.1 1.1
15.5 1.0***
Cholesterol
9.8 1.3
17.2 1.4***
Fatty acids
4.5 0.6
8.3 0.3***
Triglycerides
20.6 6.9
4.0 4.1*
Wax/cholesterol esters
22.6 1.0
41.0 3.3***
Total lipids
92.5 3.8
121.6 5.9***
Values represent means with SEM from five animals per group (From [25]) *Significant (p < 0.05) **Significant (p < 0.01) ***Significant (p < 0.005)
Insulin reportedly stimulates fat synthesis by adipocytes [40, 41]. In rats, STZ treatment stimulated hormone-sensitive lipase activity of the adipocytes [42]. Reductions in SC triglycerides observed in animals with experimentally induced diabetes may be due to activation of lipolysis in the sebaceous glands. Patients with senile xerosis also have reduced levels of SC triglycerides [38], suggesting that triglycerides play a role in SC moisturization. In general, acetone extraction of skin surface lipids induces a brief but significant reduction in SC hydration. In seborrheic areas, such as the face and scalp, SC hydration returns to pretreatment levels within a few hours as skin surface lipids are replenished by sebum secretion [32]. Glycerol may play a mechanistic role in sebum-induced skin hydration [43]. Studies performed in asebia J1 and 2J mice (a unique experimental model associated with profound sebaceous gland hypoplasia) showed that these mice exhibit normal homeostasis of the SC permeability barrier and normal extracellular lamellar membrane structures; however, reduced production of sebumassociated lipids resulted in epidermal hyperplasia, inflammation, and a greater than 50% decrease in SC hydration. Application of a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) failed to restore normal SC hydration: only topical glycerol – the putative product of triglyceride hydrolysis in sebaceous glands – normalized SC hydration. In fact, the glycerol content of the SC of asebia mice was 85% lower than in normal mice. These findings suggest that glycerol produced by the sebaceous glands may be a major contributor to SC hydration.
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Notably, one cannot exclude the possibility that factors besides amino acids, intercellular lipids, and sebum contribute to the reduced water content of the SC in the mouse model of diabetes. A role for other watersoluble moisturizing factors, such as lactate and urea, is speculated; however, few studies have examined the role of water-soluble substances in regulating skin surface hydration. It has also been speculated that the pathogenesis of diabetic xerosis may involve autonomic, peripheral C fiber neuropathy [1, 3, 44]. Impaired sweating is often observed both in patients with diabetes and in older adults because of the impairment of skin-temperature control [45]. The laboratory showed that lactate, an important component of sweat, plays a role in maintaining SC hydration [46]. Decreased lactate production may be a factor to bring skin dryness in the diabetic patient. Another possible mechanism of SC hydration is water movement through the epidermis. In mice with experimentally induced diabetes, a chemiosmotic alteration in the SC itself (and/or in keratinocytes beneath the SC layer) may impair the water homeostasis of the SC. Aquaporin, a vital water channel in various tissues such as the kidney, lung, retina, and cornea, may affect epidermal movement of water and glycerol. The author’s laboratory showed that aquaporin isoforms are expressed in the keratinocytes and that aquaporin-3 is inducible under hypertonic stress [47]; moreover, other investigators have reported reduced SC hydration in hairless, aquaporin-3null mice [48]. Aquaporin-3 is also involved in epidermal proliferation [49]. Therefore, the possibility exists that aquaporin modulates epidermal water movement and contributes to SC hydration.
Changes in Epidermal Proliferation and Differentiation Diabetes also affects epidermal structure, proliferation, and differentiation. The epidermis of mice with STZinduced diabetes is thinner than that of untreated controls (> Fig. 63.3). The ratio of proliferating-cell-nuclearantigen (PCNA)-positive basal cells to the total basal cells in the epidermis is significantly lower in STZ-treated mice (> Fig. 63.3); the DNA content of the epidermis is also lower in STZ-treated mice than that in controls (23.4 2.1 g/cm2 vs. 29.3 0.6 g/cm2, respectively; n ¼ 5, p < 0.05). These results suggest that diabetes inhibits the epidermal proliferation. Clinically, the corneocyte surface area is enlarged in both senile xerosis [38, 50] and in patients with
655
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Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
. Figure 63.3 Mice with streptozotocin-induced diabetes show decreased proliferation of epidermal basal cells. At 3 weeks after a single injection of streptozotocin. ***Significant (p < 0.001). Values represent means with SEM from four animals per group. Arrows: PCNA-positive cells
diabetes [24], whereas in atopic xerosis corneocyte surface area is reduced [51]. This suggests a slow epidermal turnover rate in senile xerosis and diabetic dry skin, and a rapid epidermal turnover in atopic xerosis. Mice with experimentally induced diabetes also had somewhat elevated corneocyte surface areas relative to controls (> Fig. 63.4), which suggests a reduced epidermal cell turnover rate in mice with experimentally induced diabetes, as in aged human skin. Hence, both clinical and experimental diabetes affect corneocytes in a similar manner to the aging process. Furthermore, epidermal differentiation markers in STZ-treated mice bear some similarities to those in aged skin. For example, the epidermal differentiation markers, keratin 1, keratin 5, keratin 10, and loricrin (58–90 kDa) were normal in STZ-treated animals (> Fig. 63.5). However, three loricrin-derived peptides (with molecular weights 34, 36, and 43 kDa, respectively) were observed in the SC of STZ-treated mice, with a pattern similar to that seen with SC peptides in aged mice (> Fig. 63.5). This suggests that alterations in the processing of the SC proteins that occur in the diabetic state are similar to those observed in aged skin. Loricrin, an insoluble protein of the cornified envelope [52], is produced and cross-linked in the terminal differentiation process. Loricrin-derived peptides were
extremely difficult to detect in healthy young mice, but were easily solubilized in diabetic and aged mice. Consequently, stages in the maturation, proteolysis, and/or oxidation of loricrin processing were altered in the SC of the diabetic mouse as that of aged ones. The attenuated insulin signal transduction in diabetic mouse is speculated to be important in the growth and differentiation of keratinocytes. Keratinocytes have constituent insulin receptors [23]; insulin stimulates keratinocyte migration [21] and proliferation [20] and insulin and insulin-like growth factors regulate keratinocyte differentiation [23]. For example, insulin receptor (IR)null mice exhibited lower epidermal proliferation [53]; similarly, insulin receptor substrate 1 (IRS-1)-null mice had a thin and abnormally differentiated epidermis [54]. In mice with experimentally induced diabetes, insulin signal transduction is attenuated and epidermal proliferation is reduced. Interestingly, these diabetic mice exhibited four features characteristic of senile xerosis: reduced SC hydration, reduced epidermal turnover, accumulated corneocyte layers, and decreased triglyceride content. These observations suggest that in diabetes, hyperglycemia and/or attenuated insulin signaling may promote epidermal aging. The impact of growth factors on epidermal turnover in diabetes also has been investigated. Expression of
Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
63
. Figure 63.4 Surface areas of corneocytes in mice with streptozotocin-induced diabetes. The corneocyte surface areas were measured 3 weeks after a single injection of streptozotocin and also at ages 10, 36, and 63 weeks. Values represent means with SEM from five animals per group. *Significant (p < 0.05); ***Significant (p < 0.001)
. Figure 63.5 Effects of streptozotocin on marker proteins of epidermal differentiation. Western blots of epidermal protein extracted three weeks after streptozocin treatment. K1: keratin 1; K10: keratin 10. At the same time, the SC protein was extracted and applied to Western blotting using antiloricrin antibody
nerve growth factor (an autocrine growth factor of keratinocytes [55]) is downregulated in diabetic skin, whereas the expression of nerve growth factor receptors is upregulated [56]. Decreased epidermal turnover in the hairless mouse model of diabetes may be linked to abnormal signal transduction of either insulin or nerve growth factor, or both.
Biophysical Properties of the Stratum Corneum in Patients with Diabetes Hydration, Surface Lipids, and Barrier Function of the Stratum Corneum in Patients with Diabetes Properties of the stratum corneum were examined in patients with diabetes to determine whether impairments similar to those seen in the hairless mouse model occur [29]. Patients were classified into groups with high- or low-fasting plasma glucose (FPG) (levels above and below 110 mg/dL, respectively) and into groups with high and low HbA1C (levels above and below 5.8%, respectively). FPG indicates the hyperglycemic state at the time of the measurements, while the HbA1C reflects the average hyperglycemic state in the past 7–8 weeks preceding the measurement. The comparative age ranges of patients in the low- and high-FPG groups and in the low- and highHbA1C groups were not significantly different. High-frequency conductance (HFC) of the extensor leg and volar forearm was measured for stratum corneum hydration. The content of skin surface lipids on the forehead was measured using Sebumeter. Measurements were performed in a constant-climate room at 20 C and at a relative humidity of 50%. Patients with high FPG exhibited significantly lower SC hydration on the extensor leg and volar forearm than those with low FPG (> Fig. 63.6). The group with high FPG had a significantly lower content of skin surface lipids on the forehead. When patients with high and low HbA1C were compared, those with high HbA1C had a lower content of skin surface lipids on the forehead (> Fig. 63.7). No difference in SC hydration was observed between the low- and high-HbA1C groups.
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Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
. Figure 63.6 Decreased SC hydration accompanies the real-time hyperglycemic state. Patients with diabetes were grouped according to above- and below-normal FPG (110 mg/dL) and above- and below-normal HbA1C (5.8%). Values represent means SD *p < 0.025, N.S.; not significant, □; volar forearm, ; extensor lower leg
▪
. Figure 63.7 A hyperglycemic state is associated with reduced surface lipid content of forehead skin in patients with diabetes. Patients with diabetes were grouped according to aboveand below-normal FPG (110 mg/dL) and above- and belownormal HbA1C (5.8%). Values represent means SD. *p < 0.05
These clinical findings suggest that the state of SC hydration in patients with diabetes is influenced more by the immediate or ‘‘real-time’’ hyperglycemic state rather than by the hyperglycemic state in the preceding weeks. In contrast with the aforementioned results, patients with high and low FPG exhibited similar levels of TEWL, as measured on the forearm and leg, suggesting that the water barrier function of the skin was not significantly affected. In patients with high HbA1C, TEWL measured on the volar forearm was significantly lower than in patients with low HbA1C (> Table 63.2). Therefore, hyperglycemia does not seem to impair the water barrier function of the SC, as patients with high FPG exhibited lower SC hydration without a consistent change in SC barrier function. These results are consistent with those found in mice with experimentally induced diabetes. Notably, patients with high FPG as well as those with high HbA1C had a low surface lipid content The combination of reduced SC hydration and a low surface lipid content, unaccompanied by impairment of the SC barrier function, is a phenomenon also seen in senile xerosis, a condition characterized by dry scaly skin in
Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
older adults [38]. Hence, reduced SC hydration coupled with normal barrier function, as observed in patients with diabetes, mirrors the phenomenon that develops in aged persons. SC hydration is critical to skin smoothness [57], softness [58], and surface texture [59]. Reduced sebum secretion due to impaired sebaceous gland function also may contribute to reduced SC hydration in patients with diabetes. Sebaceous glands bind insulin, but the level of binding decreases in mice with diabetes [60]. As noted earlier, rats with STZ-induced diabetes had lower sebum secretion [61]. Hence, insulin may be required for the homeostasis of the sebaceous gland; impaired insulin secretion may contribute to skin changes observed in diabetes. Future studies should be geared for assessing the potential role of natural moisturizing factors other than
. Table 63.2 Comparison of TEWL (mg/cm2/h) in patients with low and high levels of HbA1C g TEWL
(mg/cm2
/h)
HbA1C < 5.8% (n = 11)
HbA1C > 5.8% (n = 38)
p value
Volar forearm
5.8 1.4
3.6 0.3
0.017*
Extensor lower leg
3.9 0.8
3.6 0.4
0.702
Values are means SEM *Significant (From [29])
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SC amino acids and for characterizing the epidermal chemiosmotic changes.
The Influence of Aging on the Hydration State and Barrier Function of the Skin in Patients with Diabetes To examine the effect of aging on properties of the SC in above mentioned patients with diabetes, the HFC, TEWL, and skin surface lipids of patients above and below the age of 45 were evaluated. In older patients, HFC was significantly lower only on the extensor surface of the leg; however, TEWL was significantly lower at all of the measured locations (> Table 63.3). A trend to reduced skin surface lipid content on the forehead was also noted in older patients with diabetes. These findings suggest that the age-related changes in the SC properties are promoted by diabetes mellitus. In other words, a diabetic condition might enhance the aging of the SC function.
Conclusion The problem of xerotic skin is a significant issue for patients with diabetes mellitus, as it is for people with atopic dermatitis, psoriasis, ichthyosis vulgaris, and for most elderly people. Studies in hairless mice with experimentally induced diabetes and in patients with diabetes have shed light on some of the mechanisms that contribute to xerotic skin. Diabetes appears to induce functional changes in the skin similar to those that occur in older
. Table 63.3 Comparison of the functional properties of the stratum corneum between younger and older patients with diabetes
Age
Age group < 45 years (n = 23)
Age group > 45 years (n = 26)
19.3 1.7
68.2 1.5
p value 0*
161.7 17.1
148.8 11.9
0.532
HbA1C (%)
7.13 0.35
6.58 0.15
0.141
Skin surface lipid on forehead (a.u.)
64.7 9.6
40.8 9.9
0.092
Volar forearm
5.4 0.7
5.0 0.5
0.001*
Extensor lower leg
3.0 0.2
2.5 0.3
0.0001*
Volar forearm
54.7 4.4
60.0 6.4
0.511
Extensor lower leg
42.3 4.1
29.5 3.5
0.020*
FPG (mg/dL)
2
TEWL (mg/cm /h)
HFC (mS)
Means SEM *Significant (p < 0.025) (From [29])
659
660
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Dry Skin in Diabetes Mellitus and in Experimental Models of Diabetes
people. Indeed, the data strongly suggest that hyperglycemic conditions may promote epidermal aging. Declining skin hydration has clinical consequences for patients with diabetes [1–3]. For example, plantar xerosis in patients with diabetes is linked to the development of recalcitrant ulcers. Care of the feet in the diabetic patient should include moisturizing the skin of the plantar surface [62]. More research is warranted to develop better skin care approaches for patients with diabetes. Research into the moisturizing mechanisms of substances such sebum, lactate, urea, and other potential natural moisturizing factors may yield valuable insights for skin care in these patients. Moreover, avoiding lifestyle choices that contribute to adult onset diabetes will not only promote health and well-being, but could also be viewed as an anti-aging measure to maintain youthful skin.
Cross-references > Influence
of Race, Gender, Age, and Diabetes on the Skin Circulation
Acknowledgments The authors would like to thank Dr. K. Kikuchi and Dr. J. Sato of Tohoku University School of Medicine, and Y. Endo, T. Sugawara, and Dr. S. Inoue of Kanebo Cosmetics Inc. for their kind assistance and helpful technical discussions.
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46. Nakagawa N, Sakai S, Matsumoto M, et al. Relationship between NMF (lactate and potassium) content and the physical properties of the stratum corneum in healthy subjects. J Invest Dermatol. 2004;122:755–763. 47. Sugiyama Y, Ota Y, Hara M, et al. Osmotic stress up-regulates aquaporin-3 gene expression in cultured human keratinocytes. Biochim Biophys Acta. 2001;1522:82–88. 48. Ma T, Hara M, Sougrat R, et al. Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3. J Biol Chem. 2002;277:17147–17153. 49. Hara-Chikuma M, Verkman AS. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption. Mol Cell Biol. 2008;28:326–332. 50. Corcuff P, Leveque JL. Size and shape of corneocytes at various body site: influence of age. In: Leveque J-L, Agache PG (eds) Aging Skin. New York: Marcel Dekker, 1993, pp. 199–216. 51. Watanabe M, Tagami H, Horii I, et al. Functional analyses of the superficial stratum corneum in atopic xerosis. Arch Dermatol. 1991;127:1689–1692. 52. Candi E, Melino G, Mei G, et al. Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein. J Biol Chem. 1995; 270:26382–26390. 53. Wertheimer E, Spravchikov N, Trebicz M, et al. The regulation of skin proliferation and differentiation in the IR null mouse: implications for skin complications of diabetes. Endocrinology. 2001; 142:1234–1241. 54. Sadagurski M, Nofech-Mozes S, Weingarten G, et al. Insulin receptor substrate 1 (IRS-1) plays a unique role in normal epidermal physiology. J Cell Physiol. 2007;213:519–527. 55. Anand P, Terenghi G, Warner G, et al. The role of endogenous nerve growth factor in human diabetic neuropathy. Nat Med. 1996; 2:703–707. 56. Terenghi G, Mann D, Kopelman PG, et al. trkA and trkC expression is increased in human diabetic skin. Neurosci Lett. 1997;228:33–36. 57. Sato J, Denda M, Nakanishi J, et al. Dry condition affects desquamation of stratum corneum in vivo. J Dermatol Sci. 1998;18:163–169. 58. Sakai S, Sasai S, Endo Y, et al. Characterization of the physical properties of the stratum corneum by a new tactile sensor. Skin Res Technol. 2000;6:128–134. 59. Sato J, Yanai M, Hirao T, et al. Water content and thickness of the stratum corneum contribute to skin surface morphology. Arch Dermatol Res. 2000;292:412–417. 60. Jo N, Watanabe M, Kiyokane K, et al. In vivo microradioautographic study of insulin binding in the skin of normal and NIDDM mice: with special reference to acanthosis nigricans. Cell Mol Biol (Noisyle-grand). 1997;43:157–164. 61. Toh YC. Effect of streptozotocin-induced diabetes on the activity of the sebaceous glands in rats. Endokrinologie. 1982;80:56–59. 62. Pham HT, Exelbert L, Segal-Owens AC, et al. A prospective, randomized, controlled double-blind study of a moisturizer for xerosis of the feet in patients with diabetes. Ostomy Wound Manage. 2002;48:30–36.
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61 Influence of Race, Gender, Age, and Diabetes on the Skin Circulation Jerrold Scott Petrofsky . Gurinder Singh Bains
Introduction The circulation to the skin is important both for the skin’s nutrition to maintain it as a live barrier to protect the inner organs and also to support its role in thermoregulation. Because of this latter role, as much as 99% of skin circulation may be for thermoregulatory purposes. Thus, the skin has a complex control system allowing it to respond to local stimuli such as pressure and heat, and also to central sympathetic control to dissipate or save heat and to maintain blood pressure with changes in body position. This chapter will explore how the skin circulation is controlled by local and central mechanisms and how this is altered by age, race, sex hormones, and pathologies such as diabetes.
Normal Skin Circulation When people are exposed to a thermally neutral environment, skin blood flow averages about 5% of their cardiac output. However, during whole body heating, blood flow through the skin can increase to as much as 160% of cardiac output at rest or about 8 L/min [1]. In glabrous (non hairy) skin (e.g. palms, plantar aspects of the feet, and lips), cutaneous arterioles are innervated only by sympathetic adrenergic vasoconstrictor nerves [2]. Blood flow is also affected in this same skin type by local metabolites, as well as effectors such as temperature and pressure on the skin [3, 4]. In hairy (non glabrous) skin, which is present over most of the body, three separate branches of the sympathetic nervous system control skin blood flow: adrenergic vasoconstrictor nerves that reduce (constrict) skin blood vessels, and cholinergic and nitrogenic nerves that cause vasodilation of blood vessels by releasing the neurotransmitters, acetylcholine or nitric oxide, respectively [5]. In addition, as is the case for glabrous skin, local effectors, such as metabolites and changes in local skin temperature or pressure, may mediate a change in skin blood flow. Thus, in general, the control of the circulation in the skin can be divided into
two types: (1) the local response of vascular endothelial cells to metabolites and other processes (such as local pressure or shear stress on the blood vessel wall) and (2) neurogenic control through the sympathetic nervous system. Both sympathetic synapses and local effectors mediate their effects through the thin layer of cells lining the blood vessels, namely, the vascular endothelial cells. Studies conducted in the last 50 years, with drugs that specifically block vasoconstriction (e.g. bretylium tosylate) and agents that inhibit vasodilatation by blocking acetylcholine (cholinergic antagonistic agents), have been used to confirm chemical mediators at neuronal synapses [6]. Sympathetic vasodilator and vasoconstrictor nerves innervate the blood vessels by extensive terminal varicosities located on the surface of the vascular endothelial cells. The vascular endothelial cell, in turn, releases fatsoluble substances that cause the vascular smooth muscle surrounding it to relax or constrict, thereby mediating a change in the skin blood flow. Stripping the inner layer of large conduit arteries (i.e. removing the endothelial layer) eliminates vasodilation and vasoconstriction in vascular smooth muscle.
Neuronal-Controlled Vasodilatation Acetylcholine Acetylcholine, a neurotransmitter that mediates vasodilatation in the skin, is released from the terminal varicosities of the sympathetic cholinergic neurons and diffuses onto specific acetylcholine receptors on the vascular endothelial cells. Therefore, the application of atropine to the skin largely abolishes vasodilatation mediated by the sympathetic nervous system [7]. However, some sympathetic nerves must release co-transmitters in addition to acetylcholine, because a degree of active vasodilator response mediated by sympathetic nerves exists even after atropine is administered. Some studies suggest that a peptide, Vasoactive Intestinal Peptide (VIP), is involved in vasodilatation. For example, when VIP receptors in the skin are
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_61, # Springer-Verlag Berlin Heidelberg 2010
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blocked, some of the active vasodilatation is also blocked [8]. However, other studies raise doubt as to VIP’s true role in active vasodilatation in the skin [9].
Nitric Oxide A variety of different stressors can elicit an increase or decrease in skin blood flow in hairy skin. The blood flow response is controlled through a range of different mechanisms. In the 1990s, a number of laboratories demonstrated an active role for nitric oxide as a mediator of vasodilatation in the skin [10–12]. Historically, it had been postulated that a substance released from vascular endothelial cells caused vascular smooth muscle to relax [13]. This substance, originally called endothelial cell-derived relaxation factor, is now known to be several different compounds, one of which is a fat-soluble chemical, nitric oxide [14]. Several lines of evidence indicate that nitric oxide is produced by endothelial cells in both humans and animal models and over a variety of species. Nitric oxide is produced from the amino acid L-Arginine by the enzyme endothelial nitric oxide synthetase (> Fig. 61.1). When LNAME (N-nitro-L-Arginine methyl esther), an inhibitor of nitric oxide synthetase, is infused into the skin via micro dialysis in both animals and humans, the increase in blood flow due to stressors such as heat is significantly
. Figure 61.1 Synthesis of nitric oxide
attenuated, although not completely blocked [7]. During whole body heating, the bioavailability of nitric oxide increases in proportion to skin blood flow. However, nitric oxide may be generated from sources in addition to endothelial nitric oxide synthetase. For example, evidence exists that H1 histamine receptors on vascular endothelial cells generate nitric oxide during cutaneous active vasodilation [15]. It has also been suggested that the release of histamine from mast cells induced by VIP could also be involved, because histamine increases the bioavailability of nitric oxide in the skin [15, 16]. Nitric oxide, once produced, diffuses both into the blood and into the surrounding vascular smooth muscle. In smooth muscle, nitric oxide activates the soluble enzyme in the cytoplasm, guanylate cyclase, which catalyzes the production of cyclic guanosine monophosphate (cyclic GMP) (> Fig. 61.2). Cyclic GMP has several biological actions which include decreasing calcium permeability, inhibiting actomyosin ATPase activity, and increasing potassium permeability in vascular smooth muscle. These three functions, taken together, have the combined effect of relaxing vascular smooth muscle. Nitric oxide also has other effects on the endothelial cells and its environment. These autocrine and paracrine effects include those of nitric oxide, which is a potent anti-inflammatory agent on blood vessel walls, inhibiting leukocyte adhesion [17], platelet adhesion, and smooth
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. Figure 61.2 The effect of nitric oxide on smooth muscle
muscle cell proliferation [18], promoting insulin release [19], and mediating the immune response to inflammation [20]. Nitric oxide is also involved in physiologic functions outside the vascular endothelial cells. These include neuronal transmission [21, 22], pulmonary vascular remodeling [23], arterial sclerosis [24], and exercise-induced cardiac protection [25]. Impaired production or bioavailability of nitric oxide leads to endothelial dysfunction, and is the root cause of much different cardiovascular pathology including diabetes, hypertension, heart failure, and coronary artery disease [14]. Nitric oxide is derived from the bioconversion of the amino acid, L-arginine, to the amino acid, L-citrulline (> Fig. 61.1). Like all amino acids, L-arginine and L-citrulline are nitrogen-bearing compounds. The L-arginine molecule has four nitrogens: when bioconverted to L-citrulline, it loses an atom of nitrogen and of oxygen to form nitric oxide, and yields another amino acid with three nitrogens, L-citrulline. A family of enzymes called nitric oxide synthetases (NOS) produce nitric oxide in various organ systems. The enzymes include neuronal NOS, inducible nitric oxide synthetase (INOS), and endothelial nitric oxide synthetase (ENOS) [26]. ENOS is the predominant form of NOS in the vasculature [27]. There are three subunits in ENOS, a central calmodulin-binding subunit, an
oxidative end, and a reductase end. In vascular endothelial cells, ENOS is normally inactive. It is activated through a complex sequence of chemical reactions that involve the binding of nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide, and flavin adenosine dinucleotide [27]. Mediated by flavin, electrons are transferred from the carboxylate (COOH) terminal bound to NADPH to the heme of the NH2 terminus. These electrons activate oxygen. L-arginine is reduced to L-citrulline in two phases. In the first phase, L-arginine binds to ENOS. In the second phase, it is oxidized to L-citrulline and releases nitric oxide [26]. Intracellular calcium modulates the activity of ENOS through the calcium-binding subunit [28]. Intracellular calcium is mobilized through various signaling pathways and ENOS is ultimately activated by phosphorylation at one of the six phosphorylation sites [28]. Calciumactivated calmodulin increases the rate of transfer of electrons from NADPH to ENOS (> Fig. 61.3). (The complex system of reactions, used to increase calcium mobility from the extracellular to intracellular space, is discussed in another section of this chapter.) Other substances, such as proteins and free fatty acids can also modulate the activation of ENOS [28, 29]. Phosphorylation of ENOS via protein kinases is a critical step in its activation [30]. To date, six phosphorylation sites have been identified, including serine 1,177,
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Influence of Race, Gender, Age, and Diabetes on the Skin Circulation
. Figure 61.3 Reaction and cofactors in Endothelial Nitric Oxide Synthetase (ENOS)
stress. The result is impaired vasodilatation [39]. This raises cardiac work and blood pressure.
Other Vasodilators
threonine 495, protein kinase b (pkb-akt) 939, adenosine monophosphate-activated kinase, protein kinase A, and protein kinase G [31]. Multiple signaling pathways are associated with different processes in vascular endothelial cells for the activation pathways for ENOS [14, 31]. For example, the ENOS cascade can be activated by receptors for estrogen and glucocorticoids [32], insulin and Vascular Endothelial Growth Factor (VEG-F) [33], blood flow, and laminar shear stress [34, 35]. The balance between nitric oxide production and degradation determines the bioavailability of nitric oxide. When nitric oxide bioavailability is reduced, heightened vasoconstriction occurs, as seen in hypertension, cardiovascular disease, and diabetes [36, 37]. Impairments of ENOS activity can affect nitric oxide production. For example, instability in ENOS may liberate oxygen instead of nitric oxide [33]. Decreased bioavailability of L-arginine may cause ENOS to reduce production of nitric oxide in vivo [38]. Oxidative stress also reduces nitric oxide bioavailability through degradation. Oxides and superoxides degrade nitric oxide, yielding molecules that bear three oxygen atoms, such as peroxynitrate [39]. These reactive oxygen species can in turn cause cellular damage. Oxidative stress potentially can be moderated via activating NADPH oxidases or xanthine oxidases in the vascular wall [40, 41]. Diabetes and cigarette smoking may not alter nitric oxide production via NOS enzymatic pathways, yet still reduce nitric oxide bioavailability by increasing oxidative
Prostacyclin (PGI2), a prostaglandin, is another vasodilator released by vascular endothelial cells [42]. In younger people, vasodilation is mediated through the release of both nitric oxide and prostacyclin [42]. However, as people age, prostacyclin production is impaired and nitric oxide becomes the predominant vasodilator [43]. One study of younger subjects revealed that at least 60% of acetylcholine-mediated vasodilatation was preserved after inhibition of both ENOS and Cyclooxyenase (COX) [42]. Although this shows the importance of nitric oxide and prostacyclin in regulating cutaneous circulation, it points to other substances released by vascular endothelial cells, especially in younger individuals, that also mediate an increase in skin blood flow [42, 44]. For example, in studies of chronic inflammation of the skin, neuropeptides such as Substance P can be released from the sympathetic nerve terminals [45]. Substance P binds to the endothelial cell on the NK-1 receptor [45]. In rats, administration of the NK-1 receptor antagonist, CP96–345, significantly reduced the blood flow increase which occurred during sympathetic nerve stimulation [45]. Thus, Substance P may be responsible for the vasodilation seen with inflammation in rats [46] and in response to electrical stimulation of the sympathetic nerves [47] and other conditions such as electrical stimulation of the lumbar sympathetic trunk [45]. Substance P is normally expressed in small dorsal root ganglion neurons and in the skin and is upregulated in inflammatory conditions [48]. Nerve growth factors from inflamed tissue play a role in upregulating production of Substance P [48]. Because sympathetic post ganglionic neurons are affected by nerve growth factors from chronic inflammation, it has been hypothesized that upregulation of Substance P alters the normal sympathetic combination of neurotransmitters released by the sympathetic nerves [45].
Neuronal-Controlled Vasoconstriction Norepinephrine and one or more co-transmitters mediate sympathetic vasoconstriction [49]. Postsynaptic alpha-2adrenergic receptors on vascular endothelial cells have a high affinity for norepinephrine. Once these receptors bind norepinephrine, vascular endothelial cells release prostaglandin H2, a fat-soluble prostaglandin; this
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation
prostaglandin increases the permeability of the surrounding vascular smooth muscle cells to calcium and sodium, causing vasoconstriction. Other neuropeptides, such as neuropeptide Yand ATP are co-located in the pre synaptic storage vesicles with norepinephrine in the nerve terminals. In animal models, these neuropeptides have been shown to participate in altering the speed of response to noradrenergic vasoconstriction [50, 51]. Vascular endothelial cells in the skin have both alpha-1 and alpha-2 adrenergic receptors. However, some beta adrenergic receptors are also found in the skin vasculature (vasoconstriction) to sympathetic vasoconstrictor stimuli. A complete blockade of alpha-1, alpha-2, and beta adrenergic receptors fails to totally abolish cutaneous vasoconstriction induced by hypothermia. This shows that other substances, such as neuropeptides, are also involved in the vasoconstrictor response. For example, when both alpha and beta receptors in forearm skin of young subjects were blocked, the maximal vasoconstriction at this site induced by whole body cooling was attenuated by only 40% [52]. However, in subjects over the age of 61 years, blocking these receptors eliminated vasoconstriction completely. Thus, as was the case for vasodilation, the number of neurotransmitters and receptors involved in the process of vasoconstriction decreases with age. In other words, the contribution of co-transmitters to sympathetic vasocontriction by norepinephrine decreases with age.
Local Effectors of Skin Blood Flow Anoxia The smooth muscle response of blood vessels to local pressure, heat, and anoxia is also mediated by the vascular endothelial cells. For example, cutaneous reactive hyperemia occurs when the circulation in the skin is occluded for even a short period of time [53]. The pattern of the transient rise of skin blood flow and the exponential decrease back to baseline blood flow is called reactive hyperemia. The primary cause is believed to be myogenic relaxation of blood vessels; however, local mediators produced by the ischemic tissues may also play a significant role. One study implicated sensory nerves in this hyperemic response. In healthy adults, blocking sensory nerves with local anesthesia reduced the local reactive hyperemic response of the skin microcirculation. This raises the possibility of an axon–axon reflex. Mediators of this local response are nitric oxide, prostacyclin, and endothelium-derived hypopolarizing factors
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(EDHF). After ischemia, there is an increase in calcium permeability in vascular endothelial cells through a type of voltage-gated calcium channel called a TRPV-4 channel in the endothelial cell membrane. Both sensory nerves and TRPV (Transient receptor potential voltage gated) calcium channels play major roles in the EDHF component of the reactive hyperemia; these contributors to reactive hyperemia work partly independently of nitric oxide and prostaglandin-mediated pathways.
Local Heat Endothelial cells also mediate the blood flow response of the skin to temperature applied directly to the skin (local heat). When skin temperature rises, cutaneous blood vessels dilate. Maximal skin blood flow is reached when skin temperature is about 42 C sustained for 30 min. Local vasodilation is a biphasic response, with an initial rapid vasodilation followed by a sustained blood flow response. The biphasic mechanism of local cutaneous vasodilation involves both neuromechanisms (reflexes in axons) as well as generation of nitric oxide. The two mechanisms are independent of each other. When heat is first applied to the skin, sensory nerves respond with a local reflex which does not involve the spinal cord. Mediated by TRPV-1 sensory receptors, this response causes the release of Substance P from the nerve endings through a local axon axon reflex. Substance P causes relaxation in vascular smooth muscle. However, this phase lasts only a few seconds. The sustained response to local temperature is mediated by nitric oxide and is mediated through TRPV-4 calcium channels. This continuous or plateau phase is abolished by the nitric oxide inhibitor, LNAME. Because activation of the enzyme, NOS, involves heat shock proteins, heat shock protein inhibitors (HSP 90 inhibitors) reduce the magnitude of sustained increase in skin blood flow produced by local heat.
Shear Stress Shear receptors are present on the surface of the vascular endothelial cells sensing shear in blood vessels. These shear receptors activate the enzyme, NOS, through a prostaglandin intermediate. The prostaglandin released by shear receptors activates the transient receptor potential vanilloid (TRPV-4) voltage-gated calcium channels which then increase the calcium permeability of the cell membrane. These are the same TRPV-4 channels that are involved in the prolonged response to elevated skin temperature
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described earlier. In both the cases, the influx of calcium through TRPV-4 channels activates the enzyme NOS. Thus, voltage-gated TRPV-4 calcium channels are involved in a number of different processes to activate ENOS.
Vertical Pressure Conduit blood vessels as well as blood vessels in the microcirculation are susceptible to local vertical pressures and well as shear forces. Shear receptors detect increases in blood flow downstream; this allows the vascular resistance in these larger blood vessels to be reduced when blood flow rises. Other receptors in blood vessels sense vertical pressure. Vertical pressure, such as standing on the feet or applied pressure against the skin is sensed by vascular endothelial cells through TRPV-4 voltage-gated calcium channels [54–56]. Consequently, applying light pressure to the skin ( Fig. 61.5). Clinical evidence strongly suggests that estrogen plays a role in both angiogenesis and in modulating tissue blood flow. Endothelial progenitor cells or EPCs, are generally derived from bone marrow and are mobilized in response to tissue damage and/or cytokines in blood. In adults, EPCs isolated from peripheral blood create foci for neovascularization; this effect varies during the menstrual cycle [75].
Mature vascular endothelial cells in humans and a variety of species express at least two different estrogen receptors: ER-alpha and ER-beta [76]. Different genes encode ER-alpha and ER-beta. The receptors act as ligand-dependant transcription factors [77]. Human EPCs express ER-alpha but not ER-beta [78, 79]. In humans, the activity of ER-alpha receptors mediates the response of blood vessel walls to estrogen; this response includes accelerated re-endothelialization and elevated release of endothelial nitric oxide [80]. Consequently, the activity of ER-alpha contributes to gender differences in the protection from cardiovascular disease [81]. The signaling pathway by which estrogen affects levels of EPCs is not known [82, 83]. Several studies indicate that the activation of phosphoinositide 3-kinase (PI3k/akt) pathways may be involved [84, 85]. If PI3k/akt is activated by estrogen, this would in turn activate NOS [86]. Estrogen has direct effects on vascular genesis and also angiogenesis mediated by VEG-F [86]. Vascular genesis is the formation of new blood vessels in the body and is mediated by stem cells in bone marrow. Angiogenesis differs in that it involves the extension of existing blood vessels and is under the control of a compound, vascular endothelial growth factor (VEGF) released by endothelial cells. Estrogen has been
. Figure 61.5 This figure shows the limb blood flow in cc per minute per 100 mL tissue measured every other day of the menstrual cycle at the end of fatiguing isometric contractions at 20% (squares), 40% (circles), and 60% (triangles) of the subject’s strength on eight subjects (Reproduced from Petrofsky J, Al Malty A, et al. [69].)
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation
associated with wound healing in some experimental models. In animal models, male organs such as the penis have been shown to have estrogen receptors [87– 89] and estrogen will increase the rate of healing of a wound [90]. Part of the healing process is increased reactivity of VEGF induced by estrogen [91].
Role of Estrogen in Pulmonary Hypertension Studies on isolated pulmonary arteries show that estrogen upregulates nitric oxide production [92]. Both endogenous and exogenous estrogens decrease pulmonary arterial vasoconstriction under normoxic and hypoxic conditions [92, 93]. This is not surprising, because acute and chronic hypoxic pulmonary hypertension (as well as systemic hypertension) is less common and less pronounced in females than males [92, 94, 95]. However, the response to estrogen may be dosedependent. Studies on the effect of sex hormones on blood flow have produced conflicting results. For example, cutaneous vasodilatation to local warming increased in young users of oral contraceptives [5, 96, 97]. In men, testosterone may inhibit, rather than stimulate, nitric oxide-dependent vasodilatation [98]. When small doses of estrogen (a dose lower than those used in a birth control pill) were administered together with testosterone to group of subjects, the degree of vasodilation caused by local warming of the skin did not change relative to the response of untreated subjects [99]. Conversely, in young men and women, testosterone reduced the blood flow response to local heat, whereas estrogen increased the blood flow response to local heat if given in physiological concentrations [5]. However, this result was not reproduced when older people were studied [99]. It is well known that aging causes a decrease in the cutaneous vasodilator response [100]. Perhaps, aging alters the activity of estrogen receptors in vascular endothelial cells as well, although diminished production or bioavailability of nitric oxide may play a role [100]. One hypothesis is that the diminished response to estrogen and testosterone [99] in older individuals could be the result of defective nitric oxide transduction by estrogen in the aged. In people with diabetes, the nitric oxide pathway in vascular endothelial cells is defective. Not surprisingly, estrogen has little or no effect on endothelial dysfunction in post menopausal women with diabetes. Aging and diabetes both affect signalling pathways mediated by ER-alpha and ER-beta receptors on vascular endothelial cells. Hence, estrogen exerts its greatest effects on blood flow in younger women.
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The Influence of Diabetes on Skin Circulation Both Type-1 diabetes (sometimes called juvenile diabetes) and Type-2 diabetes (sometimes called metabolic syndrome) have similar effects on the autonomic nervous system. Type-1 diabetes is an autoimmune disease. Autonomic dysfunction is due to high glucose concentrations in blood plasma [101, 102]. Type-2 diabetes causes damage to endothelial cells due to high plasma glucose, oxidative and inflammatory stressors, and insulin resistance [53, 103, 104]. In Type-2 diabetes, hyperinsulinemia is common in the early disease, because the pancreas must overproduce insulin to compensate for high cellular insulin resistance. The resistance stems not from a defect in the insulin receptor itself, but from impaired signal transduction from the receptor in activating phosphatidylinositol 3-kinase. In Type-2 diabetes, insulin levels usually rise to such a degree that the pancreas is unable to sustain production and the pancreatic beta cells finally fail and insulin production decreases or stops completely over a number of years [53, 105]. Activation of phosphatidylinositol 3-kinase is the critical step in cellular activation of the glucose transporter, glut-4. The inability of the cell to transport glucose into the cytosol shifts cellular metabolism from carbohydrates to lipids [103]. Chronic lipid metabolism damages the cell due to oxidative biproducts that cause chronic cellular inflammation [106]. It was once thought that the best predictor of damage to the autonomic nervous system and blood vessels associated with diabetes was the average body burden of glucose over many months (as assessed by a measure called HbA1c). Recently, it has been shown that spikes in blood glucose concentration over the course of the day (especially post prandial) are more damaging to endothelial cells than the average concentration of glucose in the blood itself [107]. Large spikes in glucose cause immediate damage to the autonomic nervous system forcing a type of shock such that the autonomic function is impaired for over 24 h [107]. Because vascular endothelial cells are exquisitely sensitive to high glycemic concentrations, damage to these cells usually occur before Type-1 or Type-2 diabetes is clinically diagnosed [103, 108, 109]. Thus, at the time of diagnosis, young children and adults with diabetes already have autonomic damage and impaired blood flow responses to stress [103]. Damage to blood vessels of the skin is two-fold. First, in Type-2 diabetes, the sympathetic ganglia are damaged [110], although the ability of the blood vessels to constrict is unimpaired. Second, in both
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Type-1 and Type-2 diabetes, vasodilation is impaired through direct damage to endothelial cells [53, 103, 111]. Clinically, damage to the parasympathetic and sympathetic nervous systems centrally or at the ganglia is quantified by changes in the heart rate variability [103, 112]. Normally, vasomotor rhythm in the sympathetic and parasympathetic system causes the heart rate to vary continuously on a breath-by-breath and minuteby-minute basis [103]. Heart rate changes are evident onanalysis of the EKG. In diabetes, progressive damage to the sympathetic and parasympathetic ganglia diminishes the heart rate variability to such a degree that eventually little variation in heart rate occurs with a change in body position, breathing, or with exercise [103]. The loss of the skin’s ability to adequately dilate limits the response to global heat and other physiological stressors such as emotions [53]. The most damage caused by diabetes occurs directly on endothelial cells. Several pathways in endothelial cells are damaged by diabetes. First, nitric oxide is less bioavailable. In Type-2 diabetes, some studies indicate that high glycemic levels damage either the enzyme, NOS, or the TRPV-4 calcium channels [60, 104]. The result is that less nitric oxide is produced by vascular endothelial cells in response to a given vasodilator stimulus. Other studies show that diabetes reduces the bioavailability of arginine, thereby altering the ability of the endothelial cells to produce nitric oxide. Some studies point to a third mechanism in which the high free radical concentration in the body associated with both obesity and diabetes bioconvert nitric oxide into peroxynitrate, thereby reducing the bioavailability of nitric oxide as a vasodilator [103, 113]. Probably, all the three mechanisms are present to various extents in different populations of patients with diabetes. The combined effects of reduced nitric oxide bioavailability are to reduce active vasodilatation in blood vessels. Nitric oxide release can be elicited by vasodilation due to acetylcholine from the sympathetic nervous system or ENOS can be activated by stimuli like local heat, a change in local pressure or tissue osmolarity, or shear stress in the blood vessel. The response of endothelial cells to all of these stimuli is reduced in diabetes [113]. The impairment in blood flow in response to these stimuli, however, is greater than that expected by a loss in vasodilation alone. This may be due to additional diabetes-related impairments (> Fig. 61.6). Insulin is a vasodilator. In non diabetic subjects, insulin reduces inflammation and causes vasodilation as well as enhancing glucose transport. Insulin accomplishes this by activating the phosphotidylinositol 3-kinase (PI3K)/AKT pathway, activating both ENOS and glut 4 glucose
. Figure 61.6 Illustrated here is the forearm blood flow in age-matched controls (upper curve) and subjects with diabetes (lower curve) after 4 min of vascular occlusion of the arm
transport. However, insulin also activates a mitogen-activated kinase pathway (MAPK). This pathway causes vasoconstriction of the vascular smooth muscle. Normally, vasodilation overwhelms constriction and blood vessels dilate in response to insulin. But in diabetes, the MAPK pathway overwhelms the PI3K pathway and insulin, which is present in high plasma concentrations in diabetes, becomes a vasoconstrictor stimulus, further reducing blood the flow to tissue and causing additional microvascular damage. Microvascular damage to the kidneys in diabetes causes the release of renin, a renal enzyme which is involved in the regulation of sodium in the blood. Renin converts blood angiotensinogen to Angiotensin I. In most tissues in the body, angiotensin converting enzyme, converts Angiotensin I to Angiotensin II. Angiotensin II is a potent vasoconstrictor and overwhelms endothelial vasodilation as well as is pro inflammatory. It also impairs insulin signaling. Morphological changes in the endothelial cell smooth muscle interfaces are also observed in diabetes [113]. Normally, small cellular attachments (electrotonic connections) exist between endothelial cells and vascular smooth muscle. Thus, in addition to vasodilators and vasoconstrictors affecting the surrounding smooth muscle in blood vessels, there is direct electrical contact. When endothelial cells depolarize or hyperpolarize, the electrotonic connection through these gap junctions helps
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation
coordinate electrical activity between endothelial cells and the surrounding vascular smooth muscle. When vasodilator effectors bind to the endothelial membrane, the endothelial cell hyperpolarizes through an increase in potassium permeability. This increase in potassium permeability hyperpolarizes the vascular smooth muscle, making it harder for action potentials to develop and thus aiding in the process of vasodilatation [114, 115]. In people with diabetes, these electrotonic connections are destroyed; consequently, some of the ability of the endothelial cell to relax vascular smooth muscle is lost [116]. The overall effect of all of these factors is that, due to a predominant vasoconstriction, resting blood flow in the skin is reduced (by almost 66%) in people with diabetes. Consequently, blood flow responses to global and local heat are reduced; therapeutic modalities such as contrast baths, which normally cause a large increase in blood flow in the skin, are ineffective in people with diabetes [53, 104, 117]. As shown in > Fig. 61.7a, b, while the control subjects show a much greater increase in skin blood flow to a contrast bath than a constant warm bath, the response is lost in people with diabetes. The blood flow response to stressors such as electrical stimulation, when used clinically for therapy or wound healing is also diminished [118, 119]. As modulation of the skin circulation depends to a greater degree on nitric oxide in older individuals, as people with diabetes age, blood flow responses in the skin worsens disproportionately.
Lifestyle, Race, and Endothelial Function Recently, several studies have shown that lifestyle factors such as obesity [120, 121], cigarette smoking [122], as well as race [14] are associated with changes in the vascular endothelial cells connected to cardiovascular disease.
Lifestyle Adipose tissue expresses a variety of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) [120], that increase the production of C Reactive Protein (CRP) [123]. These cytokine-mediated inflammatory processes are involved in early stages of atherogenesis [124, 125], and damage occurs to endothelial and other cells in the body [126]. IL-6 and TNF-alpha cause the release of endothelial adhesion molecules and impair insulin action by interfering with the insulin signaling cascade [127]. CRP, elevated in response to
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inflammatory cytokines, impairs endothelium-dependent vasodilatation by interfering with either ENOS or endothelial nitric oxide bioavailability [128]. Endothelial damage, especially in capillaries, precedes serious cardiovascular disease [129, 130]. Vascular endothelial damage can be measured by biomarkers such as E-selectin and soluble vascular adhesion molecule (SVCAM-1) [129, 131]. Environmental factors, such as smoking, also damage vascular endothelial cells [132]. Smoking is a major risk factor for atherogenesis and vascular disease. Tobacco smoke contains more than 4,000 chemicals, many of which cause pathological changes in the endothelial cells [133]. Oxygen-free radicals from tobacco smoking increase cellular oxidation and can limit the bioavailability of nitric oxide [133]. These radicals oxidize nitric oxide to chemicals such as peroxynitrate, a superoxide that can further damage the tissue by lipid peroxidation of membranes [132]. Smoking increases the sympathetic vasoconstrictor nerve activity and thus contributes to increased vascular tone arteries. Smoking also increases the concentration of the protein fibrinogen, resulting in prothrombotic effects [134]. Thus, smoking has multiple effects on the health of vascular endothelial cells [135]. Other environmental agents, such as air and water pollutants, may impair endothelial vascular function. Recent studies showed that particulate matter from diesel engines can activate the JNK pathway and damage endothelial function [136]. Cigarette smoke [137] and air pollution, in general [138], exert their effects through the ENOS pathway.
Race The impact of race is complex. Studies on the effect of race on the vasculature, have measured the reactivity to various challenges such as vascular occlusion [14]. This was done by examining the effect of ischemia on blood flow in the vessels in the skin (by using a laser Doppler imaging) or on larger arteries such as the brachial artery, by ultrasound [14]. Tests of vascular function also employ agonists of sympathetic activity (such as acetylcholine or methylcholine) to activate muscarinic receptors on vascular endothelial cells. This increases intracellular calcium, increases ENOS activity, and releases nitric oxide [139, 140]. Bradykinin also activates ENOS [140]. The impact of race on nitric oxide-dependent vasodilatation was recently reviewed [14]. A decrease in nitric oxide-dependent vasodilatation is evident in many populations. Young, healthy African American subjects exhibit a lower vasodilatory response to methylcholine and
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. Figure 61.7 (a) This figure illustrates the blood flow in the skin (Flux) measured over the experimental period in control subjects during immersion in contrast baths (triangles), continuous passive heating (diamonds), and continuous cold immersion (squares). All data are the mean SD (Petrofsky J, Lohman E, 3rd et al. [74]). (b) This figure illustrates the blood flow in the skin (flux) in subjects with diabetes during immersion in contrast baths (triangle), continuous passive heating (diamond), and continuous cold immersions (squares) (Petrofsky J, Lohman E, 3rd et al. [74])
albuterol than Caucasians. Among African Americans, 30% of deaths in men and 20% of deaths in women are caused by hypertension, a finding probably linked to a lower vasodilatory response to stress. The incidence of asthma [141] and Type-2 diabetes [142] is also elevated in African Americans. A higher incidence of Type-2 diabetes also occurs in Asians and Native Americans and these groups exhibit impaired vascular responses to vasodilators relative to Caucasians [143]. However, factors other than racial background play a role in the impairment of vascular response to stress. For example, Europeans of African descent exhibit a higher degree of dysfunction in nitric oxide production in endothelial cells than do African Americans [144]. A few studies have examined the mechanisms that affect endothelial-dependent vasodilatation in African Americans and European Americans is impaired. Some studies suggest that in African Americans, ENOS has a higher activity than in European Americans [145]. However, in African Americans, production of reactive oxygen species is also elevated. Consequently, nitric oxide is more
readily bioconverted to peroxynitrate, which reduces nitric oxide bioavailability in this ethnic group [146]. Other races have gene polymorphisms that affect skin blood flow. Asians, for example, have a genetic polymorphism due to what is called the ‘‘thrifty’’ gene. The gene developed in populations where food supply was limited due to famine. It allows fat to be stored easily when food is available. The thrifty gene controls the production of peroxisome proliferator activated receptor (PPAR), a nuclear subtransmitter that upregulates carbohydrate metabolism in the cell. This single nucleotide polymorphism plays a role in the development of diabetes by increasing insulin resistance when high fat to carbohydrate diets are consumed [147, 148]. The defect in this gene may explain why Asians have a lower tolerance for foods that are high in fat. South Asian men exhibit lower brachial artery dilation in response to occlusion, lower vasodilatory responses to acetylcholine, higher levels of insulin resistance, and higher CRP, an index of endothelial damage when compared with Caucasian men of a similar age with similar anthropometric measurements [149]. Thus, in
Influence of Race, Gender, Age, and Diabetes on the Skin Circulation
Asians, the polymorphism in the thrifty gene alters carbohydrate metabolism such that ingesting even a single high fat meal impairs nitric oxide production and hence, tissue blood flow [150, 151]. Typically, an increase in the plasma concentration of free fatty acids after ingesting a high fat meal induces proinflammatory cytokines [152] and reactive oxygen species within the vascular walls [153]. This in turn activates nuclear factor kappa beta and generates reactive oxygen species [151]. The reactive oxygen species converts nitric oxide to peroxynitrate and other superoxides, reducing the bioavailability of nitric oxide after a high fat meal [151, 152]. In addition, free fatty acids induce protein kinase C, which inhibits PI3K, thereby inhibiting the activity and activation of ENOS, especially in Asians compared with Caucasians [154]. Because of genetic polymorphisms, Asians are more susceptible to vascular endothelial impairment. However, the effect of environmental factors should not be discounted. For example, cardiovascular disease and vascular endothelial dysfunction is increasing among native Japanese with westernized lifestyles and in Japanese men who have moved to the United States [155]. Other minority populations within the United States, such as Pima Indians, also have a higher rate of endothelial dysfunction and a higher incidence of diabetes. Markers of endothelial dysfunction, such as insulin resistance and lowgrade inflammation (elevated CRP and other cytokines), are common in Native Americans such as Pima Indians [156]. Other markers of endothelial dysfunction, such as E-selectin, Von Willebrand factor, and soluble intracellular adhesion molecule-1 (Sicam-1) are elevated in Pima Indians, compared with Caucasians living in similar area. (Interestingly, similar markers of endothelial dysfunction have been reported in Koreans.) [157] However, the genesis of endothelial dysfunction in Native Americans populations is poorly understood [156]. Because smoking is more prevalent among Asians, it is likely that smoking-related free radicals block the release of nitric oxide; moreover, free radicals cause oxidative damage in vascular smooth muscle [122]. Children exposed to passive smoking exhibit elevated oxidative markers [158]. The mechanisms by which oxidative stress damages vascular smooth muscle are unknown [122]. Not all alterations in ENOS have deleterious effects. For example, in Sherpas, modification of nitric oxide metabolism allows greater vasodilatation in systemic and pulmonary arteries during hypoxia than seen for other races [159]. Thus, this population is more able to tolerate high altitude than people of other races due to a specific polymorphism in the gene for ENOS [159].
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A recent study in populations in the north and south of India described a genetic polymorphism in the gene for ENOS on chromosome 7q35 through 36, which was associated with an impaired production of nitric oxide in response to stress [160]. This polymorphism resulted in heightened susceptibility to endothelial dysfunction, hypertension, and diabetes. Several other studies have been conducted on south Indian populations [161, 162] and northern Indian populations with similar results [163].
Aging and Circulation Aging leads to the natural senescence of organ systems, including the kidney [164], the autonomic nervous system [165], and the heart [166]. Although many physiologic changes occur as people age, one important factor contributing to decreased tissue function is diminished production of the potent vasodilator, nitric oxide (a) [167]. The sensitivity of beta adrenergic receptors also diminishes with age [168], which reduces the ability of the sympathetic nervous system to respond to stress. Indeed, damage to the microcirculation is a common denominator for all of the age-related changes in organ function. Aging principally affects three tissues associated with circulatory control. The first is the vascular endothelial cells. The second is sympathetic nervous system, which affects control of the peripheral circulation. The third is the dermis of the skin [169]. The function of endothelial cells diminishes gradually, accelerating in the later years. Disorders such as diabetes accelerate endothelial dysfunction [170]. Because diabetes incidence rises with age, the interaction of age and diabetes becomes important clinically. This will be elaborated in the next section. In general, both the parasympathetic and sympathetic nervous systems are affected by age. Most studies show only small decreases in blood pressure and diminished heart rate variability during orthostatic stress (e.g. change in body position from sitting to standing) as people age [171]. This might indicate that age has a limited effect on the autonomic nervous system. However, if additional stressors, such as heat exposure during orthostatic stress, are placed on the autonomic nervous system, the reduction in blood pressure is much more pronounced in older people during the same orthostatic stress [172]. Although overall autonomic function diminishes with age [173], a decrease in baroreceptor sensitivity also occurs [174], masking sudden changes in body position not properly detected by the autonomic nervous system.
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Specifically, for the vascular endothelial cell and the sympathetic nervous system, there is evidence that exist that age increases vasoconstriction, and reduces vasodilation [175]. Therefore, vasoconstrictor muscle tone predominates; the blood flow at rest and during an autonomic stress is lower [175]. The mechanism by which vasodilatation is lost appears due to several factors. First, nitric oxide release from skin blood vessels diminishes with the age [175]. Second, beta adrenergic receptor sensitivity diminishes with age due to structural changes in the receptor which render it insensitive to catecholamines [168]. This further reduces sympathetic activity. Finally, aging also reduces the sensitivity of the parasympathetic nervous system.
Interaction Between Age and Diabetes Diabetes, like age, is associated with damage to the autonomic nervous system [176]. Damage to autonomic nerves, causing orthostatic intolerance, can occur before clinical symptoms of diabetes are manifest [177]. Damage to the parasympathetic nervous system results in loss of heart rate control, especially during orthostatic stress [178]. The damage usually occurs at the autonomic ganglia and also at the peripheral nerve endings, where microcirculation is critical. When patients with diabetes are subjected to an orthostatic challenge (e.g. going from sitting to standing) in a thermally neutral environment,
25% of diabetic patients show a drop of 20 mm Hg (0.266 kPa) or more in mean blood pressure [179]. However, as autonomic stressors (such as a greater room temperature) are added, the systolic blood pressure falls in almost all patients with diabetes during standing [180]. The mechanism in these patients appears to be an inability to vasodilate [181]. As with aging, in patients with diabetes, the damage appears to be two-fold. First, ganglionic damage impairs the ability of the autonomic nerves to generate impulses; second, the ability to generate adequate amounts of nitric oxide is impaired [182]. These factors affect both arterial and venous circulations [183]. Thus, diabetes seems to cause damage to the autonomic nervous system and to endothelial cell function, similar to that which occurs normally with aging. For this reason, diabetes is thought to accelerate the aging process by causing more severe loss of autonomic function. Neuronal damage can be so pronounced as to cause lesions of the spinal cord [184]. In addition to nerve and circulatory damage, another factor also seems to come into play with both aging and diabetes. This is thickness of the dermal layer of the skin, which decreases with age [169]. In older adults, thinning of the skin is a consequence of a thickening in the stratus corneum and a thinning in the dermal layer. Aging not only reduces skin blood flow but also alters the skin structure, including collagen composition and skin thickness [185]. As the dermal layer is thinner, this implies that
. Figure 61.8 Illustrated here is the relationship between age and the blood flow in the arm at rest with arm temperature stabilized at 37 C. In control subjects (diamonds) and subjects with diabetes (squares), the line through the figure is the regression line calculated by the method of least squares. The regression equation for the control subjects was resting flow= 0.0306 age + 3.5106. The regression equation for the subjects with diabetes was resting flow = 0.011age + 1.4087 (Petrofsky JS et al. [170])
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. Figure 61.9 Illustrated here is the relationship between age and the blood flow in the arm with arm temperature stabilized at 37 C. The blood flow here is the flow after 4 min of occlusion as the area under the blood flow curve for 2 min. In control subjects (diamonds) and subjects with diabetes (squares), the line through the figure is the regression line calculated by the method of least squares. The parallel lines represent the data after 2 weeks (2w), 4 weeks (4w), and 3 months administration of rosiglitazone, a PPAR agonist (Petrofsky JS et al. [170])
the vasculature in the skin in older people is reduced. This is also the case in people with diabetes. Thinner skin would increase susceptibility to injury, and as is the case for people with diabetes, makes the skin harder to heal. For example, as shown in > Fig. 61.8, with both diabetes and age, resting blood flow is less. However, at the same age, diabetes causes a greater reduction in blood flow than age alone. The administration of Rosiglitazone for 6 months did reverse the diabetes’ effect on resting and post occlusion blood flows (> Fig. 61.9), but not the age effects, pointing to different mechanisms for endothelial damage. Moreover, the layer of subcutaneous fat also becomes thinner with age [169, 185, 186]. The foot becomes particularly susceptible to injury, as the reduction in padding over the bone may make the foot more susceptible to lesions during gait. The impact of dermal thinning may be particularly important in people with diabetes. It has been assumed that the reduction in blood flow observed in older people and people with diabetes is solely due to impaired nitric oxide synthesis [60, 187, 188]. However, thinner skin also has a lower density of blood vessels, which may account for some of the reduction in blood flow through the skin. Because people with diabetes have a higher body mass index (BMI) and because BMI correlates with the level of subcutaneous fat, people with diabetes whose weight is normal may have a very thin layer of
subcutaneous fat. A thin person with diabetes would be more susceptible to foot injuries caused by gait.
Conclusion The status of blood vessels in human populations is complex and altered by many factors. Thus, many factors alter the endothelial function.
Cross-references > Dry
Skin in Diabetes Mellitus and in Experimental Models of Diabetes
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57 Melanoma and Skin Aging Salina M. Torres . Marianne Berwick
Introduction The interaction between skin aging and melanoma derive from the contributions of chronologic aging and photoaging, and the subsequent changes that occur in each scenario in the skin. The resultant changes in skin dynamics and their impact on melanoma development and progression are the focus of this chapter. During the past 50 years, the world has seen an increase in the incidence and mortality of cutaneous malignant melanoma (CMM). The American Cancer Society estimates that in 2009, 68,720 Americans will be diagnosed with melanoma, resulting in approximately 8,650 deaths [1]. Several studies have found tumor thickness to be the most important prognostic factor in cutaneous malignant melanoma [2, 3]. Tumor thickness, referred to as Breslow thickness, is the depth of a melanoma lesion measured from the basement membrane of the epidermis to the deepest identified melanoma tumor cell [4]. The 5-year survival rate for patients diagnosed with early melanoma, Breslow thickness of 4 mm, the survival rate is about 40% [5, 6]. In addition to thickness, ulceration and mitotic index are also strong prognostic indicators [7, 8]. Risk factors for melanoma development include age, phenotype, genetic and family history, numbers and types of nevi, and immune system status [9]. Melanoma, like other cancers, is a complex disease: Lachiewicz and colleagues describe it as a heterogeneous cancer with tumors with different biological mechanisms having different survival patterns [10]. Aging is a significant risk factor in the development of skin cancer. The incidence of skin tumors has been found to increase with age; with more than one million new cases each year; basal cell carcinoma, squamous cell carcinoma, and melanoma combined account for almost half of all cancer diagnoses [11]. Over 3 decades (1969–1999), mortality rates from melanoma increased 157% in men aged 65 and older [12]. The continual exponential increase in the incidence of skin cancer during adulthood strongly relates risk to chronologic age; further, basal cell carcinoma, squamous cell carcinoma, and melanoma are strongly associated with photoaging [13]. The age-adjusted
incidence rate of malignant melanoma is increasing faster than any other cancer. Combined with the fact that populations of adults of advanced age continue to increase because life expectancy continues to increase and individuals born in years with high birth rates are growing older, the actual numbers of adults diagnosed with melanoma will continue to be a significant public health concern. Independent of the population structure, it should be noted that some proportion of the increase is due to better methods of, and attention to, early detection. However, as the mortality rate among males continues to increase, early detection is not the entire answer. Although younger Americans are experiencing improved survival and stabilizing incidence rates of melanoma, older individuals continue to encounter increasing melanoma incidence and mortality [14]. Older adults tend to develop different subtypes of melanoma, have reduced access to medical specialists, and have comorbidities that affect their ability to undergo treatment for advanced disease [15]. The age–cancer relationship is dependent on increasing opportunities for cancer to develop from old or aging cells because these cells have had more time to acquire tumorigenic mutations [16]. In the case of melanoma among older individuals, melanocytes have been exposed for decades to ultraviolet (UV) radiation and other environmental exposures, accumulating mutations that have bypassed cellular DNA repair mechanisms.
Epidemiology of Melanoma in Relation to Chronologic Age Several epidemiologic studies have been conducted to evaluate the relationship between age and melanoma. Several of these studies are listed in > Table 57.1. Lachiewicz and colleagues utilized the National Cancer Institute Surveillance, Epidemiology and End Results (SEER) data for analyses of 51,704 non-Hispanic white adults diagnosed with a first invasive CMM between 1992 and 2003 [17]. The objective of their study was to compare anatomic site of melanoma with prognosis. In their analyses,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_57, # Springer-Verlag Berlin Heidelberg 2010
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. Table 57.1 Epidemiologic studies evaluating the relationship between age and melanoma Study population
Age group(s)
Findings
Reference
Florida population of melanoma patients n = 442
Separated into Geriatric patients with melanoma had a 65 or >65 worse prognosis
Austin et al. (1994) [19]
Non-Hispanic white adults diagnosed 1969–1999
20–65+
Fivefold increase in the incidence in men 65 years of age
Geller et al. (2002) [12]
Adults diagnosed 1988–1999 n = 23,068
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Older age independently associated with greater risk of death from melanoma
Reyes-Ortiz et al. (2006) [21]
Cutaneous head and neck melanoma patients diagnosed 1994–2002 n = 2,218
66 (mean age) Increased age had a significantly higher risk Golger et al. of death (2007) [34]
Non-Hispanic white adults diagnosed 2000–2004 n = 48,673
20–80+
Age-specific incidence rate of melanomas increased with age
Lachiewicz et al. (2008) [10]
Non-Hispanic white adults diagnosed 1992–2003 n = 51,704
20–65+
Older age predicted faster rates of melanoma-related death
Lachiewicz et al. (2008) [17]
older age was found to independently predict more aggressive rates of melanoma-related death [18]. Further analyses of age-specific incidence patterns in melanoma cases in non-Hispanic white adults diagnosed between 2000 and 2004 revealed a peak in age-specific incidence rates among patients 70–79 years of age [10]. The incidence rate in this age group was 5.9 times higher than in patients aged 20–29 years. Age-specific incidence rate curves and age distribution curves were plotted for all cases of melanoma as well as by anatomic site. The curve for age-specific incidence rates of all cases of melanoma showed a rapid increase until age 55–59 at which point it continued to rise at a slower rate before an eventual decline; the age distribution plot for all cases of melanoma displayed early- and late-onset peak frequencies at ages 54 and 74 years, respectively [10]. Trunk melanoma agespecific incidence rates increased until age 55–59, then plateaued, and subsequently declined [10]. Similar to the age distribution plot for all cases of melanoma, the age distribution for trunk melanoma demonstrated an earlyonset peak around 44 and 54 years in females and males, respectively. Melanoma on anatomical areas that are continually exposed to UV radiation, the face and ears, exhibited age-specific incidence rates that drastically increased with age and peaked in the age distribution plot at age 78 [10]. These two reports of SEER data analyses reveal an association between age and melanoma mortality and incidence. Lachiewicz and colleagues hypothesized that their findings of a multimodal age distribution of melanoma in their population are indicative of a
‘‘divergent pathway’’ model, originally proposed by Whiteman and colleagues [18]. In this model, people with inherently low propensity for melanocyte proliferation develop melanoma after chronic sun exposure to habitually exposed sites like the face and ears (lateonset); in contrast, people with a high propensity for melanocyte proliferation develop melanoma on anatomical sites with less intermittent solar damage and/or unstable melanocytes like the trunk (early-onset) [18]. The existence of divergent age-specific incidence patterns provide support for the idea that more than one causal pathway exists for melanoma and the existence of distinct melanoma genotypes [10]. Epidemiologic and animal studies have demonstrated that epidermal melanocytes are predominantly initiated and transformed by exposure to sunlight in early life [18]. These findings led Whiteman and colleagues to conclude that melanoma development is dependent upon host phenotype and environmental conditions [18]. The divergent pathway model of melanoma development is important to consider when evaluating the relationship between age and melanoma. Nevus-prone/early-onset individuals are hypothesized to have melanocytes initiated by sunlight followed by proliferation to a neoplasm without additional sun exposure; these tend to occur on the trunk of the body [18]. In the case of nevus-resistant/late-onset individuals, melanocytes from this phenotype are hypothesized to require heavy doses of ongoing sun exposure to develop melanoma; these tend to occur on sun-exposed anatomical sites among older ages. Understanding the
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differing etiologies of melanoma and their relationship to sun exposure at various points throughout the life of an individual is a crucial component for the development of appropriate prevention strategies. Intense UV exposure among older individuals is thought to be responsible for the fast increasing incidence of melanoma over other cancers [19]. Austin and colleagues evaluated the role of age as a prognostic factor for malignant melanoma in a population of 442 melanoma patients residing in Florida. They determined that increasing age was a significant predictor for disease-free survival, with a worse prognosis seen in older melanoma patients. A large population-based survey in Germany was conducted to understand the epidemiology of nevi and signs of skin aging. Nevi are of interest as they are the strongest indicators of melanoma risk [15]. The prevalence of signs of skin aging like dermal elastosis, Cutis rhomboidalis nuchae, Morbus Favre Racouchot, lentigines solaris, lentingines seniles, and actinic keratosis increased significantly with age. In contrast, after age 25, the prevalence of nevi (including atypical nevi) declined significantly with age. The decrease in nevi with age is thought to be due to induction of new antigens by sunlight or cellmediated immunity, each of which elicits an immune response capable of eliminating the nevi [15]. Findings from such studies confirm speculations that signs of skin aging are frequent and increase with age in contrast to common and atypical nevi, which decrease with age, all of which are associated with melanoma risk. The American Joint Committee on Cancer (AJCC) established a large international melanoma database, which was analyzed to determine the effect of patient age as a prognostic factor for melanoma survival [20]. Age was the third most important determinant of prognosis, following thickness and ulceration in this population of 13,581 patients with localized melanoma as well as in a group of 4,750 patients with regional nodal metastasis. Several factors have been identified to explain differences in prognosis in older patients. Some of which include undertreatment with increasing age because of narrower surgical margins and/or the interference of other medical conditions, difficulty with skin self-examination because of failing eye sight or poor health, differences in reporting signs and symptoms as compared to younger populations, inaccessibility of adequate health care, and lack of a social network to provide support for at home health care maintenance among those being treated for melanoma [14]. It has been well established that socioeconomic status (SES) influences cancer survival. SES impacts access to
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health care, insurance status, exposure to carcinogenic agents, cancer screening practices and attitudes, all of which can have an impact on survival. In general, SES affects survival from melanoma through biological factors of the tumor, stage or thickness at diagnosis, host factors, and treatment [21]. Reyes-Ortiz and colleagues utilized SEER data from 23,680 melanoma patients aged 65 and older diagnosed between 1988 and 1999 [21]. They sought to determine the association between SES and survival in older melanoma patients. Findings from this study revealed that subjects residing in lower-income areas had significantly lower 5-year survival rates than subjects residing in higher-income areas [21]. Further, an interaction effect between SES and ethnicity and melanoma survival was found in this population, which was demonstrated by 5-year survival rates in nonwhites that improved to a greater extent than in whites as income increased. Similar to other reports, this study found older age to be independently associated with greater risk of death from melanoma. The lower survival in older melanoma patients has been attributed to older patients who are screened less, present with later stages of melanoma, have a greater percentage of ulcerated melanomas, and tend to have more melanomas with a high potential for metastases; increasing the probability of recurrence and mortality [14, 21]. This study provides another demonstration of the association between age and melanoma, and also illustrates the interaction between other demographic factors commonly associated with aging (i.e., SES) that impact melanoma.
Biologic Factors The relationship between age and cancer is a result of the interaction of several biologic and environmental factors: decreased DNA repair capacity, decreased immune function, and in the case of skin cancer cumulative exposure to ultraviolet radiation [22]. Elderly patients have a propensity to have thicker lesions, which correlates with a poorer prognosis, especially in cases that are diagnosed at an advanced stage [23]. Increased incidence, long diagnostic delays, and poor prognosis experienced by elderly melanoma patients are of great concern. DNA repair mechanisms and cell cycle regulation are the cell’s means of handling UV-induced DNA damage. Cumulative DNA damage over decades mainly attributed to UV radiation along with age-associated decreases in DNA repair capacity are thought to play a central role in progression to melanoma [13]. Moriwaki and colleagues sought to demonstrate in vitro the association between DNA repair capacity and aging by measuring the ability of cells from
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individuals of different ages to repair new UV-induced DNA damage and establishing a cellular model for agerelated changes in the processing of damaged DNA [24]. Their study of primary skin fibroblasts from donors aged 3–96 years and their ability to repair damage in plasmid DNA identified an age-related decline in post-UV DNA repair capability and a corresponding increase in postUV mutagenesis. Earlier studies by the same research group demonstrated a 0.6% per annum decline in postUV DNA repair capacity of circulating T-lymphocytes in control populations over 4 decades from 20 to 60 years of age [25]. Findings from this study indicate that the ability to process new UV-induced DNA damage decreases with age and that this reduction in repair followed by an increase in DNA mutability is evident in cultured cells of the skin and blood [24]. The mirrored increase in DNA mutability has been hypothesized to arise from accumulation of DNA damage over time, a decreased ability to process new DNA damage or a combination of both [24]. In a similar study, Yamada and colleagues demonstrated an age-associated decline in nucleotide excision repair of UV-induced cyclobutane-type pyrimidine dimmers (CPD) [26]. In their study UV-exposed skin biopsies of younger men removed CPDs within 4 days after irradiation compared to biopsies of older men in which CPDs were removed within 7–14 days following irradiation. Their results suggest that age and its concomitant decline in DNA repair capacity is linked to high-risk of UV-associated skin cancer [26]. Further, long-lasting DNA damage has the potential to influence mutational events required for tumor progression [24]. Age/aging is an intrinsic component of many initiating events in melanoma. Alterations in the melanocyte cell cycle, aberrant signal transduction pathways, and activation/deactivation of melanoma-relevant oncogenes, all of which are associated with tumor formation, are all susceptible to biological alterations in response to aging. The interactions between melanocytes and their microenvironment, which can change with age, may play a critical role in tumor progression as well as a diminished immune surveillance as a consequence of age [11, 13]. Further, the biological response to sun exposure is dependent upon the physiological age of the skin more than the chronological age [27]. Cells have an inherent ability to repair damage to DNA. However, with age the proficiency of these repair mechanisms/enzymes declines with a concomitant increase in DNA mutability. Malignant transformation can still be avoided if cells with accumulated mutations are directed towards apoptosis via p53 mechanisms or senescence via the retinoblastoma (RB) system [11]. Melanocytes, cells responsible for the synthesis of pigment, which
provides protection from UV radiation, decrease in number, life span, and response to growth factors with age [28]. Further, melanocyte mutations in the B-RAF kinase initiate proliferation and the formation of nevi, and this cellular stress activates the RB/p16INK4a pathway resulting in irreversible growth arrest and senescence to prevent further proliferation and acquisition of additional mutations [11]. This mechanism explains why nevi stops growing once they reach a certain size and why they rarely develop into melanomas [16]. In melanocytes, senescence functions in vivo as a potent tumor suppressor mechanism [11]. Senescence can be thought of as a double-edged sword, although senescence of melanocytes affords these cells the capacity to suppress tumorigenesis, their cellular physiology is changed. Senescent melanocytes have been found to have altered melanin chemistry, increasing their susceptibility to DNA damage [11]. Specifically, in vitro investigations of senescent human melanocytes have revealed a reduction in the microphthalmia-associated transcription factor (MITF), a transcription factor critically involved in commitment, proliferation, and survival of melanocytes, as well as a down-regulation of dopachrome tautomerase, an enzyme involved in the melanin pathway [11]. The consequences of these alterations and resultant susceptibility to DNA damage in senescent melanocytes remain to be elucidated. Although these cells no longer proliferate, they may play a role in melanoma progression.
Immunology of the Skin and Age Immunosenescence refers to decreased immune competence as a result of advancing age. Immunologic competencies of the skin in general are difficult to assess because of the numerous influences on the immune system that affect individuals to different extents. Evaluating these competencies in the context of age adds another level of complexity. Underlying diseases or comorbidities, such as diabetes mellitus, malnourishment, increased scratching, or pruritic desiccated skin, alter immune function; thus, their individual contribution to the skin immune system is difficult to distinguish. The influence of age-associated physiologic changes on the immune system cannot be evaluated separately from intrinsic changes such as decreased DNA repair capacity and/or extrinsic changes like UV exposure [29]. Skin that is chronically sun-exposed exhibits immunologic changes that are not encountered in all aging individuals [29]. Thus it is difficult to assess whether an immunologic parameter associated with melanoma progression is associated with aging, chronic sun exposure, or both.
Melanoma and Skin Aging
The effects of decreased immune function on the skin as a consequence of aging are thought to be related to changes in skin-specific immune cells. The number of Langerhans cells, skin-specific antigen-presenting cells, in the epidermis decreases by 20–50% with age. In addition, the antigen-presentation function of these cells has also been shown to be reduced with age [28, 29]. The functional capacity of T cells is also known to be altered with age. Specifically, a decrease in the proliferative response, cytolytic activity, and repertoire of the T-cell antigen receptors has been demonstrated [29]. Keratinocytes, which provide barrier function, mechanical protection, cytokine production, and cell signaling, decrease in proliferation and differentiation with age (reviewed in [28]). In addition, the barrier function in response to injury is compromised with age and cell signaling, and growth factor response is decreased with age in keratinocytes. Alterations in the immune system of the skin in response to aging affect detection and removal of abnormal skin cells, which may favor the emergence of skin cancers [11].
Photoaging The effect of long-term UV exposure and sun damage superimposed on intrinsically aged skin is referred to as photoaging [28]. Cutaneous malignancies are one of the many clinical hallmarks associated with photoaging. Ultraviolet radiation causes damage indirectly by inducing the formation of free radicals as well as directly causing cellular injury [28]. UV radiation causes molecular and genetic changes, vascular alterations, immuno suppression, and has effects on pigmentation and the extracellular matrix. Several inherent cellular mechanisms exist to minimize UV-induced damage, among them DNA repair and apoptosis. However, these mechanisms decline with age and after a lifetime of assault, these devices may fail, making melanocytes vulnerable to the deleterious effects of UV exposure leading to photoaging and melanoma [28]. Solar elastosis, a sign of photoaging that is positively associated with age, is the deposit and accumulation of elastotic material in the upper and middle dermis that is thought to absorb sunlight [9] (> Fig. 57.1). Since solar elastosis increases with skin age, it has been considered as a surrogate for the cumulative dose of absorbed UV radiation. This protective biological response to longterm sun exposure has been found to be a favorable prognostic indicator in melanoma [2, 30]. The presence or absence of solar elastosis can be used to differentiate between melanomas that developed following higher
57
cumulative absorbed doses of UV from those that develop after a lower dose of UV, respectively [9]. A study of 1,200 patients diagnosed between 1980 and 1990 at a Melanoma clinic in North Carolina was conducted to evaluate the relationship between elastosis as a result of long-term cumulative UVexposure and cutaneous melanoma. The presence of solar elastosis was significantly related to patient age and melanomas with elastosis were found to occur at later stages than melanomas without elastosis [9]. Vollmer speculated that the results suggest that (i) elastosis is simply a surrogate for older age and thus accounts for the reason why melanomas with elastosis occur at older ages, (ii) elastosis may not be involved in the development of melanoma, or (iii) the etiology of melanomas with elastosis differs from those without. In this study, once melanoma developed, elastosis did not appear to affect thickness, mitotic rate, ulceration, or overall survival when compared to tumors in which elastosis was not present. Thus, the importance of elastosis appears to be prior to melanoma development and is associated with age. In contrast, two population-based studies have measured solar elastosis. In Western Australia, Heenan et al. found a dose–response association between solar elastosis and improved 5-year survival from melanoma [2]. In 2005, Berwick et al. reported a 50% decrease in risk of dying from melanoma associated with the presence of solar elastosis [30]. The contradictory evidence presented could be a results of study design (hospital-based vs population-based), analytic technique (univariate vs. multivariate), or some other important factor that was not measured in the studies. In any case, the role of sun exposure in photoaging and the development of melanoma is not straightforward and requires more in-depth study.
Lentigo Maligna Melanoma Older individuals are disproportionately affected by lentigo maligna (LM) melanoma. Solar lentigines, also referred to as ‘‘age spots,’’ are benign pigmented lesions that are commonly present in older Caucasian individuals. The presence of these lesions predisposes these individuals to a higher incidence of a preinvasive form of melanoma associated with chronic sun exposure, known as lentigo maligna [11]. Lentigo maligna (LM) usually present as large, flat, discolored patches on the face, the in situ component of this lesion can remain dormant for many years before the vertical growth phase nodular component develops [23]. This subtype of melanoma is often evaluated separately in epidemiologic studies because of its specificity for elderly people, slow growth, and link with cumulative sun exposure [31].
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. Figure 57.1 Scoring of the degree of solar elastosis. Two independent pathologists examined representative areas of solar elastosis on hematoxylin-and-eosin (H&E)-stained sections of normal skin surrounding the melanomas at 100–200¥ magnification. The following scoring system was used: CSD 0: absence of elastotic fibers; CSD 0+: rare elastotic fibers discernible only at 200¥ magnification. CSD 1: scattered elastotic fibers lying as individual units, not as bushels, between collagen bundles; ‘‘ ’’ or ‘‘+’’ classifiers were used to indicate whether the elastotic fibers were scarcely or densely scattered. CSD 2: densely scattered elastotic fibers distributed predominantly as bushels rather than individual units; the ‘‘ ’’ classifier was used to indicate that bushels were present, but elastotic fibers distributed as individual units predominated; the ‘‘+’’ classifier was used when larger aggregates of bushels formed, but preserving the outline of individual bushels instead of forming amorphous deposits. CSD 3: amorphous deposits of blue-gray material with lost fiber texture; ‘‘ ’’ only focal formation of amorphous deposits; ‘‘+’’ very large agglomerates of diffuse basophilic material. All images were taken with the 40¥ objective except for the lower right, which shows an overview with the 10¥ objective (Reprinted from Landi et al. [35]. With permission from AAAS)
Melanoma and Skin Aging
A double case–control study was designed to compare risk factors for LM and other melanomas in elderly people; in general, this study revealed that the risk factors for LM were similar to those for other melanomas (sunburns, light skin, and tendency to freckle). Contrary to other melanomas, this epidemiologic study revealed the absence of an association between lentigo maligna and the presence of nevi and/or genetic propensity to develop nevi [31]. These findings agree with those of an earlier study conducted by the same research group, in which their case–control study designed to identify epidemiologic factors associated with skin aging characterized by multiplication of senile lentigos, also did not identify a relationship with the number of nevi [32]. A correlation between these studies and the decrease in the number of nevi and an increase in the incidence of lentigo maligna seen in older individuals is evident. Although associated with chronic sun exposure, the risk of lentigo maligna has not been found to increase with the cumulative dose of sun exposure in epidemiologic studies [32]. Another form of melanoma, the most rare, acral lentiginous melanomas (ALM) are not related to ongoing sun exposure, in fact they usually develop on the skin of the lower limb. In a comprehensive study of 1,413 acral lentiginous melanomas using the SEER data from 1986 to 2005, 78% of these melanomas were found on the skin of the lower limb with 22% on the skin of the upper limbs. These melanomas, although rare and unlikely associated with sun exposure, are somewhat more aggressive than other melanomas. In this study the mean age of diagnosis was 62.8 years and a significant increase in incidence was seen with each year of advancing age [33]. In addition, the proportion of ALMs is greatest in people of color – being more common among blacks [33].
Conclusion Skin aging and melanoma are uniquely intertwined in a complex continuum of cancer. Advanced chronologic age and skin age have been demonstrated biologically and epidemiologically to be associated with melanoma development and progression. This intricate relationship needs to be taken into consideration in future investigations of melanoma as well as during development of prevention and intervention strategies. The observed increase in melanoma incidence along with the growing population of elderly adults establishes the importance of early detection in elderly populations as well as a further understanding of the etiology of melanomas in this population.
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Cross-references > Carcinogenesis: > Neoplastic
UV Radiation Skin Lesions in the Elderly Patient
References 1. American Cancer Society. (2009) Cancer Facts and Figures 2009. http://www.cancer.org/docroot/STT/STT_0.asp 2. Heenan PJ, English DR, Holman CDJ, Armstrong BK. Survival among patients with clinical stage I cutaneous malignant melanoma diagnosed in Western Australia in 1975–1976 and 1980/1981. Cancer. 1991;68:2079–2087. 3. Rosso S, Sera F, Segnan N, Zanetti R. Sun exposure prior to diagnosis is associated with improved survival in melanoma patients: Results from a long-term follow-up study of Italian patients. Eur J Cancer. 2008;44:1275–1281. 4. Breslow A. Thickness, cross-sectional area, and depth of invasion in the prognosis of cutaneous melanoma. Am Surg. 1970;66: 527–531. 5. Finley JW, Rodriguez LM, Letourneau R, Driscoll D, Kraybill W. Pathologic and clinical features influencing outcome of thin cutaneous melanoma: correlation with newly proposed staging system. Am Surg. 2000;66:527–531. 6. Buzaid CM, Ross MI, Balch CM, et al. Critical analysis of the current American Joint Committee on Cancer staging system for cutaneous melanoma and proposal of a new staging system. J Clin Oncol. 1997;15:1039–1051. 7. Balch CM. Cutaneous melanoma: prognosis and treatment results worldwide. Semin Surg Oncol. 1992;8:400–414. 8. Gershenwald JE, Thompson W, Mansfield PF, et al. Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage I or II melanoma patients. J Clin Oncol. 1999;17:976–983. 9. Volmer RT. Solar elastosis in cutaneous melanoma. Am J Clin Pathol. 2007;128:260–264. 10. Lachiewicz AM, Berwick M, Wiggins CL, Thomas NE. Survival differences between patients with scalp or neck melanoma and those with melanoma of other sites in the Surveillance, Epidemiology, and End Results (SEER) program. Arch Dermatol. 2008;144: 515–521. 11. Desai A, Krathen R, Orengo I, Medrano EE. The age of skin cancers. Sci Aging Knowl Environ. 2006;9:pe13. 12. Geller AC, Miller DR, Annas GD, et al. Melanoma incidence and mortality among US whites, 1969–1999. JAMA. 2002;288: 1719–1720. 13. Yaar M, Gilchrest BA. Ageing and photoageing of keratinocytes and melanocytes. Clin Exp Dermatol. 2001;26:583–591. 14. Sweeter SM, Geller AC, Kirkwood JM. Melanoma in the older person. Oncology. 2004;18:1187–1196. 15. Schafer T, Merkl J, Klemm E, Wichmann HE, Ring J, KORA study group. The epidemiology of nevi and signs of skin aging in the adult general population: results of the KORA-survey 2000. J Invest Dermatol. 2006;126:1490–1496. 16. Sage J. Making young tumors old: a new weapon against cancer? Sci Aging Knowl Environ. 2005;33:pe25. 17. Lachiewicz AM, Berwick M, Wiggins CL, Thomas NE. Epidemiologic support for melanoma heterogeneity using the Surveillance,
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Epidemiology, and End Results program. J Invest Dermatol. 2008;128:243–245. Whiteman DC, Watt P, Purdie DM, Hughes MC, Hayward NK, Green AC. Melanocytic nevi, solar keratoses, and divergent pathways to cutaneous melanoma. J Natl Cancer Inst. 2003;95:806–812. Austin PF, Cruse CW, Lyman G, Schroer K, Glass F, Reintgen DS. Age as a prognostic factor in the malignant melanoma population. Ann Surg Oncol. 1994;1:487–494. Balch CM, Soong SJ, Gershenwald JE, et al. Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer Melanoma Staging System. J Clin Oncol. 2001;19:3622–3634. Reyes-Ortiz CA, Goodwin JS, Freeman JL, Kou YF. Socioeconomic status and survival in older patients with melanoma. JAGS. 2006;54:1758–1764. Syrigos KN, Tzannou I, Katirtzoglou N, Georgiou E. Skin cancer in the elderly. In Vivo. 2005;19:643–652. Kaplan RP. The aging skin. Comp Ther. 1991;17:59–67. Moriwaki SI, Ray S, Tarone RE, Kraemer KH, Grossman L. The effect of donor age on the processing of UV-damaged DNA by cultured human cells: reduced DNA repair capacity and increased DNA mutability. Mutat Res. 1996;364:117–123. Wei Q, Matanoski GM, Farmer ER, Hedayati MA, Grossman L. DNA repair and aging in basal cell carcinoma: a molecular epidemiology study. Proc Natl Acad Sci USA. 1993;90:1614–1618. Yamada M, Udono MU, Hori M, Hirose R, Sato S, Mori T, Nikaido O. Aged human skin removes UVB-induced pyrimidine dimmers from the epidermis more slowly than younger adult skin in vivo. Arch Dermatol Res. 2006;297:294–302.
27. Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol. 2008;12–16. 28. Rabe JH, Mamelak AJ, McElgunn PJS, Morison WL, Sauder DN. Photoaging: mechanisms and repair. J Am Acad Dermatol. 2006;55:1–19. 29. Sunderkotter C, Kalden H, Luger TA. Aging and the skin immune system. Arch Dermatol. 1997;133:1256–1262. 30. Berwick M, Armstrong BK, Ben-Porat L, Fine J, Kircker A, Barnhill RL. Sun exposure and mortality from melanoma. J Natl Cancer Inst. 2005;97:1–5. 31. Gaudy-Marqueste C, Madjlessi N, Guillot B, Avril MF, Grob JJ. Risk factors in elderly people for lentigo maligna compared with other melanomas. Arch Dermatol. 2009;145:418–423. 32. Monestier S, Gaudy C, Gouvernet J, Richard MA, Grob JJ. Multiple senile lentigos of the face, a skin ageing pattern resulting from a life excess of intermittent sun exposure in dark-skinned Caucasians: a case-control study. Br J Dermatol. 2006;154:438–444. 33. Bradford, PT, Goldstein AM, McMaster ML, Tucker MA. Acral lentiginous melanoma incidence and survival patterns in the United States, 1986–2005. Arch Dermatol. 2009;145:427–434. 34. Golger A, Young DS, Ghazarian D, Neligan PC. Epidemiologic features and prognostic factors of cutaneous head and neck melanoma. Arch Otolaryngol Head Neck Surg. 2007;133:442–447. 35. Landi, et al. MC1R Germline Variants Confer Risk for BRAF-Mutant Melanoma. Science 28: Supporting Online Material, 2006. http:// www.sciencemag.org/cgi/content/full/1127515/DC1
Malignant Skin Conditions
55 Neoplastic Skin Lesions in the Elderly Patient Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach . Isaac M. Neuhaus
Introduction As the proportion of the aged in the US population increases, so does the burden of cutaneous disease [1]. Cutaneous diseases of the elderly represent significant morbidity, with many patients having multiple conditions [2]. Hyperproliferative disorders of the skin are strongly associated with exposure to solar radiation and are commonly present in older adults [3]. Although nonmalignant growths are far more common than malignant ones, skin cancers represent about 6% of all dermatology visits [4]. The prevalence of cutaneous malignancies increases with age [5] (> Table 55.1). The most common cutaneous neoplasms are basal cell carcinoma (BCC) (> Figs. 55.1 and > 55.2), squamous cell carcinoma (SCC) (> Fig. 55.3), and malignant melanoma. These three types of skin cancer account for almost half of all human cancers [6]. Increases in skin cancer rates have paralleled a cultural shift towards recreational ultraviolet (UV) exposure [7]. As a result, skin cancers are appearing at alarming rates in young adults. Because the risk of recurrence of skin cancer is directly related to the length of time past initial diagnosis, dramatic leaps in skin cancer rates in young people may well precede a steep increase of future skin cancer in old age [8]. The major factors contributing to skin cancer in humans are lifetime cumulative exposure to cancer-causing agents including UV radiation, decreased melanocyte protection, diminished DNA repair, and decreased immunosurveillance [9]. Although the contribution of UV exposure to some types of skin cancer is a hotly debated topic, more than 90% of all skin cancers are believed to have direct origin in exposure to the sun’s UV rays [7]. A major function of the skin is the absorption of UV radiation. Virtually all ultraviolet-B (UVB) is absorbed by the upper layers of the epidermis, with only 20% reaching the dermo-epidermal junction. Ultraviolet-A (UVA) penetrates deeper, releasing up to 50% of its energy in dermal stratum papillare [10]. Despite its inability to penetrate beyond the epidermis, UVB is far more mutagenic than UVA [10]. Induction of squamous cell
carcinoma has been observed to be 1,000 times more efficient with UVB (300 nm) than with UVA (380 nm) [11]. Constitutive levels of melanin in the skin, an inherited contributor to UV risk, play a major role in the capacity of UV insult to affect DNA mutation. Twenty percent of Caucasians, more accurately described as Fitzpatrick skin types I, II, and III, will suffer from some form of skin cancer over their lifetimes [12]. People with darker skin, Fitzpatrick types IV and V, benefit from the substantial protection that melanin density offers: a 500-fold level of protection from UV radiation (based on skin cancer rates) between white and black skin [13]. The appearance of a cutaneous malignancy is the culmination of an accumulation of genetic abnormalities, some inherited and some acquired. The types of mutations and the number accumulated will define malignant potential [14]. Genetic abnormalities caused by UV radiation represent a spectrum of damage. The ability of UV light (UVL) to cause point mutations has been long recognized [15]. More recently, UV-induced deletions [16] and micronuclei [17] have also been observed in irradiated skin cells. In addition, the presence of aneuploidy has been observed in every type of skin cancer; aneuploidy increases as the tumor progresses and robustly correlates with the malignant potential of the lesion [14]. Epidermal cells bear the brunt of environmental insult to the body and multiple mutations occur in every epidermal cell every day. The vast majority will be quickly repaired and do not lead to malignant transformation. In cases where accumulation of unrepaired genetic abnormalities occurs in a particular cell over time, impaired normal function can result [14]. The accrual of genetic damage in particular genes – those related to cell cycle control, ongoing DNA repair, upregulation of proliferation, or induction of apoptosis – can result in cell transformation with subsequent tumor initiation [14]. Ultimately, transformation is a multistep process requiring distinct damage events, creating the typical latency period between initial insult and appearance of a cancerous lesion [10].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_55, # Springer-Verlag Berlin Heidelberg 2010
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. Table 55.1 Malignant potential in hyperproliferative diseases of the aged skin Carter DM, Balin AK. [33], Lin et al. [37], Keller et al. [38], Tschen et al. [47], International Society for Photodynamic Therapy in Dermatology [48], Mogensen M, Jemec GBE [55], Erbagci Z, Erkilic¸, S. [75] Benign
Premalignant
Malignant
Skin tags
Actinic keratosis (AK)
Basal cell carcinoma (BCC)
Senile lentigo (Lentigines)
Lentigo maligna
Squamous cell carcinoma (SCC)
Seborrheic keratoses
Leukoplakia
Bowen’s disease (SCC in situ)
Cherry hemangiomas (de Morgan’s spots)
Atypical nevi
Keratoacanthoma (SCC in situ)
Corns
. Figure 55.1 Picture of basal cell carcinoma (BCC)
. Figure 55.2 Picture of basal cell carcinoma (BCC)
UV insult causes damage with a dual action of creating mutation in the DNA and suppressing intrinsic immunity through disruption of tumor surveillance [10]. UVL acts
Malignant melanoma
as both tumor initiator and promoter [18]. Mutation in the gene that produces p53 is considered a hallmark of UV-induced genome modification. P53 in healthy cells is involved in tumor suppression, arresting the cell cycle in the G1 phase in order to allow for DNA repair before mitosis ensues [10]. Most UV-induced DNA damage is repaired by p53 [19]. If genetic damage in the cell is extensive, p53 will initiate apoptosis [20]. Mutation in p53 aborts the progression to apoptosis, permitting the mutated cell to continue to divide [10]. Cells carrying p53 mutations are ubiquitous in every type of skin cancer [21]. Another regulator of apoptosis, CD95 (fas), is also inhibited by chronic UV exposure [22]. UV exposure has also been demonstrated to increase the population of tumor-permissive macrophages, depress Langerhans cell numbers, reduce Langerhans cell function, induce cytotoxic T-cells to undergo apoptosis, and activate suppressor T-cells [18]. A resident malignancy can affect immunosupression as well. The capacity for DNA repair after UVexposure was observed to be lower in patients with basal cell carcinoma (BCC) than in controls without skin cancer [23]. In addition, in a population of nonmelanoma skin cancer (NMSC) patients, 90% exhibited a decreased immune response in delayed hypersensitivity skin testing [18]. Organ transplant recipients receiving immunosuppressive therapy have dramatically increased risk for all types of skin cancers [24]. The efficacy of DNA repair is an additional factor in carcinogenesis. Three levels of cellular DNA repair exist. Base excision repair (BER) removes lesions in DNA bases caused by oxidation, nucleotide excision repair (NER) acts as the main repair mechanism for UV-induced lesions, and mammalian mismatch repair (MMR) is responsible for removal of mismatched bases [25]. The importance of these processes in preventing carcinogenesis is reflected in the 100-fold increase in mutation frequency observed in cells that lack MMR [25] and the 2,000-fold increase in cancers in xeroderma pigmentosum (XP) patients who
Neoplastic Skin Lesions in the Elderly Patient
. Figure 55.3 Squamous cell carcinoma (SCC)
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history of the condition and can have numerous lesions (50% have greater than ten lesions [33], and some have hundreds [34]). The sudden appearance of numerous prutitic SKs may be associated with internal malignancy (sign of Leser-Trelat) [33, 38]. SKs can cause irritation and become sore, but they pose no risk of malignant transformation. Careful cutaneous exam is required to ensure that SKs do not obscure a distinct malignant skin lesion which may be missed [35]. No treatment is medically necessary but cryotherapy or curettage can be used to remove unwanted lesions [36].
Skin Tags and Angiomas
lack NER [25]. A defect in MMR recently found in tumor cells indicates that MMR is affected by a repair protein hMSH2, which is regulated by p53 [26]. Mutation in p53 produces dysfunctional hMSH2, acting as a second step towards transformation [10]. An additional factor that may influence carcinogenesis in the skin is its nutritional status. High-fat diets [27] and low levels of vitamin C [28], beta carotene [28], cruciferous vegetables [28], vitamin E [29], and selenium are associated with elevated risk of skin cancer [30]. Concurrent infection with oncogenic human viruses may also play a role. Human papillomavirus (HPV) infection, for example, appears to promote NMSC [31].
Nonmalignant Hyperproliferative Conditions Include seborrheic keratoses, cherry hemangiomas, senile lentigenes, and skin tags [32]. Almost every adult over 65 has at least one benign neoplastic growth, with seborrheic keratoses (SKs) being the most common [3].
Seborrheic Keratoses Seborrheic keratoses (SKs) are tan to black growths, which appear to be stuck on the skin and vary from a few millimeters to over 4 cm in diameter. They occur in all ages, but 88% of all those affected are over 65 [33]; the incidence in men is higher than in women. Lesions most often appear on the trunk and can occur anywhere on the body, but generally avoid the palms, soles, and mucous membranes [34, 35]. People with SKs often have a family
Skin tags (soft, flesh-colored, pedunculated papules on the neck and upper eyelids) and cherry hemangiomas (bright red nodules several millimeters in size) are both very common in older adults. Benign growths are treated for cosmetic reasons or if their location makes them susceptible to irritation [36].
Actinic Keratoses Actinic keratoses (AK) arise from UVB damage to the genetic material in keratinocytes; this damage induces clones of malignant keratinocytes that are confined to the epidermis [33]. Mutation in the p53 gene is the most important pathogenetic event [10]. Left untreated, an occurrence of AK has a minor risk of extending into the dermis, becoming invasive squamous cell carcinoma (SCC) [37]. Patients with AKs often have numerous lesions [38]. Since approximately 1 out of every 1,000 AKs progress to invasive skin cancer per year [37], patients with many AKs have a significant risk of eventually having at least one lesion becoming invasive [14, 49]. AKs represent the earliest recognizable manifestation of SCC as a premalignant lesion [39, 52]. AK is very common in elderly Caucasians (Fitzpatrick skin types I-III), with a higher prevalence in men [37]. The incidence of AK increases steadily with age, from approximately 10% in people in their twenties to more than 80% in people older than 70 [40]. These growths have a strong association with sun exposure, in terms of both geographic region and the individual’s degree of sun exposure [41]. Lesions occur almost exclusively on photodamaged sites, that is, neck, face, dorsal surface of the hands, forearms, and the bald scalp [42]. Extensive involvement of the lip can occur and is termed actinic cheilitis [43].
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Although AK lesions typically remain inactive for years, approximately 10% will eventually invade the dermis as SCC [44]. Some patients have numerous keratoses that never transform, while, for unknown reasons, some have numerous AKs that undergo rapid change simultaneously [42]. AKs are more likely to progress to SCC if mutations exist in the p16 gene (which codes an additional tumor suppression protein) [40]. The presence of AK lesions signifies a high risk for other skin cancers in the future; regular follow-up and the strongest measures of prevention should be encouraged [38]. Use of sunscreen may not only prevent further lesions, but may also hasten regression of existing ones [40]. Further details on AK are presented in > Table 55.2. AKs often multiply within a specific region of the skin, a phenomenon now considered to be field cancerization. Skin that appears clinically normal but surrounding existing AKs is believed to be genetically vulnerable, damaged skin in which lurk clinically invisible precancerous changes, with progression to AK likely [45]. Topical applications are a good choice as the entire susceptible field can be simultaneously treated [46, 47]. Patients with numerous lesions may be treated simultaneously with intermittent topical applications as follows: 5%-fluorouracil cream, diclofenac sodium, imiquimod, photodynamic therapy. Photodynamic therapy (PTD) activates photosensitizers with visible light [45] to form reactive and cytotoxic
singlet oxygen. The site is photosensitized by topical application of gamma-5-aminolaevulinic acid or its methyl esters; it is then irradiated with visible light, causing phototoxic destruction of superficial layers of skin [48]. Cure rates are as high as 91% [122–126], similar to that with cryotherapy but with improved cosmetic results [45].
Nonmelanoma Skin Cancers The incidence of nonmelanoma skin cancers (NMSC) is increasing steadily, and now represents more than 33% of all cancers in the USA [21]. Rigorous and substantial evidence supports the assertion that the vast majority of NMSCs result directly from cumulative unprotected sun exposure, particularly occurring before the age of 18 [49]: they develop primarily in pale, sun-exposed skin [50]; frequency is related to the degree of sun exposure and also directly related to latitude [51]; sun avoidance and use of sunscreen decreases the frequency of NMSC [52]; these cancers are readily produced experimentally by UV exposure [53]; and their frequency is greatly increased in XP, a condition in which the repair process for UV damage to DNA is compromised [54]. The incidence of NMSCs is approximately 20 times the incidence of melanoma [21]. The most important factor in skin cancer prognosis is early diagnosis [55], including accurate classification of the malignant lesion. It can be clinically difficult to
. Table 55.2 Differential diagnosis of actinic keratoses American Cancer Society 2004 [147] Description of lesions Flesh or tan colored papules with marked scaling and welldefined borders. Rough texture
Population & prevalence Caucasian, especially fair-skinned.
Growth
Usually appears in multiple lesions. May Onset typically wax and greater than age wane with 50 in northern latitudes, younger seasons ages in southern Prevalence: Nears 100% in Caucasian populations of aged individual with lifelong sun exposure
SCC = squamous cell carcinoma
Common sites Strong correlation with sun exposure: face, hands, forearms, bald scalp
Size of lesion Invasive Metastasis Few millimeters to more than an inch in diameter. Usually less than 1 cm
No, but up to 20% become invasive SCC
No
Danger sign Palpable nodularity under scale, infiltration, elevation, rapid growth, tenderness
Neoplastic Skin Lesions in the Elderly Patient
distinguish from proliferative AK and early invasive SCC [45], to distinguish among the different classifications of BCC, and sometimes even to distinguish among the three major classifications themselves, particularly in patients of color [56]. Ideally, histopathologic classification should accurately identify subtypes and therefore predict tumor behavior. Immunohistochemical adjuvants such as marker Melan-A improve traditional histology; the use of Melan-A increased successful differential diagnosis of melanocytic tumors from among actinic keratosis and solar lentigines also evaluated [57]. Optical coherence tomography, positron emission tomography, and computed tomography also can improve diagnostic capabilities. A new technique, computer-assisted nuclear morphometry, was able to predict recurrence and disease-free survival rates in BCC [58]. Biopsies have been relied on for prognostic information, but the advent of less invasive therapies demands noninvasive diagnostics as well [55]. Much research effort, therefore has focused on technological improvements in traditional diagnosis and prognostic methods. A comparison of common therapies for neoplastic lesions is shown in > Table 55.3. Dermatoscopic evaluation of the suspicious lesion can improve differential diagnosis [59]. Ultrasound shows promise for determining three-dimensional size and margin characteristics, as well as information about homogeneity and tissue health of inner structures [55]. Ultrasound can also be employed for identification of metastasis in the sentinel node [60]. Analysis of genetic markers also promises to increase prognostic ability, particularly with regard to obtaining tumor karyotypes [14]. The frequency of aneuploidy in skin cancer suggests that it has at least some etiologic role in carcinogenesis and therefore can indicate malignant potential [14]. Aneuploidy is generally detected through use of flow cytometry, and the level of aneuploidy correlates well with staging criteria and therefore prognosis. For example, in melanoma, while aneuploidy was found in only 3% of melanocytic nevi, it was found in 34% of level IV melanomas and 100% of level V [61]. Assessing level of aneuploidy may therefore aid in prognostic consideration.
Basal Cell Carcinoma Basal cell carcinoma (BCC) (> Figs. 55.1 and > 55.2), which arises from cells in the basal layer of the epidermis, is the most common form of skin cancer [ 10, 38]. BCCs account for 80% of all skin cancers [62]. Although clonal
55
expansion of mutant p53s is often expressed in and around BCCs on sun-exposed skin [63], these UV-fingerprint mutations are much more rare in BCC than in SCC [10]. The seminal event in the development of sporadic BCC appears to be mutation in the gene PTCH1, a hedgehog pathway gene mutation with broad control of embryonic proliferation [10]. Other regulatory defects have been observed in association with BCC lesions: mutation in the H-ras gene (also involved in cell division) occurs at increased frequency [64], mutations in c-fos (a proto-oncogene) are reduced [65], and mutations in p16, a tumor suppressor gene, are also observed [66]. Induction of IL-4 and IL-10 by UV irradiation reduces tumor surveillance [67] as well as interstitial collagenase, which facilitates invasion of the tumor into the surrounding tissue [68]. The most typical presentation of BCC is a papule or nodule with a central umbilication, distinguished by a generally pearly or translucent appearance with telangiectasia and a characteristic rolled boarder. Central crusting and bleeding is often seen [69]. Other presentations can also be seen and include an erythematous, scaly, flat patch (superficial BCC variant), or a pale scar-like lesion without (morpheaform variant) [9]. Tumors resemble hair follicle structures morphologically, so BCC is believed to be a malignant tumor of the follicular germinative cells (these germinative cells are referred to as trichoblasts) [70]. The clinical presentation of BCC in its various forms makes classification difficult and aggressiveness and recurrence difficult to predict; recurrence sometimes occurs even when excision is performed with clearly free margins [58]. Over 50% of BCC lesions that occur in people of color are pigmented versus only 6% of lesions found in Caucasians [56]. Most BCCs are visually distinguishable from squamous cell carcinomas (SCC), but biopsy should nonetheless be performed [71]. Although the BCCs rarely metastasize, they are invasive, with a high rate of recurrence [71]. BCC is strongly associated with sun exposure. Tumors occur most frequently on sun-exposed sites [23], 85% of the time on the head and neck [45]. BCC is far more common in those with fair complexions and significantly more common in those exposed to UVL, including phototherapy [23]. It has been observed to occur less frequently, however, on certain body sites that nevertheless have strong sun exposure (e.g., the dorsal hand), and its relative frequency on diverse facial sites is not strongly correlated to UVL exposure [23], even when topographical anatomy and other possible cofounders are considered [23]. Some correlation exists between a high frequency of BCC and concave shape, reduced skin tension, and marked skin folds, which may reflect areas of high matrix
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. Table 55.3 Comparison of common therapies for neoplastic lesions Treatment Curettage
Cryotherapy
Photodynamic therapy
Photodynamic therapy, cont.
Lesion
Protocol
AK
Standard
nBCC, sBCC
Standard
AK
Standard
BCC
Standard
AK
AK, cont.
Efficacy
Advantages Tissue available for histological examination
5-year no Less recurrence rate 86– expensive, 97% easier technically than Mohs
Disadvantages
Reference
Sometimes pigment changes, scarring, poor wound healing. Potential for nerve damage
[40]
Higher risk of recurrence
[91]
Preserves Possibility of dermal nerves, hypopigmentation blood vessels, collagen
[40]
Inexpensive, few side effects, quick recovery
Pain, scarring, dyspigmentation
[91]
20% ALA, two 78% cure rate treatments 14–18 h exposure, blue light, 12 months follow-up
Very good cosmetic results Useful for treatment of large areas (field cancerization)
Painful
[47]
20% ALA, two 89% cure rate treatments 14–18 h exposure, blue light 3-month follow up
Very good cosmetic results Useful for treatment of large areas (field cancerization) [47]
Painful [47]
[114]
20% ALA, two 85% cure rate treatments 14–18 h exposure, red light 2-month follow up
Very good cosmetic results Useful for treatment of large areas (field cancerization)
Painful [47]
[115]
16% MAL, 3 h 89–91% cure rate exposure, two treatments and 3-month
Very good cosmetic results Useful for treatment of large areas (field cancerization) [47]
Painful [47]
[116]
5-year no recurrence as high 96–83%
Neoplastic Skin Lesions in the Elderly Patient
55
. Table 55.3 (Continued) Treatment
Lesion sBCC #
Protocol
Efficacy
Advantages
91% cure rate
Very good cosmetic results Useful for treatment of large areas (field cancerization) [47]
Painful [47]
[117]
16% MAL, red 92% cure rate light, two treatments and 3-month follow up
Very good cosmetic results Useful for treatment of large areas (field cancerization) [47]
Painful [47]
[118]
Erythema, dryness; temporarily disfigurement and pain so high rate of noncompliance [40]
[119]
20% ALA one treatments 4 h exposure, red light
Disadvantages
Reference
5-fluorouracil topical cream (DNA synthesis inhibitor) [76]
AK
N = 21 0.5%, 78% reduction in once per day, lesions opposite side of face as control for 4 weeks
Comparatively short treatment period needed for efficacy
5-fluorouracil topical cream (DNA synthesis inhibitor) [76] cont.
AK, cont.
N = 21 5%, 60% reduction in twice per day, lesions opposite side of face as control for 4 weeks
Comparatively Erythema, dryness short treatment period needed for efficacy [119]
[119]
N = 177 0.5%, 78% reduction in once per day, lesions for 4 weeks
Comparatively Erythema, dryness short treatment period needed for efficacy [119]
[120]
N = 120 3%, twice a day, for 3 months
50% subjects cleared
Higher tolerability than 5-FU
Pruritus, erythema, dryness, relatively long treatment period for efficacy
[40, 121]
N = 195 3%, twice a day, for 2 months
33% subjects cleared
Higher tolerability than 5-FU [40]
Pruritus, erythema, dryness
[122]
N = 20 3% once per day for 3 months
9.3% cleared, 64.7 reduced in size
Higher tolerability than 5-FU [40]
Minimal irritation
[123]
Prolonged effect noted
Little to no irritation reported
[124]
Diclofenac sodium (appears to boost immunosurveillance [76])
Colchicine (mitosis inhibitor [76])
AK
AK
N = 20 1% gel 70% cleared twice a day for 10 days, monitored 2 months
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Neoplastic Skin Lesions in the Elderly Patient
. Table 55.3 Comparison of common therapies for neoplastic lesions (Continued) Treatment
Imiquimod (toll-like receptor 7 agonist, boosts immune response.)
Imiquimod (toll-like receptor 7 agonist, boosts immune response.), cont.
Lesion
AK
AK, cont.
Protocol
Efficacy
Advantages
N = 16 0.5% 87.5% cleared cream twice a day for up to three 10-day cycles
Higher tolerability than 5-FU [40]
Little to no irritation reported
[125]
N = 16 1% 75% cleared cream twice a day for up to three 10-day cycles
Higher tolerability than 5-FU [40]
Little to no irritation reported
[125]
N = 52 5% cream three times a week for up to 12 weeks.
Reveals previously undetectable AKs
Mild to severe erythema, erosions, edema vesicles, pruritus
[126]
N = 25 5% 82% cleared (figure Prolonged cream three achieved by end of effect noted time a week second cycle) for 4 weeks, 4week rest, cycle repeated three times
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[127]
N = 22 5% cream three times a week for 8 weeks, opposite of face as control
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[128]
N = 436 5% 45% cleared at cream twice a 24 weeks week for 16 weeks.
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[129]
N = 286 5% cream three times a week for 16 weeks.
57% cleared at 24 weeks
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[80]
N = 492 5% cream three times a week for 16 weeks
48% cleared at 24 weeks
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[129]
84% cleared
33% reduction in lesions
Disadvantages
Reference
Neoplastic Skin Lesions in the Elderly Patient
55
. Table 55.3 (Continued) Treatment
Imiquimod (toll-like receptor 7 agonist, boosts immune response.), cont.
Imiquimod (toll-like receptor 7 agonist, boosts immune response.), cont.
Mohs micrographic surgery
Lesion
Protocol
Efficacy
Advantages
Disadvantages
BCC (80% superficial, 20% nodular
N = 35 5%, once or twice daily three times a week for 16 weeks
Three times a week: 100% clearance at 16 weeks. Twice a week clearance dropped to 60%
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[130]
sBCC
N = 99 5%, once or twice daily for 6 weeks
Twice daily, three times a week: 100% clearance at 6 weeks (once daily, three times a week clearance dropped to 70%)
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[131]
N = 128 5%, once or twice daily 3–5 times per week for 12 weeks
Twice daily, five times a week: 100% clearance at 12 weeks (once daily, three times a week clearance dropped to 52%)
Reveals previously undetectable AKs [126] Prolonged effect noted [141]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[132]
N = 364 5%, five or seven times per week for 6 weeks
Five times weekly 82% clearance, seven times weekly 79% clearance
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[133]
N = 99 5%, 71% clearance once daily for 6 weeks
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[134]
N = 92 5%, 76% clearance once daily for 12 weeks
Reveals previously undetectable AKs [126] Prolonged effect noted [127]
Mild to severe erythema, erosions, edema vesicles, pruritus. Efficacy proportional to intensity of site reaction
[134]
BCC
Standard
5-year no recurrence rate greater approaches 100% [91]
Lowest risk of recurrence, good cosmetic result
Bony invasion, risk of loss of function or deformity, ongoing positive margins, surgical procedure
[76]
SCC
Standard
5-year no recurrence rates 92%
Lowest risk of recurrence, good cosmetic result
Bony invasion, risk of loss of function or deformity, ongoing positive margins, surgical procedure
[91]
nBCC
Reference
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Neoplastic Skin Lesions in the Elderly Patient
. Table 55.3 Comparison of common therapies for neoplastic lesions (Continued) Treatment
Lesion
Protocol
Efficacy
Advantages
Disadvantages
Reference
Radiation therapy
BCCs
Standard
5-year no recurrence rates 92.6%
Useful for large tumors or tumors in difficult locations, older patients
Risk of inducing [91] malignancy in younger patients, not cosmetically favorable
Radiotherapy
SCC
Standard
Tumor control rates 70–100%
Useful when surgery refused or impossible
Cartilaginous areas [91] susceptible to necrosis, not advised in immunocompromised patients
AK = actinic keratoses, ALA = aminolevulinic acid, BCC = basal cell carcinoma, MAL = methyl aminolevulinate, nBCC = nodular basal cell carcinoma, sBCC = superficial basal cell carcinoma 5-FU = 5-fluorouracil # Not recommended for nodular or morpheaform BCC due to tendency to increase rate of recurrence
metalloproteinases (MMP) expression. MMPs are responsible for tissue breakdown as tumor expansion proceeds, and they are present at increased levels in and around BCCs [68]. Integrins, which regulate MMPs, have been observed to be greatly reduced in BCCs [72]. The frequent occurrence of BCC on nonexposed sites implies a potential for multiple mechanisms in BCC development [70]. Arsenic, x-rays, grenz-ray exposure, and mustard gas are known contributors to some specific BCCs [70]. The role of an additional causative agent in BCC is suggested by the observations: that sunscreens with a high skin protection factor reduce the risk of SCC, but prospective studies have found no protective effect on BCC [73]; Li-Fraumeni syndrome, caused by an inherited point mutation in p53, does not raise BCC risk [74]; and truncal BCCs on the trunk appear to be specifically associated with variations in the gene for the enzymes glutathione S-transferase (three forms) and cytochrome P450 [70]. It is considered likely that immunosuppression may play a significant role in the development of BCC, as BCCs occur frequently in immunosuppressed patients (only SCCs have a higher prevalence in this population). In fact, the risk of BCC in organ transplant recipients is considerably higher (reported at anywhere from 6–100% higher [70]) than in the general population. Basal cell tumors, particularly morpheaform BCC, also have dramatically elevated mast cell indices which do not correlate to levels of sun exposure [75]. The observation that elevation of mast cell indices does correlate with cigarette use supports the hypothesis that BCC development is
multifactorial, with some part of its etiology susceptible to influence by lifestyle factors [75]. Further details on BCC are presented in > Table 55.4. The rate of recurrence of basal cell carcinomas is directly related to tumor size, location, and histologic subtype as well as the presence of residual transformed cells after treatment [76]. The chosen therapy will depend on the patient’s age, lesion size and site, histologic subtype, and whether it is a primary lesion or a recurrence [37]. Surgical excision with suturing has a recurrence rate of less than 5% [48, 116]. The recurrence rate drops to 1% [48, 116] with Mohs micrographic surgical excision. Mohs surgery evaluates horizontal sections with 100% margin control. The neoplasm is mapped and additional sections are taken until clear margins are achieved. Imiquimod appears to be a potentially useful adjuvant in BCC: one study found no recurrence after 34 months when imiquimod was used as adjuvant therapy after Mohs [77], and another observed 94% of lesions to have histologically cleared at 3 months after curettage [78]. Another study evaluated treatment of BCC with three cycles of curettage against treatment with electrodessication followed by 5% imiquimod cream for 1 month. At 8 weeks, 1/11 (9%) of imiquimod-treated group had residual tumor compared to 4/11 (36%) of controls [79]. Patients with a previous BCC should be fastidiously examined by a physician yearly for signs of recurrence [38]. Photodynamic therapy (PTD), which activates photosensitizers with visible light [45] to form reactive and cytotoxic singlet oxygen, has shown potential efficacy in early clinical trials [45]. The site is photosensitized by
Neoplastic Skin Lesions in the Elderly Patient
55
. Table 55.4 Differential diagnosis of basal cell carcinoma (Ericson et al. [45] and American Cancer Society 2004 [147]) Population & Prevalence
Type of Growth
Description of Lesions
80% of all nonmelanoma skin cancers
Small, domeshaped pimplelike growth often pearly in color. Usually single lesion
90% Relatively Caucasian, slow especially fair-skinned. Onset greater than 50 years, men higher incidence than women
Head, neck account for 85%, 30% of these on nose. Most subtypes strongly associated with sun exposure
1–2 cm
Yes
Nodular
Smooth, flesh colored, translucent nodule with pearly border, telangiectasias, and slight central umbilication. Sometimes accompanied by itch
60% of BCC Caucasian, especially fair-skinned
Slow Usually appears as single nodule Nests push into dermis
Good correlation with sun exposure
Few millimeters to a few centimeters
Yes, No even bone, nerves, cartilage
Superficial
Slightly raised, erythematous, scaling patch, with threadlike periphery and atrophy in center
Caucasian, especially fair-skinned.
Slow to ulcerate. Nests restricted to epidermis
Trunk and other nonexposed sites. Not correlated with sunexposure
As large as Not greater readily than 4 cm in diameter
Morpheaform
Indurated Caucasian, yellow to flesh- especially colored plaque fair-skinned with poorly defined margins. Resembles a scar
Infiltrate, Tumor nests diffuse and irregularly spread, often subcutis
Good correlation with sun exposure
Growth
topical application of gamma-5-aminolaevulinic acid or its methyl esters and then irradiated with visible light, causing phototoxic destruction of superficial layers of skin [48]. Cure rates are similar to that with cryotherapy but with improved cosmetic results [45]; efficacy, however, has been limited to superficial BCC [45].
Common Sites
Size of Lesion
Invasive Metastasis
Yes
Rarely, when metastasis occurs it appears in lymph nodes, lungs Recurs in 40–50% within 5 years
Danger sign Ulcer that won’t heal and bleeds when scratched
Ulcer that won’t heal and bleeds when scratched
No
Ulcer that won’t heal and bleeds when scratched
No, but recurrence common
Scat that ulcerates, oozes, or bleeds when scratched
Squamous Cell Carcinoma Squamous cell carcinoma (SCC) is the second most common cutaneous neoplasm. SCC has significant risk of metastisis and accounts for 20% of deaths from skin cancer [80]. SCC arises from the keratinizing malpighian cells of
555
556
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Neoplastic Skin Lesions in the Elderly Patient
the squamous layer [14] of the skin and mucous membranes [46]. P53 mutation is thought to be the primary mutagenic event [10]. Other tumor suppressor genes mutated in SCC include p16 (INK4a) and p14 (ARF). SCC is associated with chronic, long-term photodamage and is most commonly seen in exposed skin and mucous membranes [71]. Chronic sunburn is more strongly implicated in SCC than sunburns in childhood [81]. Other risk factors include prior trauma, frostbite, psoralen plus ultraviolet (PUVA) therapy, exposure to ionizing radiation or chemical carcinogens, viral oncogenesis, and chronic immunosuppression [38]. SCC often presents as a firm, discrete nodule, or plaque which arises on an erythematous elevated base. With time, the tumor can progress and become fungating, exophytic, with overlying crust. SCC in situ more typically presents as an erythematous scaly patch or plaque with clear margins. Both forms of SCCs are commonly associated with surrounding actinic damage. Clinicians should keep in mind that, though relatively rare in black patients, SCC is nevertheless the most common skin cancer in that population and more likely to occur in damaged or chronically inflamed skin in sun-exposed areas [82]. Like all skin cancers, diagnosis is often delayed in more heavily pigmented skin [83].
SCC In Situ Two forms of SCC may represent developing SCC in situ. Bowen’s disease lesions occur on nonexposed sites; they become more aggressive SCCs than those which arise from AK [46]. An association with internal malignancy typically signals arsenic occupational exposure as an etiological agent [84].
. Figure 55.4 Kerathoacanthoma
keratoacanthomas as a well-differentiated variant of SCC that arises from an alternative follicular-based, etiologic pathway, as distinct from SCCs that more commonly arise from solar keratoses. Further details on SCC are presented in > Table 55.5. Treatment for squamous cell carcinoma (SCC) employs many similar treatment options as for BCC. Surgical excision is the treatment of choice. As with BCC, the use of Mohs surgery for treatment of SCC results in a lower recurrence rate as compared to standard excision (3% vs. 8%) [38]. In the rare case of extensive SCC, topical treatments may be considered. Follow-up should be frequent and thorough, beginning at 3 months. Most recurrences (70%) occur within the first 2 years [38] and should be managed aggressively [71]. Use of high SPF sunblock has been persuasively demonstrated to reduce the risk of squamous cell carcinoma [86].
Melanomas Keratoacanthoma Keratoacanthomas, 55.4) whose classification as SCC is more controversial, are single dome-shaped nodules with a central keratin plug that typically arise in a hair follicle in a sun-exposed location [83]. These tumors grow rapidly for about 8 weeks, then remain static for about the same period before spontaneously regressing [37]. Recurrences are common, however, and typically more invasive than the initial tumor [37]. Once thought to be benign and self-limiting, keratoacanthomas both clinically and histopathologically resemble well-differentiated SCCs [83] and are now believed to represent SCC in progress [71]. The term infundibular squamous cell carcinoma [85] has been proposed to classify
Malignant Melanoma
(> Fig.
Malignant melanoma is the ‘‘near epidemic’’ directly related to exposure to solar radiation [9]. It accounts for about 80% of the fatalities related to skin cancer [87]. Older patients often are first diagnosed with advanced stages of the disease: consequently, lesions exhibit a higher grade at presentation, which results in a worse prognosis [88]. Melanoma is classified according to the TNM system, which evaluates the thickness of the tumor (known as the Breslow depth), the amount of ulceration present, the evidence of presence in the lymph nodes, and the presence of metastatic lesions in other tissues (perineural or perivascular involvement) or organs. Stage III reflects
Neoplastic Skin Lesions in the Elderly Patient
55
. Table 55.5 Differential diagnosis of squamous cell carcinoma Tschen et al. [47] Type of growth Squamous Cell Carcinoma (unspecific)
Description of lesions Crusted or scaly patches with red inflamed based, nonhealing ulcer, or enlarging growth, typically heaped-up, cauliflower appearance
Population & prevalence
Growth
Caucasian, Rapid especially fairskinned Higher incidence in men than women Average onset 60 years
Common sites Primarily head 75%, back of hands arms 15%, other 10% Strong correlation with sun exposure
Size of lesion 1–5 cm
Invasive
Metastasis
Danger sign
Yes
Yes to lungs or lips, nose, lymph ears etc. nodes Lips, ears, vulva, and penis have higher rates of metastasis Skin – 3% Lip – 11% SCC in burn scars, radiation scars, ulcers, up to 30%
Prevalence: 250,000 new cases/year, 2,500 deaths
Verrucous
Warty growth
Caucasian, Relatively especially fair- slow skinned
Plantar foot, Less anogenital than region 1 cm No correlation with sun exposed sites
Bowen’s disease (SCC in situ)
Round or oval Greater than erythematous 60 years plaques with well- of age defined margins and scaling. Loss of polarity gives wind-blown appearance. 55% occurs as multiple growths
KeratoLarge nodular acanthoma growth with epidermal lipping around keratinfilled crater. Usually appears as single nodule
Caucasian, onset in 60s or 70s. Higher incidence in men than women
AK = actinic keratoses, SCC = squamous cell carcinoma
Grows gradually with continuous peripheral extension, typically present for many years before suspicion of danger arises
Usually lower half of body: more often on nonexposed sites. Some cases related to arsenic exposure
As large as several inches
Grows very rapidly for about 6–8 weeks, then stops
Face, back of hands. Strong association with sun exposure
Greater Yes than 1 in diameter
No, but about 5% becomes invasive SCC
When invasive, more likely to metastasize than SCC arising in AKs
Rarely, but often recurs
Occurrence on nonexposed sites carries association with internal malignancy
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Neoplastic Skin Lesions in the Elderly Patient
evidence of in-transit or regional nodal metastasis or both; the presence of distant metastasis is defined as Stage IV [89]. Once melanoma has metastasized, a 5-year survival is about 10% [90]. Tumor thickness is the most important prognostic factor [21]. Staging by sentinel lymph-node biopsy, which identifies micrometastatic disease, has become common practice over the last few years as a prognostic tool, replacing elective lymphnode dissection. Significant survival advantage has yet to be proven in large randomized multicenter trials, and effective adjuvant therapies for patients demonstrated to carry micrometastatic disease are lacking [91]. The tumor site, however, has some prognostic influence, with location on the lower legs, upper arms, and head being associated with better outcomes than on the chest, back, and shoulders [92]. Women, on the other hand, have a better long-term prognosis than men [93]. This malignancy arises from melanocytes in basal layer of epidermis [14]. Although malignant melanomas may arise within two premalignant states, lentigo maligna or an existing mole, most arise de novo. About 25% of malignant melanomas arise from a formerly benign mole [87]. Risk factors for malignant melanoma include a light complexion, family history of melanoma, a history of sunburns as a child, and carriage of a high number of melanocytic or clinically atypical nevi [81]. Lentigo maligna are large brown macules on sunexposed skin that resemble large, permanent freckles. Lesions enlarge, but very slowly. Lentigo maligna melanoma, typically seen in older patients, arises from lentigo maligna on sun-exposed areas when malignant melanocytes penetrate the dermis [90]; however, most patients of advanced age carrying lentigo maligna lesions will die before any malignant changes occur [9, 75]. Superficial spreading melanoma, the most common form of malignant melanoma occurs primarily on the torso (men) and legs (women). Nodular melanoma enlarges rapidly in a typically darkened papule, although unpigmented nodules occasionally may arise. Acral-lentiginous melanoma, common in blacks (though rare in other ethnic groups) develops primarily on palmar, plantar, or subungual skin [90]. A melanotic melanoma is a nonpigmented lesion which is frequently misdiagnosed clinically as a BCC. It presents as a pink or flesh-colored nodule. The prevalence of melanoma at specific anatomic sites depends on age; nearly 80% of melanoma lesions in those over 80 occur on the head and neck. Although the risk of any one lesion becoming malignant is small, premalignant conditions should be watched carefully [71].
The exact role of solar radiation in the pathogenesis of malignant melanoma has been controversial for decades [94] and is still being debated [46, 91]. The majority opinion is that solar exposure is the principal carcinogen in melanoma, supported by the observation that its incidence is much higher in lighter-skinned individuals [95]. A correlation exists between sun exposure and melanoma incidence [92, 93]; this correlation is strengthened by association with gender-specific patterns of exposure (trunk in men, lower extremities in women) [96]. XP patients, who carry a mutation that eradicates repair of UV-induced damage to the DNA, are 1,000-fold more likely than the general public to develop melanoma [97]. UV-specific mutations P16/INK4a and ras are frequently observed in melanoma cells [98]. Comparison of melanoma risk between blacks and whites suggests that 96% of melanoma in men and 92% of melanoma in women could be contributed to UV exposure [99]. Persuasive arguments against a principal role of sunlight in the development of melanoma also exist, particularly by comparing data that support a role of UV in the etiology of BCC and SCC but are lacking or conflicting with respect to malignant melanoma. For perspective, the following observations support the role of sunlight in the carcinogenesis of BCC and SCC: they occur primarily in pale, sun-exposed skin [50]; they have a strong association with the degree of sun exposure and latitude [51]; they are effectively prevented by sun protection [44, 45]; readily UV-induced experimentally [53]; and are dramatically increased in patients with XP, in which UV-specific DNA repair is compromised [54]. By comparison, these lines of evidence are lacking with regard to melanoma. The variation of risk for melanoma is more ethnic [98–100] than pigmentary [100]; 75% of the lesions occur on nonexposed sites [95], especially on the feet in dark-skinned subjects [99, 100]; and the correlation of melanoma incidence to latitude is small and inconsistent in major geographic areas such as Europe and the USA [101]. Moreover, in numerous studies, melanoma incidence and mortality rates are inversely related to sun-exposure levels; moreover, neither does melanoma risk correlate strongly with the use of tanning beds [102], nor is melanoma readily induced by UV exposure in the lab [103]. Although, melanoma incidence is higher in XP patients, the elevation is far lower than for nonmelanoma skin cancers. It has also been observed that albino African-Americans rarely have cutaneous malignant melanoma, although BCC and SCC are prevalent in this group [94]. In addition, very little solar elastosis is found around
Neoplastic Skin Lesions in the Elderly Patient
CMM lesions in African-American albinos [94]. Epidemiological data records that the incidence of melanoma decreases in association with occupational limitation of sun exposure as well as higher national gross domestic product (GDP)[94]. Case-controlled studies indicate that intermittent sun exposure with sunburn, particularly in childhood, may be a primary risk factor for melanoma. This would offer some explanation for apparent contradictions in the literature regarding UV exposure as a risk factor [104]. The role of intermittent sun exposure and childhood sunburn is postulated to explain the lack of correlation between overall sun exposure and melanoma; however, a strong association of lesion site with burn site would be expected, yet is not observed [46, 99, 101]. The efficacy of sunblock in preventing malignant melanoma is still unproven. The incidence of melanoma has risen despite widespread use of high sun protection factor (SPF) sunblock since the early 1980s [105]. The comparative rarity of this form of skin cancer has hindered extensive prospective studies [86], and even retrospective data are limited. Consequently, the available data are inconclusive. In many studies, sunscreen had no demonstrable preventative power, and in others sunscreen use actually seemed to increase melanoma risk [86]. One explanation may be that chemical screens are primarily designed to avoid sunburns, blocking UVB while admitting virtually all of UVA radiation. Sunscreen use may thus allow people to spend more time in the sun than they would have otherwise, thereby increasing exposure to potentially mutagenic UVA [105]. More than one molecular mechanism may lead to the formation of malignant melanoma [106]. It has long been recognized that some melanomas arise from precursor nevi, while some arise spontaneously [106]. In addition, it has been observed that those with chronic solar exposure have a lower risk of melanoma compared to those with intermittent exposures [106]. Indeed, the B-raf oncogene (BRAF) mutation, although quite common in lesions on intermittently exposed skin, is quite rare on areas with extensive chronic sun damage, which strongly suggests divergent molecular pathways [107]. In black skin, melanoma typically does not occur in sun-exposed sites, but does appear on less pigmented sites [108]. It has been hypothesized that after UV exposure, the most severely damaged keratinocytes undergo apoptosis, while the remaining cells upregulate DNA-repair capacity [9]. The skin tans and thickens, providing additional protection. Subsequent exposure, if frequent, will perpetuate adaptive protection. Sporadic high-dose
55
exposures, however, may cause substantial damage but not apoptosis, so that mutated melanocytes survive and continue to divide. Certain mutations may enable such melanocytes to cross the basement membrane into dermis, where they proliferate and give rise to junctional nevi as a prelude to full-scale melanoma [21]. Further details on malignant melanomas are presented in > Table 55.6. The best treatment for melanoma is early detection, followed by biopsy to determine both the nature and depth of penetration of the lesion [90]. If the melanoma is still confined to the epidermis, surgical excision has a good prognosis [71]. If the melanoma has invaded the dermis and spread, surgical removal may be supplemented with additional therapies [89]. Adjuvant chemo- and radiotherapies have recently been joined by immunobased therapies like alpha interferon, particularly in cases where metastasis has reached the lymph nodes, and has resulted in significant improvement in remission and long-term survival rates [84, 120]. Surgery for melanoma metastases using 18-fluorodeoxyglucose positron emission tomography (FDG-PET) and FDG-PET computed tomography (FDG-PET/CT), both capable of providing highly sensitive tumor demarcation, provides for precise surgical margins [33, 35, 86]. Three 12-month randomized controlled trials of high-dose regimens of interferon alpha demonstrated a significant increase in recurrence-free survival in stage III disease, so adjuvant interferon in intermediate and high-risk CMM patients should be standard care [91]. One cytostatic drug, temozolomide (TMZ), has demonstrated significant improvement in progression-free survival in CMM [91]. Management requires adroit use of the available adjuvant diagnostic and therapeutic options. Chemotherapy, immunostimulants, and vaccines have failed as adjuvants in stage II–III disease. Interferon staves off relapses, but does not influence overall survival [60]. More accurate initial characterization of the tumor can greatly influence ultimate outcome, and emerging techniques show significant potential to improve diagnostic capabilities. Topical adjuvants can increase cure rates and are particularly beneficial when field cancerization has occurred.
Merkel Cell Carcinoma Merkel cell carcinoma (MCC) arises from Merkel cells in the epidermis. It represents a particularly aggressive form of melanoma. Regional metastasis occurs in up to two thirds of patients, with distant metastasis in one third of
559
560
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Neoplastic Skin Lesions in the Elderly Patient
them [41]. MCC is often initially misdiagnosed as other small-cell tumors [38] but histological and immunocytochemistry can assist in correct diagnosis [38]. Merkel cell carcinoma recurs in up to 44% of patients, usually within 4 months. The 3-year survival rate, with
appropriate excision and adjuvant chemotherapy, is only 55% [38]. AKs are commonly treated by application of liquid nitrogen (cryotherapy) as an effective method of eradication [109], with an 8-year cure rate of 98.8% [37].
. Table 55.6 Differential diagnosis of malignant melanoma Population & prevalence
Type of growth
Description of lesions
Malignant melanoma (unspecific)
Highly pigmented macule or papule, colors brown-black, red, white, gray, pink, or blue
Caucasian, especially fair-skinned. Highest incidence greater than 65 years. Incidence equal in men and women, although women better survival rate. Prevalence: 54,200 cases/ year, 7,600 deaths in the USA in 2003 [21]
Superficial spreading melanoma
Multicolored pigmented lesion with notched irregular margin, associated with acute sun exposure
Caucasian, especially fair-skinned. over 65 in men, younger in women
Large flat variegated plaque of brown-black. Usually single lesion. Associated with chronic sun exposure
Caucasian, especially fair-skinned
Lentigo Maligna (Melanotic freckle)
Growth
Slowly, up to 24 months before onset of invasive behavior
Sunexposed sites, trunk significantly more common in men, legs in women
Very slow, decades before becomes invasive
Head and neck account for 92%, most common site cheek; strong correlation with sun exposure
Prevalence: 70% of all malignant melanomas
Prevalence: 5% of all melanomas
Common sites
Size of lesion
May reach more than 3 cm
Invasive
Metastasis
Danger sign
Yes
Yes
Appearance of blue pigment in lesion, increase in size, peripheral halo of pigment, ulceration, hemorrhagic exudation, local satellite nodules
Yes
Yes, but less Formation of likely than nodule with other forms lesion, ulceration
Yes
Yes
Appearance of blackmacules or papules within lesion, induration, ulceration, nodule formation
Neoplastic Skin Lesions in the Elderly Patient
55
. Table 55.6 (Continued) Type of growth
Description of lesions
Acrallentiginous melanoma
Flat tanbrown stain with irregular borders. Usually single lesion
Population & prevalence Men more than women, highest incidence in blacks, Asians, and Hispanics. Primarily in those older than 65 Prevalence: 10% of all melanomas
Nodular
Multicolored nodule with ulceration. Usually single lesion
Growth Grows slowly, but since occurs in occult locations often overlooked until in advanced stages
Prevalence: 15% of all melanomas
Fast, no radial growth phase
Extends along peripheral nerves
Common sites Plantar foot, more rarely genitalia, mucous membranes
Size of lesion
Invasive
Metastasis
Yes
Yes
Less than Yes 2 cm in size
Yes
Danger sign Variegation in pigment, oozing, and crusting
Associated with acute sun exposure Desmoplastic melanoma
Nodule with unpredictable pigment and irregular features. Usually single lesion
Greater than 65 years of age, men more often than women
Merkel Cell Carcinoma
Shiny, indurated, pink, bluishred, or redbrown nodule
Greater than Relatively 65 years of slowly age. Primarily Caucasians
Head, neck, extremities, buttocks, trunk
Leukoplakia
Rough white patches with sharp borders
Greater than 65 years
Oral and genital mucosa
Conclusion Cutaneous lesions are not uncommon in older adults [4]. Increasing numbers of nonmalignant cutaneous lesions in older people, along with the pigmentation changes common to old age [71], can make identification of potential malignancies difficult even for dermatologists. Although very few lesions in an older person will likely
Head and neck, strong association with sun damage
Yes, often nerves
Typically Yes 0.5–5 cm, lesions up to 15 cm reported
Yes, but less aggressively than other melanomas
Typically long delay in correct diagnosis due to unusual features
Yes, frequently
Often not diagnosed before metastasis occurs Erosion, ulceration, or fissures
become malignant, cutaneous malignancy can carry a significant risk of mortality [110]. Although most skin growths are benign, sun exposure plays a crucial role in the development and progression of several types of cutaneous malignancies. Consequently, patients should consistently be made aware of the dangers of unprotected exposure to solar radiation [49]. Current evidence suggests that sunscreen use dramatically reduces
561
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Neoplastic Skin Lesions in the Elderly Patient
the risk of SCC, but may have less ability to protect against BCC and melanoma. Relying on sunscreen alone to lower the risk of skin cancers may thus be inadvisable [86]: patients should be instructed to wear hats, sunglasses, and to avoid short sleeves or shorts if they anticipate spending an extended period of time in the sun [105]. Clinicians must emphasize the risk of recurrence to all skin cancer patients and communicate that avoidance of further UV exposure is critical [40]. Older patients should be encouraged to bring to the physician’s attention any new or changed skin lesions, but family practitioners should also assume responsibility for identifying any potentially malignant skin changes [43]. Physical examinations should include the entire surface of the skin, with special attention to places with chronic sun exposure, particularly overlooked sites like eyelids, folds of nose, earlobes, and lips [71]. The relatively low risk of skin cancer in African-Americans should not reduce importance of thorough examination, as all skin cancers carry higher mortality rates in this population due to delayed detection. Available therapies for skin cancers, particularly the NMSC, are highly effective; yet recurrence is substantial, creating significant morbidity for the patient as well as a significant burden on the health care system. These effects will only increase as the proportion of older people in the population rises [24]. Clinicians should emphasize to their patients the very real risk of recurrence, and stress that regular checkups are critical in order for patients to remain skin-cancer free [40].
Clinical management of skin malignancies has relied primarily on cryotherapy or surgical excision and on avoidance of sun exposure. Although excision results in a 95% overall cure rate, skin malignancies still kill about ten thousand Americans each year. Sunscreen, although a practical and safe approach to preventing many lesions with malignant potential, is not universally effective; moreover, and, because sunscreen use promotes longer UV exposure times, it may actually increase the risk for some skin cancers [111]. For at least some malignancies, carcinogenesis involves additional factors besides sun exposure [23, 64]. Confounding factors in all studies of sunscreen efficacy are that sunscreen application is difficult to both verify and quantify, and that latency periods (believed to be as much as 4 or 5 decades) are longer than any studies yet completed [111]. The relative contributions to the process of carcinogenesis of suninduced DNA damage, a compromised immunologic response to nascent changes in the cell, and the individual’s personal risk factors, continue to be investigated on a molecular level; better understanding of the actual pathologic mechanisms of cancer formation will undoubtedly lead to better therapeutic approaches. Numerous efficacious topical treatments have been developed over the last decade or so, but none yet exceeds the efficacy of traditional excision. Use of these topical medicaments as adjuvants may increase survival rates. Immunotherapies, vaccines, adoptive immunotherapies, manipulation of cytokines, blockage of signaling pathways, and modulation of T-cell function, have been, so
. Table 55.7 Comparison of etiologic and diagnostic parameters in cutaneous malignancy Estimated lifetime risk in fair-skinned Lesion individuals
Increased risk in fairskinned individuals as compared to Blacks
Increased risk in OTR patients
Percentage containing p53 mutations
Percent containing aneuploid cells
Percentage on sunexposed skin
AK
100% in some Virtually nonexistent in geographical areas blacks [135] [41]
50% [136]
80% [137]
69% [14]
80% [40]
BCC
33% [138,139]
80-fold [82]
100-fold [70]
56% [140]
Up to 40% [14]
80% [141]
SCC
11% [138,139] cancer: clinical
65-fold [56]
82-fold [142]
90% [21,143]
Up to 80% [14]
82% [144]
CMM
1.3% USA [145]
50-fold [21]
8-fold [18]
20% [35]
100% of Stage 5 [14]
Varies by age group, 80% in those over 80 years of age [146]
4% AUS [145]
AK = actinic keratoses, AUS = Australia, BCC = basal cell carcinoma, CMM = cutaneous malignant melanoma, OTR = organ transplant recipient, SCC = squamous cell carcinoma, US = United States
Neoplastic Skin Lesions in the Elderly Patient
far, largely unsuccessful [18]. Monoclonal antibodies as yet have also not lived up to initial expectations, although only a small fraction of possible antigens have been studied. The ongoing identification of critical molecular events in carcinogenesis provides many more potential approaches both for targeted therapies and for preventative treatments [112]. Improvement in therapies remains somewhat elusive, yet many novel approaches to better diagnosis and for predicting of malignant potential have been described. A comparison of etiologic and diagnostic parameters for cutaneous malignancies is shown in > Table 55.7. Although of significant benefit in research, it is yet to be seen if these new laboratory tools will become costeffective for clinicians [55]. Early diagnosis has enabled success in maintaining mortality rates for melanoma constant even though incidence has soared. Early diagnosis should remain the frontline focus – the diligent and meticulous examination of the older patient’s skin – yet few clinicians do this [113]. Consistent proactive surveillance of aging skin will not only prolong life but also significantly improve the aged patient’s quality of life.
Cross-references > Melanoma
and Skin Aging
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Neoplastic Skin Lesions in the Elderly Patient treatment of primary nodular basal cell carcinoma. Australas J Dermatol. 2006;47:46–48. 79. Spencer JM. Pilot study of imiquimod 5% cream as adjunctive therapy to curettage and electrodesiccation for nodular basal cell carcinoma. Dermatol Surg. 2006;32:63–69. 80. Szeimies R, Gerritsen MP, Gupta G, et al. Imiquimod 5% cream for the treatment of actinic keratosis: results from a phase III, randomized, double-blind, vehicle-controlled, clinical trial with histology. J Am Acad Dermatol. 2004;51:547–555. 81. Kennedy C, Bajdik CD, Willemze R, et al. The influence of painful sunburns and lifetime sun exposure on the risk of actinic keratoses, seborrheic warts, melanocytic nevi, atypical nevi, and skin cancer. J Invest Dermatol. 2003;120:1087–1093. 82. Garrett AB. Skin cancer in people of color. In: Snow N, Mikhail GR (eds) Mohs Micrographic Surgery. Madison: University of Wisconsion Press, 2004. 83. Mueller CS, Reichrath J. Histology of melanoma and nonmelanoma skin cancer. In: Reichrath J (ed) Sunlight, Vitamin D and Skin Cancer. Austin: Landes Bioscience, 2008. 84. Akhdari N, Amal S, Ettalbi S. Bowen disease. CMAJ. 2006;175:739. 85. Kossard S, Tan K, Choy C. Keratoacanthoma and infundibulocystic squamous cell carcinoma. Am J Dermatopathol. 2008;30: 127–134. 86. Gallagher RP. Sunscreens in melanoma and skin cancer prevention. CMAJ. 2005;173:244–245. 87. American Academy of Dermatology: Bullous Disease http://www.aad. org/public/Publications/pamphlets/bullous.htm. cited December 29, 2008. 88. Giles GG, Armstrong BK, Burton RC, et al. Has mortality from melanoma stopped rising in Australia? Analysis of trends between 1931 and 1994. BMJ. 1996;312:1121–1125. 89. McCartney RA. Malignant Melanoma In: Gale Encyclopedia of Cancer. Farmington Hills: Thomson Gale Publishing, 2002. 90. Beers MH, Berkow BR. Dermatologic disorders: malignant tumors. In: Beers MH, Berkow R (eds) The Merck Manual of Diagnosis and Therapy. Whitehouse Station: Merck Research Laboratories, 2002. 91. Rass K, Tilgen W. Treatment of melanoma and nonmelanoma skin cancer. In: Reichrath J (ed) Sunlight, Vitamin D and Skin Cancer. Austin: Landes Bioscience, 2008. 92. Gillgren P, Brattstro¨m G, Frisell J, et al. Effect of primary site on prognosis in patients with cutaneous malignant melanoma. A study using a new model to analyse anatomical locations. Melanoma Res. 2005;15:125–132. 93. Cochran AJ, Elashoff D, Morton DL, et al. Individualized prognosis for melanoma patients. Hum Pathol. 2000;31:327–331. 94. Moan J, Porojnicu A, Dahlback A. Ultraviolet radiation and malignant melanoma. In: Reicharth J (ed) Sunlight, Vitamin D and Skin Cancer. Austin: Landes Biosciences, 2008. 95. Armstrong BK, Kricker A. The epidemiology of UV induced skin cancer. J Photochem Photobiol B. 2001;63:8–18. 96. Tsao H, Sober AJ. Ultraviolet radiation and malignant melanoma. Clin Dermatol. 1998;16:67–73. 97. Kraemer KH, Lee MM, Andrews AD, et al. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm. Arch Dermatol. 1994;130: 1018–1021. 98. Hussein MR. Ultraviolet radiation and skin cancer: molecular mechanisms. J Cutan Pathol. 2005;32:191–205. 99. Armstrong BK, Kricker A. Skin cancer. Dermatol Clin. 1995;13: 583–594.
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100. Le Marchand L, Saltzman BS, Hankin JH, et al. Sun exposure, diet, and melanoma in Hawaii Caucasians. Am J Epidemiol. 2006;164: 232–245. 101. Crombie IK. Variation of melanoma incidence with latitude in North America and Europe. Br J Cancer. 1979;40:774–781. 102. Gallagher RP, Spinelli JJ, Lee TK. Tanning beds, sunlamps, and risk of cutaneous malignant melanoma. Cancer Epidemiol Biomarkers Prev. 2005;14:562–566. 103. Setlow RB, Woodhead AD, Grist E. Animal model for ultraviolet radiation-induced melanoma: platyfish-swordtail hybrid. Proc Natl Acad Sci USA. 1989;86:8922–8926. 104. Menzies SW. Is sun exposure a major cause of melanoma? Yes. BMJ. 2008;337:a763. 105. Garland CF, Garland FC, Gorham ED. Rising trends in melanoma. An hypothesis concerning sunscreen effectiveness. Ann Epidemiol. 1993;3:103–110. 106. Rivers JK. Is there more than one road to melanoma? Lancet. 2004;363:728–730. 107. Maldonado JL, Fridlyand J, Patel H, et al. Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst. 2003;95: 1878–1890. 108. Garrett AB. Skin cancer in people of color. In: Snow N, Mikhail GR (eds) Mohs Micrographic Surgery. Madison: University of Wisconsion Press, 2004. 109. Callen JP, Bickers DR, Moy RL. Actinic keratoses. J Am Acad Dermatol. 1997;36:650–653. 110. Kurban RS, Kurban AK. Common skin disorders of aging: diagnosis and treatment. Geriatrics. 1993;48:30–1, 35–6, 39–42. 111. Berwick M. Counterpoint: sunscreen use is a safe and effective approach to skin cancer prevention. Cancer Epidemiol. Biomarkers Prev. 2007;16:1923–1924. 112. Xie J. Molecular biology of basal and squamous cell carcinomas. In: Reichrath J (ed) Sunlight, Vitamin D and Skin Cancer. Austin: Landes Bioscience, 2008. 113. Wartman D, Weinstock M. Are we overemphasizing sun avoidance in protection from melanoma? Cancer Epidemiol Biomarkers Prev. 2008;17:469–470. 114. Piacquadio DJ, Chen DM, Farber HF, et al. Photodynamic therapy with aminolevulinic acid topical solution and visible blue light in the treatment of multiple actinic keratoses of the face and scalp: investigator-blinded, phase 3, multicenter trials. Arch Dermatol. 2004;140:41–46. 115. Sandberg C, Stenquist B, Rosdahl I, et al. Important factors for pain during photodynamic therapy for actinic keratosis. Acta Derm Venereol. 2006;86:404–408. 116. Pariser DM, Lowe NJ, Stewart DM, et al. Photodynamic therapy with topical methyl aminolevulinate for actinic keratosis: results of a prospective randomized multicenter trial. J Am Acad Dermatol. 2003;48:227–232. 117. Wennberg AM, Lindholm LE, Alpsten M, et al. Treatment of superficial basal cell carcinomas using topically applied deltaaminolaevulinic acid and a filtered xenon lamp. Arch Dermatol Res. 1996;288:561–564. 118. Horn M, Wolf P, Wulf HC, et al. Topical methyl aminolaevulinate photodynamic therapy in patients with basal cell carcinoma prone to complications and poor cosmetic outcome with conventional treatment. Br J Dermatol. 2003;149:1242–1249. 119. Loven K, Stein L, Furst K, et al. Evaluation of the efficacy and tolerability of 0.5% fluorouracil cream and 5% fluorouracil cream applied to each side of the face in patients with actinic keratosis. Clin Ther. 2002;24:990–1000.
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120. Weiss J, Menter A, Hevia O, et al. Effective treatment of actinic keratosis with 0.5% fluorouracil cream for 1, 2, or 4 weeks. Cutis. 2002;70:22–29. 121. Wolf JEJ, Taylor JR, Tschen E, et al. Topical 3.0% diclofenac in 2.5% hyaluronan gel in the treatment of actinic keratoses. Int J Dermatol. 2001;40:709–713. 122. Rivers JK, Arlette J, Shear N, et al. Topical treatment of actinic keratoses with 3.0% diclofenac in 2.5% hyaluronan gel. Br J Dermatol. 2002;146:94–100. 123. Fariba I, Ali A, Hossein SA, et al. Efficacy of 3% diclofenac gel for the treatment of actinic keratoses: a randomized, double-blind, placebo controlled study. Indian J Dermatol Venereol Leprol. 2006;72:346–349. 124. Grimaıˆtre M, Etienne A, Fathi M, et al. Topical colchicine therapy for actinic keratoses. Dermatology (Basel). 2000;200: 346–348. 125. Akar A, Bu¨lent Tas¸tan H, Erbil H, et al. Efficacy and safety assessment of 0.5% and 1% colchicine cream in the treatment of actinic keratoses. J Dermatolog Treat. 2001;12:199–203. 126. Stockfleth E, Meyer T, Benninghoff B, et al. A randomized, doubleblind, vehicle-controlled study to assess 5% imiquimod cream for the treatment of multiple actinic keratoses. Arch Dermatol. 2002;138:1498–1502. 127. Salasche SJ, Levine N, Morrison L. Cycle therapy of actinic keratoses of the face and scalp with 5% topical imiquimod cream: an open-label trial. J Am Acad Dermatol. 2002;47:571–577. 128. Persaud AN, Shamuelova E, Sherer D, et al. Clinical effect of imiquimod 5% cream in the treatment of actinic keratosis. J Am Acad Dermatol. 2002;47:553–556. 129. Lebwohl M, Dinehart S, Whiting D, et al. Imiquimod 5% cream for the treatment of actinic keratosis: results from two phase III, randomized, double-blind, parallel group, vehicle-controlled trials. J Am Acad Dermatol. 2004;50:714–721. 130. Beutner KR, Geisse JK, Helman D, et al. Therapeutic response of basal cell carcinoma to the immune response modifier imiquimod 5% cream. J Am Acad Dermatol. 1999;41:1002–1007. 131. Marks R, Gebauer K, Shumack S, et al. Imiquimod 5% cream in the treatment of superficial basal cell carcinoma: results of a multicenter 6-week dose-response trial. J Am Acad Dermatol. 2001;44: 807–813. 132. Geisse JK, Rich P, Pandya A, et al. Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: a double-blind, randomized, vehicle-controlled study. J Am Acad Dermatol. 2002;47:390–398.
133. Sterry W, Ruzicka T, Herrera E, et al. Imiquimod 5% cream for the treatment of superficial and nodular basal cell carcinoma: randomized studies comparing low-frequency dosing with and without occlusion. Br J Dermatol. 2002;147:1227–1236. 134. Shumack S, Robinson J, Kossard S, et al. Efficacy of topical 5% imiquimod cream for the treatment of nodular basal cell carcinoma: comparison of dosing regimens. Arch Dermatol. 2002;138:1165–1171. 135. Spencer J. Actinic Keratoses. http://www.emedicine.com/derm/ topic9.htm. cit. 136. Ulrich C, Busch JO, Meyer T, et al. Successful treatment of multiple actinic keratoses in organ transplant patients with topical 5% imiquimod: a report of six cases. Br J Dermatol. 2006;155:451–454. 137. Ortonne J. From actinic keratosis to squamous cell carcinoma. Br J Dermatol. 2002;146(61):20–23. 138. Weinstock MA. Epidemiologic investigation of nonmelanoma skin cancer mortality: the Rhode Island Follow-Back Study. J Invest Dermatol. 1994;102:6S–9S. 139. Weinstock MA. Epidemiology of nonmelanoma skin cancer: clinical issues, definitions, and classification. J Invest Dermatol. 1994; 102:4S–5S. 140. Soehnge H, Ouhtit A, Ananthaswamy ON. Mechanisms of induction of skin cancer by UV radiation. Front Biosci. 1997;2: d538–51. 141. Leman JA, McHenry PM. Basal cell carcinoma: still an enigma. Arch Dermatol. 2001;137:1239–1240. 142. Moloney FJ, Comber H, O’Lorcain P, et al. A population-based study of skin cancer incidence and prevalence in renal transplant recipients. Br J Dermatol. 2006;154:498–504. 143. Zhang H, Ping XL, Lee PK, et al. Role of PTCH and p53 genes in early-onset basal cell carcinoma. Am J Pathol. 2001;158: 381–385. 144. Scotto J, Fears TR, Fraumeni JF. Incidence of non-melanoma skin cancer in the United States. Bethesda: US Department of Health and Human Services, National Institutes of Health, 1983. 145. Rigel DS, Carucci JA. Malignant melanoma: prevention, early detection, and treatment in the 21st century. CA Cancer J Clin. 2000;50:215–36, quiz 237–40. 146. Hoersch B, Leiter U, Garbe C. Is head and neck melanoma a distinct entity? A clinical registry-based comparative study in 5702 patients with melanoma. Br J Dermatol. 2006;155:771–777. 147. Detailed Guide to Skin Cancer. Basal and Squamous cell. June, 2008. American Cancer Society electronic publication. http:// www.cancer.org/docroot/CRI/CRI_2_3x.asp?dt=51.
Part 2
Disease State/Conditions with Aging
Diseases Associated with Aging
54 Non-neoplastic Disorders of the Aging Skin Miranda A. Farage . Kenneth W. Miller . Enzo Berardesca . Howard I. Maibach
Introduction
Xerosis
The skin is the barrier between vulnerable internal tissues and a plentitude of environmental factors with the potential to negatively impact those tissues [1]. Degenerative processes inherent to aging produce the characteristic thinning, drying, and sagging of elderly skin [2] as well as a progressive deterioration in skin function [3]. This process is based to some extent on ethnicity and gender [4]. However, most visible changes in aged skin [4] as well as most pathological changes are due to a lifetime of exposure to external environmental insult [1]. Degenerative processes that occur in aging skin and their clinical significance are shown in > Table 54.1. It is estimated that 7% of all physician visits by the elderly involve skin disorders [5], and that treatable (but often untreated) cutaneous diseases occur in more than 50% of otherwise healthy older adults [5]. Most people over 65 years have at least two skin diseases worthy of treatment [6], and 10% of those over 70 years of age have more than ten concurrent complaints [7]. Although the stigma of ‘‘looking old’’ drives a $2 billion industry in the USA alone [8], skin aging is far from just a cosmetic complaint. Disorders of the skin, particular xerosis and pruritus, account for as many as 80% of skin complaints in the aged [9]; they produce significant suffering for those afflicted and significantly reduce quality of life in the later years [10]. Effective management of skin disease in old age must take into account the frailness of some patients as well as personal and social issues that affect them [11, 12].
As the skin ages, the epidermis becomes thinner, the water and lipid content of the skin diminish, and sebum production and sweating decline. This results in drier skin (> Fig. 54.1) [13]. Along with these changes, the process of keratinocyte maturation and adhesion degenerates. In combination, these changes result in skin that is dry, rough, and scaly, a condition called xerosis [14]. Xerosis (or asteatosis) is the most common dermatosis in older patients, occurring at equal rates in men and women [9], with incidence as high as 85% [15]. By age 70, nearly all adults are affected [16]. Because xerosis is aggravated by low humidity [14], its onset is often linked to the initiation of home heating [16], leading to the nickname ‘‘winter itch’’ [17]. Surface irritants, such as harsh soaps and other cleansers, can further aggravate the problem [14]. Xerotic skin causes considerable discomfort and distress in the older patient. Moreover, xerosis, and other common disorders that compromise skin integrity, may increase skin permeability to environmental allergens. This factor is now recognized to contribute to recalcitrant skin disorders in the elderly, such as nummular eczema. The key to managing xerosis in the aging patient is first to rehydrate the skin and then to lock in hydration [18]. A relative humidity level of 60% is required for atmospheric moisture to supplement stratum corneum hydration [19]; the use of room humidifiers, especially in the winter months, is advisable [16]. Applying humectant moisturizers also can increase epidermal water content [18]. Bathing, which has traditionally been discouraged in those suffering from xerosis, actually rehydrates the stratum corneum [20]. Tepid baths should be indulged in [19] soaking for at least 10 min to enable the stratum corneum to absorb water [15]. Use of soaps should be minimal and limited to gentle, nonirritant preparations [13]. Moisturizers should be applied liberally immediately
The Problem of Dry Skin The two most common conditions of dry skin are xerosis and pruritus.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_54, # Springer-Verlag Berlin Heidelberg 2010
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Non-neoplastic Disorders of the Aging Skin
. Table 54.1 The clinical implications of aging skin Physiological change
Pathological change
Clinical significance
Thinning of epidermis and dermis
Increased vulnerability to mechanical trauma, especially shearing and friction
Increased incidence of skin tears and decubitus ulcers
Flattening of dermal papillae
Increased risk of blister formation
Increased susceptibility of infection
Slowdown in epidermal turnover rate; decreased ratio of proliferative to differentiated keratinocytes
Delayed cellular migration and Increased time to re-epithelialization proliferation Longer healing times after injury or surgery Decreased wound contraction
Longer healing times after injury or surgery
Decrease in elastin fibers
Loss of elasticity
Decrease in vascularity and supporting structures in dermis
Blood vessels fragile, easily broken
Lax skin, wrinkling, Skin easily bruised (Senile purpura)
Decreased wound capillary growth
Increased risk of wound dehiscence
Decrease in vascular plexus, blunted capillary loops
Loss of thermoregulatory ability
Hypothermia, heat stroke
Changes in and loss of collagen and elastin fibers
Decreased tensile strength, lower skin layers more susceptible to injury
Increased risk of pressure damage to elderly skin, decubitus ulcers
Delayed collagen remodeling
Longer healing times after injury or surgery
Impaired inflammatory response
Impaired wound healing
Impaired delayed contact hypersensitivity reaction
Increased risk of severe injury from irritants and contact sensitizers
Decreased production of cytokines
Diminished immune function
Decrease in numbers of Langerhans cells
Increased susceptibility to photocarcinogenesis, false negative results in patch tests for delayed contact hypersensitivity
Impaired neurological responses
Reduced sensation
Increased risk of thermal or other accidental injury
Decreased skin thickness
Loss of cushioning and support Increased risk of pressure damage, decubitus ulcers Increased susceptibility to skin tears, bruising
Impaired immune response
Atrophy of sweat glands
Decreased production of vitamin-D precursor
Osteoporosis and bone fractures
Decreased sweating
Impaired thermoregulation; hypothermia Dry skin, xerosis
Reduced stratum corneum lipids
Decreased ability to retain water
Dry skin, xerosis
Structural changes in stratum corneum
Altered barrier function
Variable response to topical medications, altered sensitivity to irritants
Reduced water movement from dermis to epidermis
Reduced epidermal hydration
Dry skin, xerosis
Decrease in melanocytes
Loss of ability to tan, more susceptible to solar radiation
Cutaneous neoplasms
Graying hair
Loss of self-esteem
> Table
54.1 summarizes findings from Refs. [5, 19, 118, 134, 135].
Non-neoplastic Disorders of the Aging Skin
. Figure 54.1 Xerosis: Skin that is dry, rough, and scaly
after bathing. This fills the spaces between the keratinocytes with lipid [18], and encourages corneosome degradation of the corneodesmosome [21]. Petrolatum products also help to prevent transepidermal water loss (TEWL), thus maintaining skin hydration [22, 23]. Lotions containing ammonium lactate have proven effective in the treatment of xerosis [24]. Topical corticosteroids can be prescribed for especially severe cases [14].
Pruritus (Itch) Pruritus is a common dermatological problem in the aged, with reported prevalence as high as 29% [25]. It is more common in men than in women [26]. Itching, which can be intense, may be accompanied by sensations of tingling or burning [27]. Severity often increases at night, which may be due to a nocturnal rise in internal body temperature [28]. Chronic scratching causes the skin to become thickened and hyperpigmented, a process known as lichenification [29]. Lichen simplex chronicus, the result of this process, is a skin condition on itchy sites accessible to scratching, and is marked by the appearance of lichenified scaly plaques [16] or thick raised papules with linear excoriations from scratching [30]. Lichen simplex chronicus is more prevalent in women than in men and generally clears quickly where scratching is avoided [30]. Xeroris is the most common cause of pruritus associated with a thickened and cracked stratum corneum [31]. In one survey, xerosis accounted for almost 40% of all patients with pruritus [9]. Patients with pruritus have clinically drier skin than matched controls; the severity of the itch is directly linked to the degree of xerosis, skin surface conductance, and presence of intracorneal
54
adhesions. This suggests that an abnormality of keratinization may be involved [31]. The causes and mechanisms of itching are diverse. One study in 149 elderly patients identified the most common disorders associated with senile pruritus, in order of prevalence, as xerosis, inflammatory eczematous disorders, lichen simplex chronicus, skin infections, psoriasis vulgaris, urticaria, reactions to various drugs, and insect bites [9]. Therefore, elucidation of the cause of an individual patient’s suffering, which can drive some patients to consider suicide, can be very challenging [29]. Effective management of pruritis requires a systematic approach to discovering the causative factors [29]. About half of pruritus cases occur without any physical signs, making diagnosis difficult [25]. Nevertheless, the patient’s subjective complaints are accompanied by nocturnal scratching: A direct correlation has been established between the severity of itching that the patient reports and measurable nocturnal limb movement [25]. Several types of medications produce pruritus in varying degrees. These include: antibiotics, diuretics, nonsteroidal antiinflammatory drugs (NSAIDs), and calcium channel blockers [25]. Resolution of symptoms may take several weeks after drug withdrawal [32]. Idiopathic pruritus increases in both frequency and severity with increasing age [31]; 69% of idiopathic pruritus in one study involved patients older than age 60 [25]. Generalized pruritus often coincides with the onset of emotional or psychological stress [25]; stress acts to increase the perception of itch [25]. Older patients may have severe and intractable emotional issues such as financial pressures, chronic health issues, boredom, bereavement, and loneliness [25]; these should not be discounted as possible contributors to idiopathic itching [33]. When a patient has a sudden onset of generalized persistent itching, aggressive pursuit of the etiology should be initiated, with a minimum of complete blood count (CBC), urinalysis, thyroid evaluation, tests of renal function, and a chest X-ray to rule out evidence of other internal disease (> Table 54.2) [34]. The origin of pruritus, in fact, often proves to be multifactorial [35]. Treatment depends on the specific source of each patient’s symptoms. Treatment for pruritus of xerotic origin has been described above. Topical and systemic corticosteroids and antihistamines, as well as topical cooling agents and anesthetics have been employed. Physical therapies include phototherapy, acupuncture, thermal stimulation, and transcutaneous electrical nerve stimulations (TENS) [29]. Oral antihistamines are the most commonly prescribed therapy for pruritis [35]. Systemic corticosteroids
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Non-neoplastic Disorders of the Aging Skin
. Table 54.2 Pruritus screen for underlying disease Laboratory analysis Complete Blood Count (CBC)
Abnormal finding
Possible etiology of pruritus
Anemia
Lymphoma/leukemia, myelodysplastic syndrome, iron deficiency
Eosinophilia
Drug reaction, helminthic infestation
Lymphopenia
Leukemia, Acquired Immune Deficiency Syndrome (AIDS)
Polycythemia
Polycythemia vera
Plasma viscosity
Out of normal range
Myeloma, infection, malignancy
Ferritin
Iron deficiency
Anemia
Electrolytes
Out of normal range
impaired renal function
Liver function test
Out of normal range
Obstructive jaundice (gallstones, tumor, primary biliary cirrhosis)
Thyroid function
Out of normal range
Hypothyroidism, hyperthyroidism
Autoantibodies
Out of normal range
Thyroid disease, primary biliary cirrhosis
Renal function
Elevated blood creatinine levels
Diabetes mellitus
Chest X-ray
Mass
Carcinoma of the bronchus
Mediastinal lymph nodes
Lymphoma
> Table
54.2 summarizes findings from References [11, 29]. AIDS acquired immune deficiency syndrome.
are used in approximately 30% of cases [35]. Treatment decisions must take into consideration that antihistamines may be sedating in the older patient, and that corticoids can accentuate atrophy in skin already compromised by sun damage [36]. In addition, topical preparations used to treat pruritus may cause contact sensitivity, which should be suspected if a weeping, vesicular, crusting dermatitis develops [37]. Resolution of the older patient’s symptoms can at times proves elusive. Histamine release can induce a perpetuating cycle [38]. Senile insomnia makes night time scratching more common.
. Figure 54.2 Eczema (Atopic Dermatitis) – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
Inflammatory Scaling Dermatoses Inflammatory dermatoses are common and include a variety of clinical conditions such as eczema, contact dermatitis, and seborrheic dermatitis. Accurate histological diagnosis of the underlying conditions, which can sometimes be difficult, is important for successful clinical management.
Eczema Atopic eczema is less common in older adults, but nummular (discoid) eczema, gravitational eczema, and asteatotic eczema occur almost exclusively among the aged [1].
Epidermal inflammation with predominant intracellular edema is common to these conditions. Edematous fluid collects in surface vesicles, which may weep (> Fig. 54.2) [39]. Diagnostic difficulties arise when there are secondary manifestations caused by scratching or by infection. However, the three hallmark signs of eczematous inflammation should be recognized: erythema, scaling, and vesicles [40]. Eczematous inflammation is virtually always accompanied by itching [40]. Most eczematous diseases, if left untreated and neither scratched nor irritated, will
Non-neoplastic Disorders of the Aging Skin
resolve without complication. Unfortunately, this situation is almost never achieved [40], as virtually all patients will scratch (even during sleep [40]), and attempts at selftreatment with over-the-counter (OTC) topical drugs is very common [41]. The clinical development of eczema has been divided into three phases, although the disease may present at any phase. The acute phase of eczema includes erythematous lesions and pain, heat, and tenderness [42]. The skin may sting, burn, or itch intensely and vesicles and blisters may develop [42]. Epidermal thickening is present in the acute phase of all forms of eczema [39]. Skin biopsies show inflammatory cells and swelling. The subacute phase of eczema does not exhibit pronounced swelling but exhibits reddened, scaly, and fissured skin that appears parched or scalded [40]. The subacute phase tends to be characterized by itching rather than pain [42]. Chronic eczema is defined by the presence of lesions for more than 3 months [40]; itching is pronounced, and scratching often exacerbates existing lesions [42]. In the chronic stage, the skin is thickened and lichenified, often with marked excoriation and/or fissuring [40]. Symptoms tend to worsen in the winter months due to low humidity in the home or office [43].
Asteatotic Eczema Xerotic skin, particularly in men, may exhibit stratum corneum fissuring in a cracked porcelain pattern, particularly on the lower legs. This condition, initially termed ‘‘eczema craquele´,’’ is now known as asteatotic eczema [13]. Usually, it first appears on the shins in a polygonal pattern resembling a dried-up riverbed [44]. Fissures can be deep enough to disrupt dermal capillaries, causing bleeding [13]. The affliction is often accompanied by intense itching [14]; repetitive scratching can then result in secondary lesions [13]. The cause of asteatotic eczema is believed to be a combination of intrinsic, environmental, and lifestyle factors. Skin dries naturally with old age, as keratin synthesis and sebaceous gland activity wanes [2]; low humidity and cold intensify dryness and exacerbate the condition in northern winters [45]. Frequent bathing in hot water with use of degreasing soaps, combined with infrequent use of emollients, can promote the condition [40]. Treatment consists primarily of avoiding soaps and hot baths, but indulging in warm baths, and applying alpha-hydroxy acid preparations after bathing to preserve skin hydration [45]. Short-term use of a steroid ointment, such
54
as triamcinolone (for 4–5 days), is indicated in more serious cases [45]. In most patients, asteatotic eczema is an irksome, but not a dangerous condition, although if not adequately treated, it can compromise skin integrity and increase the risk of contact dermatitis or infection [45]. In some cases, however, the disorder may be the external manifestation of a more serious internal problem. Asteatotic eczema may be linked to a deficiency of zinc or essential fatty acids [46, 47], thyroid disease [48], and various forms of cancer [49, 50]. In a recent prospective observational study of 68 patients hospitalized with asteatotic eczema, concurrent cancers were discovered in 47% of the patients [51]. These concurrent cancers were strongly associated with specific clinical characteristics of the eczema: deep red fissures, widespread lesions (particularly on the trunk), and resistance to topical steroid treatment were significantly associated with internal malignancy [51].
Nummular (Discoid) Eczema Nummular eczema occurs most often in elderly men, usually on the extremities. The presentation of nummular eczema varies widely, from a sudden onset of florid patches of erythema with vesicles and swelling, to slow-growing, dry patches of scale [52]. Lesions are coin-shaped macules, papules, or vesicles that ooze and crust over, and are superimposed on scaly or raw inflamed skin [53]. Lesions, which vary greatly in number, are generally between 1 and 5 cm in diameter and may be mistaken for ringworm or psoriasis [52]. Scale is usually thin and sparse [40]. Papules or vesicles eventually coalesce to form nummular plaques that within a few days become dry and scaly, often with central clearing. Plaques eventually turn brown and flatten into macules, which may persist as hyperpigmented areas [54]. Nummular eczema often recurs, with new lesions appearing in the same area as former ones [54]. The lesions are frequently secondarily infected [54]. The pathophysiology of nummular eczema is not well understood. The association with xerotic skin [55], irritant, or atopic skin reactions [56, 57], recent loss of skin integrity [54], and interferon or necrosis factor-alpha blocking therapies [58, 59] is beginning to shed light on the possible origins of nummular eczema. Xerotic skin of the elderly may cause, particularly in winter or other adverse environmental conditions, a cracking and fissuring of the skin’s surface [55]. Initial dryness and skin damage produces pruritus, and subsequent scratching exacerbates damage, with a breach of skin integrity that allows skin permeation of a variety of environmental allergens.
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Non-neoplastic Disorders of the Aging Skin
The penetration of environmental allergens past the stratum corneum results in the eczematous changes characteristic of nummular eczema, seemingly an expression of delayed hypersensitivity despite advanced age [55]. Significant temperature changes, low humidity, and stress may worsen symptoms [52]. In the acute phase, inflammation and itching can be alleviated by applying cool wet dressings: evaporative cooling produces vasoconstriction and serum production. Dressings should be replaced every 30 min [40]. When lesions are intense or widespread, oral corticosteroids (prednisone) can be added at a dosage of least 30 mg bid (with no tapering) for at least 3 days; up to 3 weeks may be necessary for adequate control [40]. Antihistamine relieves itching and facilitates sleep. Systemic antibiotics are indicated when signs of secondary infection are present [40]. For subacute presentations, high-potency topical corticosteroids, applied two to four times per day are the first-line therapy [37, 40]. Topical tacrolimus is efficacious without the skin atrophy may accompany steroid use [32]. Nummular eczema is often chronic, with some cases very recalcitrant to treatment [40].
Gravitational Eczema (Stasis Dermatitis) Gravitational eczema (also known as stasis dermatitis) is a recurrent swelling with characteristic skin eruptions of the feet and ankles, particularly in older men [14], associated with disorders of chronic venous insufficiency (CVI) [60]. Risk factors are a familial disposition, prolonged standing or sitting, and concomitant vascular disease [18]. In USA, up to six million people have CVI, while approximately 500,000 have venous ulcers [60]. The prevalence of CVI is increasing [60]. Initial skin damage in gravitational eczema results from microvascular changes; later, pathology is often associated with bacterial vasculitis [61]. Venous insufficiency in these patients produces a cyanotic erythema of the distal extremities, either unilaterally or bilaterally [14]. Impairment of the main veins and venules causes pulmonary hypertension; microthrombi that form in the capillaries cause microinfarctions and micronecrosis [62]; the resulting endothelial damage produces interstitial tissue edema [62], which, in turn, promotes an itchy inflammation with a greater risk of ulceration [36]. The condition may be acute, subacute, chronic, or recurrent [40]. Subacute dermatitis begins with edema of the ankles [63]. Typically, inflammation begins in winter as the skin of the lower extremities becomes dry and scaly [40]. Brown staining of the skin, caused by
deposition of hemosiderin by extravasated red blood cells, may spread slowly [62]. Scratching induces eczematous inflammation, often self-treated with medications. Potential contact sensitizers in these treatments can exacerbate and prolong the inflammation [40]. In the acute phase, pruritic plaques appear most commonly in the medial perimalleolar area [14]. They are characterized by poorly demarcated erythema and possible scaling [14]. Weeping and crusting may occur and a vesicular eruption – the id reaction – may appear on the palms, trunk, and extremities [40]. Untreated, the disorder creates hyperpigmented areas with focal purpura, which may ulcerate [64]. These ulcers, often extremely painful, are distinctly marginated erosions that heal with ivory-white plaques called ‘‘atrophie blanche’’ [65]. Gravitational eczema can be persistent, recurring even after apparently successful treatment [1]. Chronic inflammation begins when episodic inflammation further compromises the integrity of affected tissues, typically presenting as a cyanotic red plaque over the medial malleolus [40]. This development is typically followed by a condition called lipodermatosclerosis with a characteristic production of fibrous scar tissue of the reticular dermis made up of collagen bundles [66]. The resulting scleroderma-like hardening of skin is a consequence of intense proteolytic activity which degenerates the extracellular matrix of both dermis and epidermis. The result is ulceration, a refractory condition in which the epidermis is completely destroyed, and the matrix structures of the upper dermis partially degraded [66]. At this point, the physiologic mechanisms are no longer able to repair damage [62]. Lipodermatosclerosis typically begins on the medial side of the ankle then spreads, possibly as far as thighs and trunk [67]. The ulcerated tissues of gravitational eczema occasionally give rise to secondary neoplasms, although prevalence rates are uncertain [68]. Vascular ulcers should be monitored for malignant growth. In an evaluation of 85 cases of histologically confirmed malignancies that occurred as complications of ulcerated legs, 98% were observed to be squamous cell carcinomas (SCC) and 2% basal cell carcinomas (BCC). SCC ulcers in the leg appear as rolled, partially keratinized lesions with eversion raised borders; they have a 30% metastasis rate [62]. Any ulcer that, with treatment, continues to resist healing should be suspect [62, 69]. Stasis ulceration is responsible for prolonged hospitalization of many older people who are otherwise healthy [70]; effective treatment depends on considering the whole pathophysiological process [63]. Support stockings, which act to minimize edema, have been the
Non-neoplastic Disorders of the Aging Skin
mainstay of prevention; they are effective in the short term, but do not represent a long-term solution [63]. Once edema develops, it must be treated promptly to prevent rapid deterioration of the skin. Legs should be elevated and compression continued [37]. Topical corticoids of mild to moderate potency are commonly prescribed. Topical medications should be used judiciously to avoid further complication by contact sensitization. (Potential sensitizers may be present in moisturized, medicated dressings; preservatives; and topical antimicrobials and antibiotics [71].) The cutaneous scale prevalent in this disorder can harbor abundant microbial life. It should be treated by soaking followed by a gentle rub with emollient to soften and lift it off [72]. Oral antibiotics are appropriate and effective where ulceration has resulted in infection [37]. Because often this disease is recalcitrant to standard therapies, much research effort is being devoted toward new approaches to promote healing, such as maggot debridement therapy [73], honey (as a natural antiseptic) [74], cellulose, and collagen dressings [75], therapeutic ultrasound [76], mesh grafts with vacuum-assisted closure [77], hyperbaric oxygen therapy [78], and the stimulation of cytokine release by dermal dendrocytes [61]. In severe and recalcitrant cases, a surgical approach may be indicated. In a prospective, randomized, multicenter trial of 80 chronic leg ulcer patients treated by ambulatory compression therapy, as well as subfascial, endoscopic, perforating vein surgery combined with superficial vein ligation, produced a higher healing rate than in patients treated with compression only (83% vs. 73%), although the difference was not significant [79]. Seventy-two percent of surgically treated patients remained ulcer-free after 29 months, compared to only 53% in the compression-treated group; this difference did not reach statistical significance.
Contact Dermatitis Contact dermatitis, which includes both irritant and allergic reactions, occurs in as much as 11% of the elderly population (> Figs. 54.3 and > 54.4) [18]. In both types, the appearance of lesions is accompanied by burning and itching, which can be intense [80]. Older patients generally display less inflammation with vesiculation, but more scaling than younger subjects, and hyperpigmentation may be an early feature of the eruption [80]. Contact dermatitis tends to be more persistent in the elderly [80]. Older adults are predicted to be less susceptible to allergic contact dermatitis [81], as they have less ability to mount a delayed-type hypersensitivity reaction because
54
. Figure 54.3 Contact dermatitis caused by nickel – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
. Figure 54.4 A severe reaction to rubber – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
of reduced numbers of Langerhans cells [82] and T cells, and lower vascular reactivity [81]. Despite this, several investigators found an increase in positive patch tests in older patients [41, 83, 84], with higher rates in women than in men [9]. This phenomenon is due to the frequent use of common topical medicaments by older people [84]. For example, up to 81% of patients being treated for chronic leg ulcers exhibit allergic reactions to topical medications [41]. Patch testing before prescribing topical medications may be beneficial, especially in high-risk patients, such as those being treated for dermatitis or ulceration of the lower extremities [85]. Decades of exposure to potential sensitizers [86], and increased use of medications and topical products, account for the high prevalence of allergic contact sensitivity in the geriatric population [87]. Common
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Non-neoplastic Disorders of the Aging Skin
allergens include lanolin, paraben esters, dyes, plants, balsams, rubber, nickel, and topical medications [16]. Patch testing of older patients should include components of medicaments and dressings, dental prostheses, and medications for ocular disease [85]. While aged patients are susceptible to allergic contact dermatitis, they may be less likely to develop irritant contact dermatitis [88] because of limited occupational exposure to irritants [1] and because these patients are less able to mount an inflammatory response [5]. Irritant contact dermatitis present as erythema, with edematous plaques and potentially, vesicles [16]. It begins at the site of contact, but may spread. In the elderly, signs of irritant contact dermatitis may represent thermal injury [5]. Patients should be advised not to use strong soaps and to avoid contact with household cleaners and other products that have the potential to irritate their skin [5]. Management of contact dermatitis requires first identification and removal of the offending agent [88]. Treatment consists of administering topical corticosteroids [89, 90] adding oral antihistamines where needed [88–90].
Seborrheic Dermatitis Seborrheic dermatitis is a common inflammatory disorder that is thought to result from chronic infection of the lipid-rich areas of the skin. Malassezia yeasts are believed to be the causative organism; these fungi are components of the endogenous skin microflora to which some individuals apparently have an abnormal host response [91]. Its incidence in older patients is reportedly as high as 31% [18]. Seborrheic dermatitis occurs slightly more often in men than in women. It affects areas of the skin where sebaceous glands are most prominent: the eyebrows, paranasal area, pre and postauricular regions, presternal and intrascapular areas, scalp (dandruff is a form of this disorder), axillae, and groin [92]. Affected women tend to have more severe symptoms than men [93]. Seborrheic dermatitis is characterized by inflammatory changes (erythema) with greasy red-brown papules [34] covered by scaly yellow flakes and plaques [92]. Chronic dermatitis with pruritus, resembling psoriasis, may develop in seborrheic areas [92]. The disorder disproportionately affects patients with neurological disorders like Parkinson’s disease, epilepsy, and diseases or trauma of the central nervous system [94]; the association with neurological disorders is unclear [92]. The dermatitis may intensify during times of increased stress and fatigue [70]. Seborrheic dermatitis does not have serious physiological consequences, but can be distressing. Therapies include
anti-inflammatory preparations (e.g., steroids or calcineurin inhibitors), keratolytic agents (e.g., pyrithione zinc, sulfur, coal tar, salicylic acid), and antifungal medications (e.g., ketoconazole); keratolytic agents and antifungal medications are often administered in the form of medicated shampoos. Shampoos should be regularly applied by being lathered abundantly, rubbed into scalp, and left on for about 5 min [92]. Ciclopirox olamine, piroctone olamine, and climbazole are also used outside USA [92].
Cutaneous Expression of Autoimmune Disorders Older adults have a higher frequency of autoimmune disease, possibly linked to the senescence of the immune system [95]. The prevalence of polypharmacy in the elderly also increases the risk of drug-induced autoimmune cutaneous eruptions [95]. Cutaneous autoimmune eruptions include bullous pemphigoid, benign mucous membrane pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, and lichen sclerosis (LS).
Bullous Pemphigoid Bullous pemphigoid occurs in people over 60 and exhibits no ethnic or gender difference in prevalence [96]. It is a life-threatening disorder whose incidence is increasing for reasons yet unknown [97]. This autoimmune disease produces chronic eruptions of multiple bullae, either on otherwise normal skin, or on an urticarial base (> Figs. 54.5 and > 54.6) [98]. It may initially present as hives accompanied by intense itching [99]. Blisters result when basal epidermal keratinocytes detach from the dermis at the level of the lamina lucida [100]. Cutaneous blisters are large, tense, and tough enough to resist minor trauma [34]. About a third of people with this condition have oral blisters [37, 64], which are exquisitely painful. The disease may be preceded by a long period of generalized itching or eczema. If large areas of denuded skin occur, a significant loss of fluids and vital electrolytes may result [67]. Bullous pemphigoid is associated with circulating and tissue-associated [37, 98] antibodies to hemidesmosomal proteins, which are present in the basement membrane of stratified squamous epithelia [101]. The antibodies are produced specifically against a 230 kDa antigen located in the lamina lucida region of the dermo-epidermal junction, and produce separation of the epidermis from the dermis with a subsequent blister [98]. Bullous pemphigoid is
Non-neoplastic Disorders of the Aging Skin
. Figure 54.5 Bullous pemphigoid on trunk and extremities – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
. Figure 54.6 Bullous pemphigoid on the hand – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
confirmed by histology, immunofluorescence, electron microscopy, and molecular biology techniques [98]. Potent topical corticosteroids have been employed for first-line therapy, with tetracycline, alone or with nicotinamide, for patients who cannot tolerate corticosteroids or as corticosteroid-sparing therapy following steroid use [98]. In recent research, however, methotrexate
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produced better results with fewer side effects compared to prednisone [102].
Benign Mucous Membrane Pemphigoid Benign mucous membrane pemphigoid, an autoimmune disease in which the antibodies produced attack the attachment fibrils of the basement membrane, occurs primarily in the older adults. Blisters and erosions appear primarily in the mouth, on the conjunctivae, and in the nose. In 20–30% of cases, blisters also occur on the head, neck, and upper trunk. The disease can lead to blindness due to optic keratitis caused by occlusion of the lacrimal ducts. If left untreated, within 3–5 years both eyes may be affected. Patients diagnosed with mucous membrane pemphigoid should be promptly referred to an ophthalmologist. Mouthwashes containing topical steroids may be employed for oral lesions and corticoid-containing artificial tears can be used to treat the eyes [103].However, ocular disease is difficult to treat, and management usually involves systemic therapy with immunomodulators to control inflammation and prevent progression to irreversible blindness; surgical intervention may be indicated in advanced disease. Recent advances in treatment, including methotrexate, mycophenolate mofetil, monoclonal antibodies, and topical tacrolimus therapies are promising [104].
Pemphigus Vulgaris Pemphigus vulgaris is the most serious blistering disease in older adults (> Fig. 54.7). Onset typically occurs after the age of 65 [97]. Oral blisters erupt initially, followed by blistering of the trunk, limbs, face, and scalp. Lesions progressively ooze, become crusted, and lichenify (> Fig. 54.7) [37]. The condition is readily identified by the Nikolsky’s sign: lateral pressure with the thumb at the edge of the blister will produce an erosion [37]. Histological evaluation reveals intraepidermal blister formation and acantholytic cells within the lesion. Indirect immunofluorescence reveals intercellular deposition of immunoglobulin (Ig) conjugates and complement 3 (C3); on occasion, other immunoglobulins and complement components are present [37]. Serum antibodies, particularly to desmoglein-3, are helpful for diagnosis; serial titers can help monitor progress of disease [105]. Pemphigus vulgaris is a serious chronic disorder with the potential for fatality due to secondary electrolyte imbalance or secondary infection [37]. The risk of death
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Non-neoplastic Disorders of the Aging Skin
. Figure 54.7 Pemphigus vulgaris on the back – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
mucosa. Histological analysis reveals acantholysis, basal cell vacuolation and clefts, and scattered necrotic keratocytes as unique features of the erosions [109]. Direct immunofluorescence locates intercellular and basement membrane IgG and C3 both within the epidermal spaces as well as at the epidermal basement membrane. Indirect immunofluorescence demonstrates circulating antibodies specific for stratified squamous or transitional epidermal epithelium [96]. Autoantibodies against epidermal proteins are produced by the associated tumors [109]. Paraneoplastic pemphigus is often complicated by bronchiolitis obliterans, which leads eventually to respiratory failure [109]. Successful treatment depends on early detection and removal of associated tumor as well as intravenous administration of immunoglobulin [109].
Lichen Sclerosis
in people with the condition is three times that of agematched controls [97]. Treatment requires systemic therapy with corticosteroids, which should be started as early as possible [37]. Morbidity and mortality from chronic corticosteroid use, however, are considerable [106]; lower doses of corticosteroid (80–120 mg/day) [37], adjunct use of immunosuppressive drugs, tetracycline with nicotinamide [37], or sublesional corticosteroid injections [107] can be considered as alternative therapies. Recent studies have shown adjuvant therapies, particularly azathioprine, to be useful in reducing steroid dosage [108].
Paraneoplastic Pemphigus The elderly are especially susceptible to paraneoplastic pemphigus [101]. Paraneoplastic pemphigus occurs in conjunction with several neoplasms, most commonly chronic lymphocytic leukemia [109], but also with Castleman’s tumor, non-Hodgkin’s lymphoma, thymoma, and follicular dendritic cell sarcoma. The condition is seen primarily in those over 60 and twice as often in men than in women. Paraneoplastic pemphigus exhibits extensive and painful mucocutaneous erosions that usually arise in the oral
In older men, cutaneous issues of the genitalia are generally limited to those who are uncircumcised. However, genital lesions are not uncommon among elderly women [110]. LS is an apparently autoimmune dermatosis in women, with a predilection for the genital skin. Known formerly as kraurosis vulvae or leukoplakia of the vulva, LS produces well-demarcated, porcelain-white papules, and plaques among areas of bruising; lesions occur throughout the genital area with the exception of the genital mucosa [111]. Itching is the principal symptom, which creates the potential for secondary lichenification due to scratching [111]. LS creates rare but potentially debilitating complications. Lichenified scars or adhesions may cause the introitus to narrow or close; this interferes with micturation and intercourse, and occasionally requires subtotal or total circumcision [111]. Additionally, LS is associated with an increased incidence of invasive squamous cell carcinoma of the anogenital area [111]. The ultra-potent topical corticosteroid, clobetasol propionate, is a first line therapy [112]. It produces improvement in as many as 96% of patients [113]. There is some concern that corticosteroid use may induce oncogenic human papillomavirus (HPV), which is carried by 20% of LS patients [111].
Vascular Disorders Several vascular changes occur in aged skin. The capillaries and small vessels regress, and become more disorganized [12], blood vessel densities diminish [85], and the number of venular cross sections per 3 mm2 of skin surface in nonexposed areas is reduced by 30% [12].
Non-neoplastic Disorders of the Aging Skin
Intravital capillaroscopy measurements in 26 subjects, using fluorescein angiography and native microscopy, suggest a decrease in dermal papillary loops [85]. Because of the loss of functional capillary plexi, the maximum amount of blood pumped is reduced, although in individual capillary units the blood-flow pattern remains unchanged [98]. Loss of collagen and elastin fibers in the dermis (associated with an overall derangement of organization) decreases the tensile strength of the skin. This makes aged skin more susceptible to injury (especially the lower layers) [11, 70], and results in a collapse of structural support for the cutaneous vasculature. Where initial skin injury occurs, impaired wound healing significantly increases the risk of complication [34, 98, 99]. Vascular disorders common in the aged include pressure ulcers and rosacea.
Pressure Ulcers Elderly patients who lose functional mobility become susceptible to pressure ulcers (bedsores), a localized area of tissue necrosis that affects the skin, subcutaneous tissue, muscle, and bone. People aged 70–75 have double the risk of pressure ulcers compared to those aged 55–69 [114]. Up to 14% of patients in acute care facilities [115], 25% of patients in skilled-nursing facilities, and 12% of patients in home care suffer from pressure ulcers [116]. Pressure ulcers occur most often over bony prominences: the sacrum, ischial tuberosities, greater trochanters, heels, and lateral malleoli [64]. The first sign of pressure damage is blanchable erythema (indicating the presence of a mild, perivascular, lymphocytic infiltrate, and edema in the papillary dermis). This is followed by nonblanchable erythema, due to red blood cell engorgement of the capillaries and venules and degeneration of pilosebaceous structures and sub-cutaneous fat. At this stage there is no observable effect in the epidermis [115]. Pressure ulcer dermatitis subsequently manifests as marked redness, with scaling or bullae. Initial ulceration is due to the loss of the epidermis and acute inflammation of papillary and reticular dermis. Chronic ulcers display a diffusely fibrotic dermis [115]. Early pressure damage may go unnoticed by caregivers [117], as damage first destroys deeper structures while the surface of the skin exhibits only erythema [115]. High interstitial pressures are created at the bone/muscle interface, causing substantial deep tissue injury with relatively little superficial damage [118]. Multiple factors contribute to the formation of pressure ulcers: prolonged immobilization; pressure upon bony
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prominences, combined with shear forces; a compromised vasculature; heightened skin moisture in patients with urinary incontinence; poor nutritional status; impaired wound healing; and possible sensory deficits in the patient. Institutionalized patients are most at risk; the composition of many hospital beds produce twice the pressure required to produce necrosis in a 2-h period [115]. Immobile patients, therefore, must be regularly repositioned, a practice that caregivers find difficult to perform regularly [119]. Nutrition is also important: pressure ulcers were significantly less likely in at-risk patients taking oral nutritional augmentation than in a similar population receiving appropriate routine care without nutritional supplement [120]. Smoking also increases risk [115]. Management of pressure ulcers should focus on prevention, with nutritional supplementation, regular pressure relief, and fastidious perineal hygiene in patients who are incontinent [115]. Seating systems that provide pressure relief and blood-flow stimulation, and/or passive standing capability, reduce the risk of pressure ulcers and enhance the patient’s functioning [115].
Rosacea Rosacea is a chronic inflammatory disorder characterized by acneiform papules, pustules, and dilation of the capillaries. It appears primarily on the cheeks, nose, forehead, and chin (> Fig. 54.8). Its onset is intermittent in middle age, but later becomes persistent. Prevalence is 12% among people over age 64 [14, 16]. Hyperplasia of the
. Figure 54.8 Rosacea on the face – Reproduced with permission from the American Academy of Dermatology, ß 2008. Reprinted with permission
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Non-neoplastic Disorders of the Aging Skin
sebaceous glands may be present [14], and in more than 90% of all patients there is ocular involvement [14]. Histology reveals telangiectasias and a perivascular lymphocytic inflammatory infiltrate with dermal edema [16]. The etiology is not well understood: mites, vascular instability, and vitamin deficiencies have been proposed [16]. The pathology involves, at least in part, atrophy of the papillary dermis that allows easier visualization of dermal capillaries [16]. Triggers include spicy foods, sun exposure, some medications, and facial products that contain known irritants [121]. Recently, the observation that rosacea correlates with gastrointestinal disorders [16] has been strengthened by the association of rosacea with Helicobacter pylori (HP) infection [122]. Eradication of H. pylori when identified in rosacea patients, produced significant improvement in rosacea symptoms in 66% of treated patients, and complete resolution in 33% [123]. Moreover, rosacea also has been associated with an increase in the generation of reactive oxygen species (ROS) in vivo [124], and tentatively proposed to be an antioxidant system defect [125]. H. pylori is known to increase the generation of ROS in the gastric mucosa; further research is needed to elucidate the role of this microorganism in the progression of rosacea [126]. Often chronic, rosacea deteriorates the skin over time [37] and should be treated to avoid disfigurement (e.g., of the nasal area in men.) [121]. Patients should be instructed to avoid triggers [121]. Metronidazole is the topical treatment of choice [37]; antibiotics (topical and oral) may be useful as well. Azelaic acid, topical retinaldehyde, and vitamin C also are efficacious [121]. Speculation regarding a possible antioxidant-deficit etiology for rosacea, prompted a recent trial of a macrolide antibiotic, azithromycin, evaluated in 17 patients with papulopustular rosacea against healthy controls. Rosacea patients had higher ROS levels in facial skin biopsies at baseline than healthy controls, which normalized significantly in rosacea patients after 4-week treatment with azithromycin (P < 0.001) [127].
Viral Infection: Herpes Zoster Herpes zoster (shingles) is a reactivation of Varicella zoster (the chicken pox virus) [37]. Involvement of the major sensory nerve ganglion accompanies skin eruptions [37]. Two-thirds of cases occur in patients older than 50 [70], with highest prevalence in patients 60 years and older [16]. A tingling or itching sensation (sometimes with pain) precedes a unilateral vesicular [11] cutaneous eruption by several days [37]. Vesicles persist for up to 2 weeks,
and eventual form dry hemorrhagic crusts and with possible scarring. Secondary bacterial infections are common [16]. Reactivation sites, in decreasing order of frequency, are the thoracic, cervical, trigeminal nerves, and the lumbosacral segments [67]. Shingles is usually self-limiting. [66]. Serious sequelae occur only in immunosuppressed patients (in whom the virus can easily disseminate), or when the optic nerve is involved, which occurs more often in older patients [37]. Vesicles on the side of the nose often occur in association with corneal involvement [11]. Postherpetic neuralgia (acute chronic pain along involved nerves) is a complication in about half the patients over the age of 60 with herpes zoster reactivation. This risk of this complication increases with age [37]. Although the pain gradually abates over time, it is frequently disabling and refractory to typical pain medications. This can significantly affect quality of life in the older patient [67]. The severity and duration of postherpetic neuralgia [62, 128], particularly in older patients [129], can be reduced by prompt and aggressive treatment of the acute infection with oral antiviral drugs (such as acyclovir) before virus spreads beyond the initially damaged nerve. In a randomized, double-blind, placebo-controlled, prospective trial in more than 38,000 senior adults, a new vaccine composed of live, attenuated Varicella zoster virus reduced incidence of postherpetic neuralgia by 66.5% [130]. Morbidity due to Herpes zoster was markedly reduced with few side effects (principally mild dermatological reactions at the injection sites). Discussion of the cost effectiveness of the vaccine in the target population is ongoing [131].
Management of Cutaneous Disorders in the Elderly Management of skin disease in the elderly must take into account variables pertinent to the older patient. Accurate diagnosis is key. In the elderly, diagnosis is complicated by the use of multiple medications in this age group: drug eruptions which can simulate almost any dermatological disease (> Table 54.3) must be distinguished from cutaneous eruptions of other etiologies [32]. Diagnosis of drug reactions is critical before any underlying cutaneous disorder can be identified. Moreover, prompt withdrawal of the culprit drug may be necessary to avoid complications [132]. Topical treatment must take into account the fragility of aged skin [70]. Aged skin is compromised structural degeneration and comorbidities [72]. Consequently,
Non-neoplastic Disorders of the Aging Skin
54
. Table 54.3 Cutaneous eruptions and possible drug etiology by classification of lesions Type of rash or eruption
Possible drug etiology
Exanthems
Beta-lactam antibiotics, sulfonamides, erythromycin, gentamicin, anticonvulsants, gold salts
Eczema, lichenification
Antiarrhythmic agents, anticonvulsive agents, antituberculosis agents, gold, quinidine, methyldopa
Acne-like
Corticosteroids, bromides, iodides
Urticaria and angioedema
Converting enzyme (ACE) inhibitors, NSAIDs, opiates, curare, antibiotics (esp penicillins), blood products
Bullous
Penicillamine, bleomycin, iodides
Fixed drug
Penicillins, phenolphthalein, tetracycline, nalidixic acid, barbiturates, sulfonamides, gold salts
Exfoliation
Gold
Anticoagulant skin necrosis
Warfarin, heparin
Nodular eruption
Sulfathiazole, salicylates, oral contraceptives
Rash on sun-exposed areas
Coal-tar derivates, psoralen, chlorpromazine, tetracycline, doxycycline, NSAIDS, phenothiazines, chlorothiazide, demeclocycline, griseofulvin, oral hypoglycemics, sulfonamides.
Erythema multiforme target lesions, SJS, TEN
Allopurinol, barbiturates, dapsone, digitalis, phenobarbital, carbamazepine, phenytoin, gold, hydralazine, salicylates, sulfonamides, penicillin, quinolone, cephalosporins, NSAIDS, tetracycline, trimethoprim-sulfamethoxazole
> Table 54.3 is modified from References [136, 137]. ACE angiotension-converting enzyme; NSAIDs nonsteroidal anti-inflammatory drugs; SJS Stephens–Johnson Syndrome; TEN Toxic Epidermal Necrolysis.
second-line therapies may be more advisable at earlier treatment stages [11]. Psychosocial issues must also be considered in treatment decisions. Older patients may suffer from memory loss, impaired vision, hearing, or mobility, and sometimes, dementia [11, 12]. They may lack attentive caregivers, and adequate housing or nutrition [11]. The patient’s ability to comply with therapy should be considered, though often it is not [72]. Physicians need to consider whether compliance with the regimen prescribed is actually feasible for the patient: simple regimens should be advised whenever possible to maximize compliance [72]. Clinicians or care providers should follow-up to ensure that medications were applied as required [32].
Conclusion Diseases and disorders of the skin, increase in prevalence in older people [70]. Most people over 65 have at least one skin disorder, and many have two or more [6]. This creates substantial morbidity and mortality among older adults [14, 115, 133]. Effective management requires that primary-care providers monitor the skin condition of the
patient, and distinguish lesions that are a normal part of aging from more clinically significant lesions that may require specialized intervention [5], prompting referral to the dermatologist as needed [5]. Treatment decisions should consider the patient’s housing, presence or lack of caregivers, nutritional status, clothing, heating, mobility, hearing, and vision [11]. Prompt and thoughtful treatment of any clinically significant cutaneous disorder will substantially improve the aging patient’s quality of life in the later years [133].
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59 Non-surgical Modalities of Treatment for Primary Cutaneous Cancers Ossama Abbas . Salah Salman
Introduction
Topical Therapies
Skin cancer, which includes melanoma and non-melanoma skin cancers (NMSCs), is the most common malignancy affecting humans [1–4]. Basal cell carcinoma (BCC) represents the most common cutaneous malignancy (comprising approximately 75% of NMSCs), followed by squamous cell carcinoma (SCC), which comprises around 20% of NMSCs [1–4]. The incidence of these cutaneous malignancies increases with age, making the elderly population most prone to the development of these cancers [1–4]. Although surgery, particularly Mohs micrographic surgery, is usually the treatment of choice in the management of cutaneous malignancies in terms of margin control and cure rates, often it may not be the most appropriate because of several disadvantages [1–4]. The choice of the treatment modality to be utilized should be tailored according to specific cancer characteristics (such as type, size, location) and patient factors (age, comorbidities, use of multiple drugs, including anticoagulants) [1–4]. Not only may surgery cause major disfigurement and functional impairment, but the patient may be a poor surgical candidate, necessitating use of other modalities of treatments [1–4]. This is especially true of elderly patients who are not only characterized by an increased incidence of cutaneous malignancies, but also by an increased incidence of other medical comorbidities that may have a negative effect on surgery [1–4]. In such circumstances, alternatives to surgery may, in fact, be the preferred choice. The chapter aims at reviewing the currently available evidence on the various non-surgical therapeutic modalities for the different types of skin cancer. These include topical, intralesional, and systemic treatments, as well as physical treatment modalities. Furthermore, certain dietary and herbal supplements may also have a role in the prevention of skin cancers.
Several topical agents have been used in the treatment of cutaneous malignancies, including imiquimod, 5flurourasil, tazarotene, diclofenac, and cidofovir [1–4]. The use of these agents should be guided by the evidence on their effectiveness in the treatment of specific types of skin cancers, patient profile, and the medication’s side effects. The use of topical agents has several advantages, including ease of use, convenience (as the medication can be applied at home), ability to treat large lesions and those in critical sites, and, in general, better cosmetic results than surgery. However, several disadvantages are to be kept in mind, the most important of which is the initial irritating inflammatory response, which can affect patient’s compliance and subsequently the final results. In addition, the expensive cost of some of these agents (such as imiquimod) may limit its use.
Imidazoquinoline Compounds (Imiquimod and Resiquimod) Several studies have shown that the mechanism of action of imidazoquinoline compounds (imiquimod and resiquimod) as immunomodulators is mediated by the activation of Toll-like receptors 7 and 8 (TLR7, TLR8), which leads to the production of interferon-alpha (IFN-a) and other cytokines, including interkeukin (IL)-12 and IL-18 [1, 5]. These then mediate the anti-tumoral effect through their enhancement of the cell-mediated immunity. Also, the anti-tumoral effect is mediated through up-regulation of the Opioid Growth Factor receptor (OGFr) that, in turn, stimulates the interaction of the OGF–OGFr axis, which is an inhibitory pathway regulating cell proliferation [6]. There is now plenty of evidence on the effectiveness of the imiquimod 5% cream in the treatment of multiple
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Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
primary cutaneous malignancies. Currently, it is US Food and Drug Administration (FDA)-approved for the treatment of superficial BCCs (especially those that are smaller than 2 cm, on the trunk, neck, or extremities) and actinic keratoses (AKs) [1, 2, 5]. Different studies have shown that the rate of clinical and histological clearance of superficial BCCs treated with imiquimod 5% cream (applied 5 days or 7 days/week for 6–12 weeks) is greater than 90%. Although less evidencebased, the use of imiquimod in the treatment of nodular BCCs has also proven to be effective (clearance rate ranged between 70% and 100% based on different studies) [1, 2, 5]. As is the case of superficial BCCs, imiquimod is also FDA-approved for treating AKs of the head and neck region. This has been supported by several studies that have shown a 50% complete clearance rate of AKs (compared to 5% of AKs in patients treated with placebo) that were treated with imiquimod 5% cream (applied 3 days/ week for 12–16 weeks) [1, 2]. Although there is currently less evidence supporting the use of topical imiquimod in the treatment of other skin malignancies – including Bowen’s disease or squamous cell carcinoma in situ (SCCIS), invasive SCC, or lentigo maligna – anecdotal reports and small studies have shown that imiquimod may be quite effective [1, 2]. This can thus be a last resort in those patients who are poor surgical candidates. Adverse reactions most commonly encountered with the use of topical imiquimod 5% cream include erythema, ulceration, edema, and/or scaling, and these are usually limited to the application site. These reactions can be intense, especially with increased application frequency and especially in patients being treated for actinic keratoses or BCC [1, 2, 5]. Flu-like symptoms such as fever, fatigue, and myalgias are probable systemic adverse effects that have been reported in approximately 1–2% of patients.
5-Fluorouracil (5-FU) The mechanism of action of 5-FU derives from it being a structural analogue of thymine. This leads to the inhibition of thymidylate synthetase and thus blocking of DNA synthesis, especially in rapidly dividing cells such as tumor cells [1–4]. First used in clinical practice in the 1960s, topical 5-FU is now present in different formulations including solutions (1%, 2%, and 5%) and creams (0.5%, 1%, 2%, and 5%). Among these, the 5% cream has been approved
by the FDA for treating AKs and superficial BCCs. Its effectiveness in the treatment of AKs has been shown to be comparable to imiquimod [1, 2]. Anecdotal reports have also shown that 5-FU may be effective in the treatment of SCCIS [1]. The usual application regimen is once or twice daily for up to 4 weeks. Adverse reactions commonly described with the topical use of 5-FU include local irritation, allergic contact dermatitis, pain, erythema, edema, pruritus, dyspigmentation, and photosensitivity [1–4]. Uncommon reactions such as onychodystrophy and the appearance of telangiectasias may also occur. Rarely, systemic absorption may lead to systemic side effects such as nausea, myelosuppression, diarrhea, cardiac abnormalities, and neurologic toxicity [1–4].
Tazarotene Tazarotene is a third-generation retinoid that usually exerts its effect on keratinocyte differentiation and proliferation, mainly through its interaction with RAR-b and d receptors [1]. However, the underlying mechanism of its confirmed effect in the treatment of NMSCs in a few small studies is still not well understood [1]. One study showed that the daily use of 0.1% tazarotene gel for the treatment of BCC resulted in a complete clearance rate of 53%. The duration of treatment in this study ranged between 5 and 8 months [1]. Similarly, 0.1% tazarotene gel used daily for up to 6 months in the treatment of SCCIS resulted in a clearance rate of 47% [1]. Like the other topical retinoids, the most common adverse reaction observed with tazarotene is skin irritation, manifesting in the form of redness, scaling, dryness, and pruritus, in addition to a burning, stinging sensation. This reaction tends to be most severe during the first weeks of therapy, with gradual recession later on [1]. Other less common adverse effects include dyspigmentation and allergic contact dermatitis.
Other Topical Agents Case reports and small studies have also documented the effect of other topical agents such as cidofovir and diclofenac in the treatment of cutaneous malignancies [1]. In one study, cidofovir, which is a purine nucleotide analog of deoxycytidine, resulted in a 75% clearance rate of BCC when used as a 1% cream applied daily over a period of 2 months [1]. No significant side effects were observed in the study, and the treatment was well
Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
tolerated by patients. The underlying mechanism of action of cidofovir is thought to be an anti-neoplastic and anti-angiogenic effect. Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), usually exerts its anti-tumor effect through the inhibition of the cyclooxygenase (COX II). This is believed to inhibit angiogenesis and tumor invasion, leading to a decrease in the rate of epithelial tumor growth. One double-blind, placebo-controlled study showed that twice daily diclofenac application in the form of a 3% gel resulted in a 33% complete clearance of AKs [1]. Local irritation was observed as a side effect, but was much less severe than that observed with either 5-FU or imiquimod [1].
Intralesional Agents Multiple agents in an intralesional form have proven their efficacy in the management of cutaneous malignancies, including bleomycin, 5-FU, and interferon-a (IFN-a) [1, 7, 8]. Advantages of this form of therapy include the ease of delivery, the ability to use it as an adjuvant treatment to surgery, and good cosmetic results in general. Disadvantages include the currently sparse amount of evidence supporting their use, their high costs, and the usual need for multiple treatment sessions [1, 7].
Bleomycin Several mechanisms mediate the anti-tumor effect of bleomycin, which is a cytotoxic antibiotic produced by Streptomyces verticillatus, including its inhibition of DNA ligase preventing repair of DNA, its effect on the G2 and S phases of the cell cycle of fast-dividing cells resulting in SS DNA breakage, and also by promoting apoptosis and epidermal necrosis [1, 7]. Individual case reports have described the efficacy of intralesional bleomycin in the treatment of BCCs and keratoacanthomas [1, 7]. Adverse effects that have been described with the intralesional use of bleomycin include local pain, swelling, dyspigmentation, ulceration, superficial scarring, flu-like symptoms, and, rarely, flagellate hyperpigmentation [1, 7].
5-Fluorouracil There is now evidence that 5-FU as an intralesional preparation can be quiet effective in the treatment of BCC,
59
SCC, and keratoacanthomas [1]. In one study, intralesional injection of 0.5 mL of 5-FU/epi gel three times weekly for 2 weeks resulted in 100% clearance of BCCs. Another study on the treatment of SCCs showed that 1.0 mL weekly injection of 5-FU/epi gel for up to 6 weeks achieved a 96% clearance rate. Although excellent cosmetic results may be achieved with its use, intralesional 5-FU may be locally complicated by pain, erosion, ulceration, and dyspigmentation [1].
Interferon-a (IFN-a) Intralesional interferon-a (IFN-a) can be quite effective in the treatment of keratoacanthomas (> Fig. 59.1), BCCs, and SCCs [1, 2, 8]. This effect of IFN-a is thought to be mediated by the enhancement of cell-mediated immunity against malignant cells through increasing the antigen-presenting cell function, stimulating the activity of natural killer cells, and promoting the development of T-helper (Th)-1 response, while at the same time suppressing the production of Th-2 cytokines [1, 8]. One study showed complete clearance of all BCCs and SCCs that were treated with intralesional IFN-a given in a dose of 1 106 to 2 106 IU three times weekly for 3 weeks. Adverse reactions most commonly encountered with the use of IFN-a include flu-like symptoms and local injection-site reactions. Laboratory abnormalities may also be observed, such as elevation in hepatic transaminases and decrease in white blood cell count [1, 8]. Given that the relative contraindications for the use of IFN-a include a history of cardiovascular, renal, hepatic, or central nervous system disorders, its use in the elderly population should be undertaken with extra caution, as these patients usually have multiple comorbidities.
Systemic Agents A problem in treating transplant patients is to provide effective immunosuppression, while at the same time not promoting cancer development. Mammalian target of rapamycin (mTOR) inhibitors have both immunosuppressive and tumor suppressive functions [9]. For the most part, there are insufficient data to draw clear conclusions on the effectiveness of mTOR inhibitors against cancer in humans. However, there are hints that these drugs may be very useful in transplant recipients. Multiple groups have reported on calcineurin inhibitors (CNIs)-immunosuppressed renal transplant recipients with Kaposi’s sarcoma, demonstrating tumor regression
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Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
. Figure 59.1 Elderly woman with keratoacanthoma over the nose treated with intralesional IFN-a: (a) before treatment, (b) after one injection, and (c) after two injections
after switching from CNIs to sirolimus, which is an mTOR inhibitor [9]. Tumor regression occurred in the face of full immunosuppression with sirolimus, thus not increasing the risk for organ allograft rejection.
Physical Modalities of Treatment Many treatment modalities fall under this category, including cryotherapy, electrodessication and curettage (ED&C), radiotherapy, photodynamic therapy (PDT), and laser ablation [1, 2]. Most of these have plenty of evidence to support their use, with each of them having its advantages and disadvantages.
Cryotherapy Cryotherapy has historically been the classical alternative treatment for cutaneous malignancies when surgery is not an option. There is now plenty of evidence to support the use of cryotherapy in the treatment of AK, SCCIS, and BCC (> Fig. 59.2) [1, 2, 10]. It is suitable for the treatment of single or multiple tumors, especially in patients who are old, debilitated, using pacemakers, or maintained on anticoagulation [1, 2, 10]. Excellent cure rates, ranging from 97% to 99%, have been achieved upon treatment of these different tumor types with cryotherapy; however, cryotherapy is usually associated with higher recurrence rates (reaching up to 17%) and poorer cosmetic outcomes
when compared to surgery or PDT [1, 2, 10]. In order to ensure successful outcome, malignant lesions should generally receive cryotherapy until a clinical freeze margin of 5 mm is observed [1, 2, 10]. The major advantage of cryotherapy is its convenience and ease of use in regular dermatology offices [1, 2, 10]. Treatment of AK is usually achieved with one freeze-thaw cycle of 5–7 s, while SCCIS and BCC should be treated with two freeze-thaw cycles of approximately 40–90 s each, aiming at a temperature of 50 to 60 C at the base of the lesion [1, 2, 10]. Cryotherapy is contraindicated in patients with a history of Raynaud’s phenomenon, cold urticaria or cryoglobulinemia, as well as in treating deeply penetrating or aggressive tumors or those characterized by indistinct or ill-defined borders [1, 2, 10].
Electrodesiccation and Curettage ED&C is another physical destructive therapeutic modality that has proven its efficacy in the treatment of different NMSCs, including BCC, SCCIS, and SCC [1, 2, 11]. In order to achieve high cure rates with ED&C, selection of the appropriate patient, with low-risk tumor characteristics, is of paramount importance. Relative contraindications for treatment with ED&C include immunocompromised patients, high-risk locations (such as nose, ear, or periorificial areas), a tumor size larger than 2 cm, recurrent lesions, and an aggressive histological
Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
59
. Figure 59.2 Elderly man with BCC over the nose treated with cryotherapy: (a) before treatment, (b) during treatment, and (c) after treatment
tumor subtype [1, 2, 11]. The evidence has mainly been provided by large retrospective studies, which showed that treatment with ED&C achieves cure rates in the range of 74–100% for BCCs and 96–100% for SCCs [1, 2]. Recurrence rates of BCCs treated with ED&C are comparable to surgical excision, and range between 3.3% and 5.7% for primary BCCs less than 1 cm in size [1, 2]. Compared to cryotherapy in the treatment of SCCIS, ED&C is characterized by lower recurrence rates and shorter healing times [1, 2]. Combining ED&C with topical treatments such as imiquimod cream has been shown to have a synergistic effect, resulting in better tumor clearance and improved cosmetic outcome [1, 2]. ED&C has the advantages of being a valuable, efficient, cost-effective tool that could be the ideal alternative to surgery in the management of cutaneous malignancies, especially in a patient who is a poor surgical candidate [1, 2, 11]. Compared to surgery, the cosmetic outcome after ED&C is usually inferior. Disadvantages of ED&C include the inability to confirm tumor clearance histologically. Adverse effects observed with the use of ED&C include dyspigmentation and hypertrophic or atrophic scars [1, 2, 11]. Patients with cardiac pacemakers are better managed by electrocautery instead of electrodesiccation/ coagualtion [1, 2].
Radiotherapy Radiation represents another important alternative to surgery in the treatment of BCCs and SCCs, especially in
elderly patients or those having major medical comorbidities or large-size inoperable tumors [1, 2]. Although the use of radiotherapy has declined in recent years in order to avoid detrimental side effects of radiation and because of the appearance of better and less harmful modalities of treatment, radiotherapy can still be of significant value in the treatment of medium-sized (1–4 cm in diameter) cutaneous malignancies occurring on the face of older patients [1, 2]. Smaller malignancies are better managed with surgery, while larger tumors are better treated with a combination of surgery and radiation or by Mohs surgery [1, 2]. Radiotherapy of cutaneous cancers can usually be done using grenz rays or soft or superficial x-rays. Preservation of surrounding normal tissue is usually possible when using radiotherapy for cutaneous malignancies, because the radiation doses required for cancer eradication are not that highly damaging [1, 2]. The major advantages of using radiotherapy include preservation of normal uninvolved tissue (especially for large tumors or those in difficult locations), minimal patient discomfort, the ability to be performed on outpatient basis, and as the ideal alternative to surgery in the treatment of elderly patients who are poor surgical candidates or who are physically or psychologically handicapped [1, 2]. While radiotherapy is considered to be curative for lentigo maligna, BCC, SCC, and keratoacanthoma, its use for the treatment of invasive melanoma, Kaposi sarcoma, and lymphomas is only palliative [1, 2]. Contraindications for the use of radiotherapy include chronic radiodermatitis, verrucous carcinoma, previously
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Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
irradiated cutaneous malignancies, genodermatoses such as xeroderma pigmentosum, intra-oral tumors, tumors penetrating into cartilage or bone, and those found in scars of burns, chronic leg ulcers, or osteomyelitis [1, 2]. Although several studies have shown that radiotherapy may be comparable to surgery in the clearance rate achieved for the treatment of different cutaneous malignancies (BCC, SCCIS, keratoacanthoma, SCC), the former is usually associated with higher recurrence rates [1, 2]. A 5% to 15% 5-year recurrence rate occurs when radiotherapy is used for the treatment of BCCs and SCCs. Radiotherapy can also be utilized for the treatment of recurrent cancers when surgery is not an option, or it can complement (adjuvant to) surgery in treating lesions (both BCCs and SCCs) that cannot be completely removed by surgery due to their size or location [1, 2]. Another excellent indication for radiotherapy is a large lentigo maligna, in which the results can be at least as good as those achieved with surgery; however, cosmetic outcomes are usually better in such cases with radiotherapy than they are with surgery [1]. BCCs, SCCs, and keratoacanthomas are best treated with soft or superficial x-rays, and the regimen can be variable (a typical course would be a 2–4 Gy daily dose for up to 20 days) [1, 2]. Classical treatment for lentigo maligna includes the use of 5–10 doses of 10–20-Gy grenz rays depending on the lesion size [1]. There are early and late adverse effects in the use of radiotherapy. The former include transient erythema and desquamation, while the latter include hypopigmentation, telangiectasia, atrophy, and fibrosis. Although this may indicate that radiotherapy leads to worse cosmetic outcomes than surgery, radiotherapy may actually achieve better cosmetic results than surgery for tumors in special locations, such as the lower lip, nasal tip, nasal ala, and eyelid [1, 2].
Photodynamic Therapy Not only is topical photodynamic therapy (PDT) being widely used for the treatment of Aks, but plenty of evidence is currently accumulating to support the use of PDT in the treatment of NMSCs [1, 2, 12, 13]. In PDT, a photosensitizing compound, such as 5-aminolevulinic acid (ALA) and the methyl ester of ALA (mALA), applied to the skin gets converted to protoporphyrin IX upon absorption. Protoporphyrin IX then preferentially accumulates in the intracellular membranes of organelles, such as lysosomes and mitochondria, within the tumor cells. Upon activation by a light source in the
417–750 nm wavelength range, protoporphyrin IX goes into a higher energy state, leading to the generation of reactive-oxygen species (including singlet oxygen), which damage and induce apoptosis of tumor cells [1, 2, 12, 13]. Most studies on topical PDT have been done to test its effectiveness in the treatment of Aks, and these have shown that the clearance rates (up to 90%) are similar or maybe even better than those achieved with cryotherapy or 5-fluorouracil [1, 2]. Studies on BCCs have shown that PDT achieved results comparable to cryotherapy in the treatment of superficial BCCs as also comparable to simple excision in the treatment of nodular BCCs (complete responses in up to 90%), although the recurrence rates were slightly higher with PDT [1, 2, 12, 13]. However, the cosmetic results are usually better with PDT than with either cryotherapy or surgery [1, 2, 12, 13]. This is because the damage is limited to the tumor cells of epithelial origin that get preferentially sensitized, while surrounding tissues are usually not affected as much. PDT is also effective in treating SCCIS, with one study showing a complete response rate of 88%. Other cutaneous tumors, such as pigmented morpheaform or infiltrative variants of BCC and metastatic melanoma, are considered to be poor responders to PDT [1, 2]. Common adverse effects observed with PDT include stinging, itching, and burning during treatment, subsequently followed by erythema and edema [1, 2, 12, 13].
Laser Ablation Laser ablative (carbon dioxide (CO2) or Erbium:YttriumAluminum-Garnet (erbium:YAG) lasers) vaporizes tissue reaching the level of the papillary dermis, and in so doing limits the injury and decreases scarring and the risk of permanent altered pigmentation [1, 14]. When considering treatment of cutaneous malignancies with laser ablation, the physician should consider the type of malignancy, as well as its location and the skin phototype of the patient. The advantages of using laser ablation in the treatment of primary cutaneous malignancies include the ability to treat large surface areas, the hemostatic nature of the procedure, the prophylactic effects, and the added potential cosmetic result of rejuvenation [1, 14]. Disadvantages include risk of scarring, expensive costs of the procedure, and the inability to treat hyperkeratotic or elevated lesions. Several studies have shown that laser ablation is quite effective in the treatment of AKs (reducing the number by
Non-surgical Modalities of Treatment for Primary Cutaneous Cancers
up to 94%), superficial and nodular BCCs (up to 97% clearance), and SCCIS [1, 14]. However, laser ablation should not be used for thick hyperkeratotic lesions. Laser ablation as a full-face resurfacing procedure has also been shown to prevent the appearance of new NMSCs [1, 14].
Chemical Peels Not only has chemical peeling proven its efficacy in improving photodamaged skin, but a few studies have also shown satisfactory effects in the treatment of AKs (> Fig. 59.3) [1, 15, 16]. Chemical peeling involves the controlled application to the skin of one or more exfoliating agents, resulting in different levels of peeling (superficial, medium-depth, deep) depending on the agents used and techniques followed. Advantages of using chemical peeling in the treatment of AKs include, in addition to the reduction in the number of lesions, the diffuse nature of the treatment and the added skin rejuvenation effect [1, 15]. In fact, one study showed that medium-depth peeling (Jessner’s solution followed by 35% trichloroacetic acid) was as effective as 5% 5-FU cream applied twice daily for 3 weeks. Disadvantages of chemical peeling include prolonged healing time and the self-pay nature of the procedure. Adverse effects depend on the depth of injury reached by the chemical peel, and include persistent erythema, secondary infection, dyspigmentation and scarring, as well as cardiac, renal, and hepatic toxicity (associated with deep phenol peels) [1, 15, 16].
59
Dietary and Herbal Effects Recently, there has been great interest in the presumed benefits provided by dietary modifications and herbal supplements in the prevention or even treatment of cutaneous malignancies [3]. Extensive work has been done, with only a few studies showing significant benefit. In one study, a low-fat diet was associated with significantly lower incidence of actinic keratosis development compared to a high-fat diet. Other studies on animal models have shown that the polyphenols from black and green tea may inhibit UV-induced photocarcinogenesis.
Conclusion Elderly people constitute a special, expanding patient population that is more prone to develop cutaneous malignancies. Although surgery is usually considered the optimal management option for the treatment of cutaneous malignancies, elderly patients are, not uncommonly, found to be poor surgical candidates because of multiple causes, including age and associated medical comorbidities, among others. In such cases, alternative modalities of treatment may be of great benefit. The details of the different modalities should be known, as well as the characteristics of the tumor (size, type, and location) and the patient profile, in order to choose the best modality. In future, more large randomized controlled trials are needed to reach standard guidelines for the use of the different modalities, and also to test new therapies that are continuously emerging.
. Figure 59.3 Elderly woman with extensive AKs over the face treated with chemical peeling: (a) before treatment, (b) during treatment, and (c) after treatment
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References 1. Tull S, Nunley K, Sengelmann R. Nonsurgical treatment modalities for primary cutaneous malignancies. Dermatol Surg. 2008;34(7): 859–872. 2. Neville JA, Welch E, Leffell DJ. Management of nonmelanoma skin cancer in 2007. Nat Clin Pract Oncol. 2007;4(8):462–469. 3. Chakrabarty A, Geisse JK. Medical therapies for non-melanoma skin cancer. Clinics in dermatology. Clin Dermatol. 2004;22(3):183–188. 4. Martinez JC, Otley CC. The management of melanoma and nonmelanoma skin cancer: a review for the primary care physician. Mayo Clin Proc. 2001;76:1253–1265. 5. Papadavid E, Stratigos AJ, Falagas ME. Imiquimod: an immune response modifier in the treatment of precancerous skin lesions and skin cancer. Expert Opin Pharmacother. 2007;8(11):1743–1755. 6. Zagon IS, Donahue RN, Rogosnitzky M, et al. Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function. Exp Biol Med (Maywood). 2008;233(8):968–979. 7. Saitta P, Krishnamurthy K, Brown LH. Bleomycin in dermatology: a review of intralesional applications. Dermatol Surg. 2008;34(10): 1299–1313. 8. Kim KH, Yavel RM, Gross VL, et al. Intralesional interferon alpha-2b in the treatment of basal cell carcinoma and squamous cell carcinoma: revisited. Dermatol Surg. 2004;30:116–120.
9. Gaumann A, Schlitt HJ, Geissler EK. Immunosuppression and tumor development in organ transplant recipients: the emerging dualistic role of rapamycin. Transpl Int. 2008;21(3):207–217. 10. Kuflik EG. Cryosurgery for skin cancer: 30-year experience and cure rates. Dermatol Surg. 2004;30:297–300. 11. Sheridan A, Dawber R. Curettage, electrosurgery, and skin cancer. Australas J Dermatol. 2000;41:19–30. 12. MacCormack MA. Photodynamic therapy in dermatology: An update on applications and outcomes. Semin Cutan Med Surg. 2008;27(1):52–62. 13. Morton CA, McKenna KE, Rhodes LE. Guidelines for topical photodynamic therapy: update. Br J Dermatol. 2008;159(6):1245–1266. 14. Iyer S, Friedli A, Bowes L, et al. Full face laser resurfacing: Therapy and prophylaxis for actinic keratoses and non-melanoma skin cancer. Lasers Surg Med. 2004;34:114–119. 15. Hantash BM, Stewart DB, Cooper ZA, et al. Facial resurfacing for nonmelanoma skin cancer prophylaxis. Arch Dermatol. 2006;142: 976–982. 16. Lawrence N, Cox SE, Cockerell CJ, et al. A comparison of the efficacy and safety of Jessner’s solution and 35% trichloroacetic acid vs 5% fluorouracil in the treatment of widespread facial actinic keratoses. Arch Dermatol. 1995;131:176–181.
60 Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma Panthea Heydari . Andia Heydari . Howard I. Maibach
Introduction Epidermal thickness is used for studying tissue weight, protein, and/or DNA content since epidermal metabolism occurs at the level of single keratinocytes [1] and varies significantly over anatomic sites but not significantly from person to person [2]. Epidermal thickness is greater in males than in females [2] and can be further evaluated in regard to risk factors for melanoma. In fact, centuries ago, heliotherapy via intense sun exposure was used to treat illness [3]. Aging, which produces dermal damage to elastic and collagen fibers that releases thickened, stiff, tangled, and degraded non-functional fibers of skin, is a continuous process that decreases rapid production of keratinocytes and is enhanced by sun exposure [4]. Outward signs of skin aging, as determined by photogoaging [5–7] or simply by sun exposure [8], are seen as a decrease in size of appendages and subcutaneous fat [8]. These changes are a result of local regulatory factors not operating as accurately as they once did in younger skin – examples include loss, thinning, and de-pigmentation of hair, decreased secretion of sebum, increased dryness, and thinning and atrophy of the epidermis [8].
Sun Exposure Sun exposure parameters have often demonstrated a positive correlation between the development of melanoma and the recollection of short-term intense UVR exposure, particularly burning, in childhood [7]; yet, Ackerman [3] suggests evidence has not yet convincingly shown that sunlight in excessive exposures is the determinant in the development of most melanomas. Photoaging is triggered by receptor-initiated signaling, mitochondrial damage, protein oxidation and consequences of telomere-based DNA damage [6]. The aforementioned variations result in differing thickness displayed by photodamaged skin.
Epidermal thickness is used as a variable to measure cutaneous melanoma (CM) risk. UVR is considered a foremost environmental cause for CM risk. The number of sunburns acquired over a lifetime, measured by acute and intense sun exposure, is also an important risk factor for CM [5]. Since sunburn measurements are easily acquired by looking at regions of excessive redness after exposure to the sun and measurement of penetration of UVR, they are commonly used by investigators for CM risk research. However, they still present the limitation of being a retrospective tool, as discussed later in the article.
Sunburns and Risk for Melanoma Elevated risk for CM was found to be associated with the history of childhood sunburns, with the highest risk groups as those exposed to sunlight in early life, even if the period of exposure was relatively brief [9–12]. In fact, a significant increase in the risk of adult cutaneous melanoma acquisition corresponded to the number of weeks spent on holiday at the beach, such as Australia or California, as a child [9, 10]. Events occurring during the first few decades of life have a special role in determining melanoma risk for that same adult (> Tables 60.1 and > 60.2). In adults older than 20, there was a significant negative correlation between age and number of sunburns per sun-year. In subjects younger than 20, there was a significant positive correlation between age and number of sunburns [13]. For this study, damage to the skin does not correspond with age, but, instead, with the amount of UV penetration and sun exposure [3, 6]. Severe or frequent sunburns in childhood conferred a two- to threefold increased risk for melanoma acquisition [6] – the number of sunburns is interpreted as an indicator of sun damage during that age frame, as influenced by sun exposure, habits, and individual sun sensitivity. Tissues that undergo postnatal development are especially vulnerable to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_60, # Springer-Verlag Berlin Heidelberg 2010
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60
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma
. Table 60.1 Odds ratio of melanoma by sunburns in childhood and severe sunburns lifelong (Autier P, Dore JF [12]) Factor
Melanoma casesa
Controlsa
186
382
1b
Sometimes
48
26
4.4
(2.5–7.5)
Often
26
12
(4.6–31.0)
Category
Odds ratio (reference category)
(95% confidence interval)
Sunburns in childhood Never
X21 (trend)
58.7; P < 0.001
Mild
21
15
Severe
41
16
X21 (trend)
3.2
(1.5–1.6)
6.5
(3.4–12.3)
43.8; P < 0.001
Severe
Never
180
328
1b 1.7
(1.1–2.6)
1.5
(0.8–2.7)
Sunburns
1
50
53
Lifelong
≥2
24
29
X21 (trend)
4.5; P = 0.04
a
Some strata do not add up to the total because of missing values Adjusted for sex and category
b
environment carcinogen exposure in childhood [10]. Within the melanoma paradigm, there are grounds for inferring that the period of peak melanocytic activity occurs in early life and might therefore be a period of vulnerability to the adverse effects of solar radiation [10]. Biologically, this finding may suggest that melanocytes in children are more sensitive to the sun or that, in the context of the multi-stage model of carcinogenesis, early exposure to the purported carcinogen (i.e. solar radiation) may increase the changes of completing the remaining stages in subsequent life periods [9]. Contradictory evidence [3] suggests that skin lesions or sunburns are not correlated to melanoma acquisition because these skin lesions occur on sites directly exposed to sunlight and are numerous, and none of these descriptions are true for melanoma.
Sun Protection and Risk for Melanoma Research on sun protection results in an increased risk for CM in adults who did not have appropriate sun
protection as children – sun avoidance during childhood would have a greater impact on decreasing melanoma risk than sun avoidance during adulthood [12]. The melanoma risk associated with a given level of sun exposure during adulthood increased with higher sun exposure during childhood; meaning that high sun exposure during childhood constitutes a significant risk factor for melanoma [12]. Adults with current low or moderate sun exposure but high childhood sun exposure may well be at higher risk to develop malignant melanoma than adults with high current sun exposure, but with low childhood sun exposure. Thus, sun protection during childhood has a greater impact on melanoma risk than sun protection during adulthood [12], ultimately reiterating the conclusion of increased CM risk with increased childhood sun exposure and sunburn [9–12]. When considering sun protection, other research [3, 14] suggests no association with overall sunscreen amount used with CM [14] or an increase in CM [3]; yet it is the never or rare use of sunscreen that increases CM risk. Some [14] suggest that the propensity to sunburn, ability to tan, skin type, density of facial freckling, hair color and
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma
60
. Table 60.2 Interaction between sunburns in childhood and severe sunburns lifelong on melanoma risk (Autier P, Dore JF [12]) Odds ratio (95% confidence interval)a Severe sunburns lifelong Sunburns in childhood Never
Never Cases
143
40
Controls
316
64
OR
1(reference)
1.4
(95% CI) Ever
Adjusted
Adjusted odds ratio (95% CI)b
Ever
1(reference)
(0.9–2.2)
Cases
37
35
Controls
13
18c
OR
8
4.9
5.3
(95% CI)
(3.9–16.5)
(2.6–9.2)
(3.3–8.6)
1(reference)
1.2 (0.8–1.8)
a
Adjusted for sex and age in decades Adjusted for sunburns in childhood or severe sunburns lifelong in addition to sex and decade c Strata do not add up because of missing values b
eye color over the course of an individual’s lifetime are all significantly associated with melanoma acquisition. Yet, the only period for which there was an association with sunscreen use and melanoma was under 5 years of age, where the risk of melanoma was doubled for those who never/rarely used sunscreen vs. often/always used sunscreen. Additional research [3] correlates sunscreen use with the development of melanoma since sunscreen might lure individuals into believing that they are allowed to be in the sun longer, increasing the length of exposure to solar radiation and thus risk for developing melanoma. Other research [15] suggests that the concept of a ‘‘critical period’’ for melanoma acquisition is not true – in fact, there is an additive effect that begins at an ‘‘early age’’ with respect to melanoma and sun exposure.
Skin Thickness Research about epidermal thickness differences in children verses adults suggests increased melanoma occurrences in individuals who have had childhood sunburns. Contradictory evidence suggests either photoaging [6, 14] or solely sunlight exposure [3] is responsible for most aging changes in skin appearance. Photoaging can be associated with mitochondrial damage (the most common deletion in mtDNA, which is tenfold less common
. Table 60.3 Epidermal thickness using backward multiple regression analyses is not significant when measuring stratum corneum and cellular epidermis thickness Stratum corneum Body site
>0.0001
Age
Ns
Gender
0.048
Cellular epidermis >0.0002 Ns >0.0001
Ns = Nonsignificant
than sun-protected areas, is found in photodamaged skin), telomere based DNA damage, protein oxidation, and receptor-initiated signaling [6]. Long-term stress and old age are also common causes of epidermal chalone mitotic depression in basal cells [16]. Epidermal thickness is important because it is a uniformly accessible measure and can be measured in relation to anatomic site, age, gender, pigmentation, blood content, and smoking [2]. Skin thickness and pigmentation does not significantly increase or decrease in specific anatomic sites of individuals as they age during adulthood [1, 2, 4, 17] (> Table 60.3), where ‘‘adulthood’’ is measured as different periods of the 20–68-year-old age group (1), the 50–72-year-old age group (2), and/or the 23–47-year-old
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age group (3). These data suggest that, as expected, the stratum corneum and stratum granulosum is significantly thicker in sun protected than in sun-exposed sites, where the latter sites show a greater variability [17]. Contradictory results [18] (> Fig. 60.1) from research done thirty years prior that take proposed cell shrinkage measurements into account suggest an increase epidermal thickness in the arms and leg sites of adults in the 15–89-year-old age group. Such research [18] suggests it is commonly believed that the epidermis in an older person is thinner than in a younger person. This may be because skin looks thinner and measurements that fail to correct for shrinkage would show an apparent thinning of epidermis with advancing age. This thin appearance occurs because skin becomes less elastic. Additional results [19] suggest no change in epidermal thickness during the young adult phase (age group 18–25), but an increased epidermal thickness during the older age group (adults over 65).
Discussion Skin cancer is a common malignancy [11] and requires great attention to be paid per patient for appropriate care. Many changes in aging skin are produced or aggravated by ionizing or ultraviolet radiation [8]. Photoaging, change accentuated by the sun, accounts for much of these age-related changes in skin appearance [6] and is represented by chronic degenerative changes including freckles (ephelides), solar lentigines, nevi, solar keratoses and melanoma [4, 28]. Chronic sunlight exposure can induce an impediment of normal maturation of human dermal collagen resulting from a degradation of mature collagen cross-links [4, 6, 8, 20]. These cells’ histological photo-damage of degradation and remodeling of collagen is related to individual cumulative ultraviolet radiation (UVR) exposure. As a function of age, nonfibrous protein and soluble collagen decrease while insoluble collagen increases, ultimately increasing total collagen
. Figure 60.1 Distribution of Average epidermal thickness on three regions are about half those recorded by negative shrinkage (Whitton 1973). Average epidermal thickness (micrometer) measured against Percentage (male and female) per 20 micrometer increments
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma
with respect to age [20]. Senescent cells have an increased resistance to apoptosis (natural cell death) and survive for very long periods of time without division, allowing for DNA and protein damage accumulation, and, eventually, cumulative cellular damage [4, 8]. Absorption accounts for more than 93% of why light does not pass through skin [21]. Excessive absorption of sunlight can create sunburns, which, some suggest, play an important role in the development of skin cancer, especially malignant melanoma [13]. In contrast to earlier views of dependence on the stratum corneum’s thickness for filtration of harmful UV rays, studies show [22] that the epidermal pigment is largely responsible for such filtration. The filtration and absorption ratios are evaluated in regards to sun exposure. Sunburns occurring during childhood are often cited as posing the greatest risk for cutaneous malignant melanoma [5]. Since melanoma is a common malignancy [11], studies are conducted for further knowledge on the disease. While skin thickness data are often difficult to interpret [23], it remains a widely used parameter to evaluate the influences of different variables on skin aging, including sunlight exposure, elasticity, and absorption [23, 24]. Whole skin thickness increases in youth [23, 24], remain constant during adulthood, and decrease in the elderly, beginning at age 70 [24]. Others maintain that photoexposed areas thicken with age whereas protected areas become thinner, or that skin thickness changes with age are related to location on the extremities or axially as opposed to sun exposure [23]. Regardless, further evaluation of skin thickness, melanoma, and childhood exposure to the sun must be acquired to better understand relations to melanoma occurrences in adulthood. The only period for which there was an association of melanoma with sunscreen use was under 5 years of age, when risk of melanoma was doubled for those who never/ rarely used sunscreen at home versus often/always [14]. Participants with many sunburns during childhood also tend to have more sunburns later in life than those with only few or no sunburns during childhood and ultimately have a higher risk for acquiring adulthood melanoma [15]. When relating epidermal thickness to age with respect to adult melanoma acquisition, conclusions suggest that since epidermal thickness measurements do not change as an individual ages [1, 2, 4, 17], skin thickness cannot currently influence the increased risk of childhood sunburns on adult melanoma. While aforementioned data suggest a relationship between melanoma and UVR exposure, some research [3] proposes that the notion of melanoma in Caucasians
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being a consequence of the effects of the dose of cumulative UVR received by the skin is without merit in prepubescent children. The association of melanoma as the result of intermittent UVR exposure over the course of years, specifically in caucasions, is baseless [3] because the melanoma acquired in these individuals were in places not in the direct path rays of the sun.
Problems with Data A possible confound when measuring epidermis thickness is shrinking of the skin during fixation, which is required to measure epidermal thickness [1, 25, 26]. To offset shrinkage, acetic acid is often used. The belief that the epidermis of an older individual is thinner than that of a younger individual is due to the inability of some researchers to correct such cell shrinkage [18]. The apparent thinning of the epidermis with advancing age occurs because skin becomes less elastic with age. The method by which sun exposure (and ultimately skin thickness) is measured and the concept of shrinkage leads to strikingly different conclusions regarding the association of sun exposure at specific ages and consequent risks of adulthood melanoma [10]. For several studies, data were acquired by asking patients to recall past circumstances. Recall and retrospective data acquisition [15, 27] can create biased responses by patients and, consequently, altered data. Additionally, interviewers and questioneers can cause patients to feel either overly comfortable or awkward, thus further skewing responses. When applicable in certain studies of adolescent or older individuals, patients also kept a diary of daily events [13], which could have allowed individuals to learn from their mistakes, showing that sun behavior changes with age and maturity [5] – again, an extraneous variable that is not consistent throughout the research. It has also been suggested that melanoma patients tend to over-report past sun-exposure habits in an attempt to explain and/or justify why they received the diagnosis of malignant melanoma [27]. The inability to keep participant environment the same could affect data in other ways as well. Some suggest [9] a significant increase in the risk of cutaneous melanoma is also associated with the number of weeks spent on holiday at the beach as an adult and as a child. However, others [10] propose that individuals who spent their youth in places similar to California (where excessive outdoor play is the norm) were at increased risk of melanoma compared with those born and raised elsewhere,
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even after many decades of co-habitation. Thus, the amount of time spent in the sun as a child appears to be a significant risk factor for melanoma – not increased age or decreased skin thickness.
Vitamin D and Sun Exposure UV exposure is the most important environmental risk factor for the development of non-melanoma skin cancer [7]. Clothing is efficient in absorbing all UV-B radiation, thereby preventing any UV-B photons from reaching the skin; additionally, a Vitamin D supplement can also be effective in melanoma prevention [7], Approximately 90% of all requisite vitamin D is attained via sunlight; thus, strict sun protection procedures to prevent melanoma may induce the severe health risk of vitamin D deficiency [7]. To help alleviate this challenge, physicians suggest daily administrations of 15 min of sunlight exposure or oral consumption of vitamin D [7]. A healthy balance must be reached and publicly advertised in order to minimize acquisition of malignant melanoma.
Future Implications Sunburns may play an important role in the development of skin cancer [13], and should be further investigated as a means to prevent melanoma occurrences. Preventing intermittent sunburns during childhood has the strongest correlation with decreased malignant melanoma occurrences later in life [9–12]. Such prevention measures include appropriate sun block usage and control of the amount of time spent in the sun and outdoors, depending on risk factors of the individual, such as sun sensitivity, white skin, fair hair, light eyes, tendency to freckle, family history of melanoma, dysplastic nevi, increased numbers of typical nevi, large congenital nevi, and immunosuppression [11, 28]. Since UVR exposure is the most important environmental risk factor for the development of skin cancer [7], it is important to protect early childhood skin from exposure to excessive sunlight to ultimately protect them from adulthood melanoma acquisition.
Conclusion In relation to the study of age thickness as a function of time, future studies would be benefited by increased standardization of skin sites tested, methodology, and sample size. Great differences in the study method,
population, and anatomic site are likely to account for markedly different results from different researchers, which obscure reasonable conclusions. Since the effect of age on thickness of skin strata is one of the more controversial topics among dermatological scientists [23, 29], further research needs to be done to synchronize data and create a standard method for both acquiring and interpreting such data.
Cross-references > Carcinogenesis:
UV Radiation
References 1. Bergstresser PR, Pariser RJ, Taylor JR. Counting and sizing of epidermal cells in normal skin. J Invest Dermatol. 1978;70(5):280–284. 2. Sandy-Moller J, Poulsen T, Wulf HC. Epidermal thickness at different body sites: relationship to age, gender, pigmentation, blood content, skin type, and smoking habits Acta Dermato-Venereologica. 2003;83(6): 410-413. 3. Ackerman B. The Sun and the ‘‘Epidemic’’ of Melanoma: Myth on Myth! New York: Ardor Scribendi, 2008. 4. Wulf HC, Sandby-Møller J, Kobayasi T, Gniadecki R. Skin aging and natural photoprotection. Micron. 2004;35(3):185–191. 5. Dennis LK, Vanbeek MJ, Beane Freeman LE, Smith BJ, Dawson DV, Coughlin JA. Sunburns and risk of cutaneous melanoma: does age matter? A comprehensive meta-analysis. Ann Epidemiol. 2008;18 (8):614–627. 6. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and therapy. Br J Dermatol. 2007;157(5):874–887. 7. Reichrath J. The challenge resulting from positive and negative effects of sunlight: how much solar UV exposure is appropriate to balance between risks of vitamin D deficiency and skin cancer? Prog Biophys Mol Biol. 2006;92(1):9–16. 8. Daniels F, Jr. Sun exposure and skin aging. NY State J Med. 1964;64:2066–2069. 9. Zanetti R, Franceschi S, Rosso S, Colonna S, Bidoli E. Cutaneous melanoma and sunburns in childhood in a southern European population. Eur J Cancer. 1992;28A(6–7):1172–1176. 10. Whiteman DC, Whiteman CA, Green AC. Childhood sun exposure as a risk factor for melanoma: a systematic review of epidemiological studies. Cancer Causes Control. 2001;12(1):69–82. 11. Rager EL, Bridgeford EP, Ollila DW. Cutaneous melanoma: update on prevention, screening, diagnosis, and treatment. Am Fam Physician. 2005;72(2):269–276. 12. Autier P, Dore JF. Influence of sun exposure during childhood and during adulthood on melanoma risk. EPIMEL and EORTC Melanoma Cooperative Group. European, Organization for Research and Treatment of Cancer. Int J Cancer. 1998;77(4):533–537. 13. Thieden E, Philipsen PA, Sandby-Moller J, Wulf HC. Sunburn related to UV radiation exposure, age, sex, occupation, and sun bed use based on time-stamped personal dosimetry and sun behavior diaries. Arch Dermatol. 2005;141(4):482–488.
Sunlight Exposure and Skin Thickness Measurements as a Function of Age: Risk Factors for Melanoma 14. Youl P, Aitken J, Hayward N, Hogg D, Liu L, Lassam N, Martin N, Green A. Melanoma in adolescents: a case-control study of risk factors in Queensland, Austrailia. Int J Cancer. 2002;98(1):92–98. 15. Pfahlberg A, Kolmel KF, Geffeller O. Timing of excessive ultraviolet radiation and melanoma: epidemiology does not support the existence of a critical period of high susceptibility to solar ultraviolet radiation-induced melanoma. Br J Dermatol. 2001;144 (3):471–475. 16. Bullough WS. The control of epidermal thickness. Br J Dermatol. 1972;87(3):187–199. 17. Huzaira M, Rius F, Rajadhyaksha M, Anderson RR, Gonzalez S. Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy. J Invest Dermatol. 2001;116(6):846–852. 18. Whitton JT, Everall JD. The thickness of the epidermis. Br J Dermatol. 1973;89(5):467–476. 19. Sauermann K, Clemann S, Jaspers S, Gambichler T, Altmeyer P, Hoffmann K, Ennen J. Age related changes of human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo. Skin Res Technol. 2002;8(1):52–56. 20. Smith JG, Jr., Davidson EA, Sams WM, Jr., Clark RD. Alterations in human dermal connective tissue with age and chronic sun damage. J Invest Dermatol. 1962;39:347–350. 21. Na R, Stender IM, Henriksen M, Wulf HC. Autofluorescence of human skin is age-related after correction for skin pigmentation and redness. J Invest Dermatol. 2001;116(4):536–540.
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22. Mitchell RE. The effect of prolonged solar radiation on melanocytes of the human epidermis. J Invest Dermatol. 1963;41:199–212. 23. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res Technol. 2005;11(4):221–235. 24. Serup J (ed). Handbook of Non-invasive Methods and the Skin. Boca Ratton: Taylor & Francis, 2006, pp. 512–513. 25. Zinabu GM, Thomas B. The effects of formalin and Lugol’s iodine solution on protozoan cell volume. Limnolog – Ecol Manage Inland Waters. 2000;30(1):59–63. 26. Ohno N, et al. Application of cryobiopsy to morphological and immunohistochemical analysisE´vessels. Cancer. 2008;(113): 1068–1079. 27. Westerdahl J, Olsson H, Ingvar C. At what age do sunburn episodes play a crucial role for the development of malignant melanoma. Eur J Cancer. 1994;30A(11):1647–1654. 28. Elwood JM, Whitehead SM, Davison J, Stewart M, Galt M. Malignant melanoma in England: risks associated with naevi, freckles, social class, hair colour, and sunburn. Int J Epidemiol. 1990;19(4):801–810. 29. Linos E, Swetter SM, Cockburn MG, Colditz GA, Clarke CA. Increasing burden of melanoma in the United States. J Invest Dermatol. 2009 Jul;129(7):1604–1606.
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75 Animal Models Sara Flores . Farzam Gorouhi . Howard I. Maibach
Introduction Skin aging is influenced by a combination of biological, physiological, and environmental factors. While in vitro models and bioengineering techniques can reproduce one or more of these factors, they cannot encompass the numerous components existing in living tissue. Animal models mimic the combination of influences contributing to skin aging in humans. Though many potential animal models exist, studies on skin aging use relatively few lab species. The following describes some of the experiments that utilized animal models. They are divided into two categories, actinic and intrinsic aging experiments. Hopefully, it will serve as a starting point and resource for those wishing to develop further knowledge and interventions.
Actinic Aging Most experiments utilize rodentia because they possess the characteristics needed for studies on skin aging. Mice and rats in particular are useful for numerous reasons. First, genes in mouse and man are >99% conserved. In addition, the ability to add to and selectively alter the mouse genome increases opportunities for using the mouse to understand the genetic basis of human health and disease [1]. Therefore, the mouse provides a good model for many human diseases, including skin disorders. The first attempt to observe connective tissue damage via UV radiation utilized the Dublin Imprinting Control Region (ICR) Albino Random Bred mouse. The albino mouse, a laboratory strain of the house mouse, Mus musculus, is characterized by a mutation in the Tyr locus on chromosome 7, which codes for tyrosinase, an enzyme important for the proper production of melanin. In 1964, Sams et al. shaved the backs of mice and subjected them to long-term exposure with UV, which produced the first report of observed elastosis on the dorsal backs of the animals [2]. The study did not differentiate between results of UVA and UVB exposure but instead recreated the effects of long-term exposure to sunlight in humans, a feat that had failed up to this point in other animal models, such as the guinea pig. However, the study
used doses of UV radiation exceeding those normally experienced by humans, and the tumors developed by the mice were thought different from those produced in man. Thus, scientists concluded that the albino mouse was not a suitable model for elastosis and turned to the hairless mouse. Due to events mentioned above and a report by Winkelmann et al., which discussed another experiment that produced the same tumors in hairless mice as those seen in humans, hairless mice became the animal of choice [3]. Hairless mice possess mutant alleles at the hairless (hr) gene, which lies at the 70 Mb position of chromosome 14 [4]. The strains have proven valuable in various photobiologic investigations ranging from phototoxicity, photoimmune effects, carcinogenesis, and UV-induced DNA damage [5]. In addition, the UVinduced changes are comparable to those in human skin and are therefore very relevant. The action spectrum for the acute responses to UV radiation edema in the hairless mouse is comparable to that for sunburn erythema in humans, and the time courses for both responses are similar [6]. Further, the UV-induced connective tissue damage that occurs is largely analogous to that in man [7]. These mice occur in two varieties, albino and pigmented [1], and the option of pigment can add another dimension to experimental possibilities. However, the most commonly used hairless mouse for photoaging is the albino Skh-hairless [8]. Elastosis has been the most common method for assessing damage caused by long-term UV exposure. The first study designed to produce elastosis in a hairless mouse was performed by Berger et al. in 1980 using the naked (Ng/–) albino strain [9]. Since then, numerous experiments have recreated elastosis from exposure to ultraviolet radiation. Johnston et al. exposed hairless mice either to UVA or UVB radiation on alternate days to assess the consequences of long-term exposure on the connective tissues of the skin. Hydroxyproline and desmosine were used to interpret collagen and elastin concentrations. Desmosine content of the skin, which was used as an index of cross-linked elastin, was increased in mice treated with UVA or UVB. In contrast, collagen content, measured as hydroxyproline, was the same in all
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_75, # Springer-Verlag Berlin Heidelberg 2010
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treatment groups [10]. Despite the similarities in collagen content, the experiment demonstrated through biological assays that collagen synthesis via prolylhydroxylase (PH) was impaired with UVA exposure and thus may lead to decreases in collagen synthesis with increased time and dermal atrophy. Studies on elastosis caused by UV radiation continued with histochemical studies by Kligman et al. [7], and with electron microscopy, biochemical and immunochemical experiments [11]. The former studies emphasized the effects of UV light on elasticity, a dermal component. Some studies demonstrated the effects of photodamage on the viscosity of the skin, an epidermal component. Fujimura et al. treated female HR/ICR hairless mice topically with 1,25-dihydroxyvitamin D3 to assess its effect and contribution to photo wrinkling [12]. 1,25-dihydroxyvitamin D3, or 1,25dihydroxycholecalciferol, is the active form of the vitamin produced in the kidneys. In humans, it is made from 25OH-cholecalciferol, which arises from cholecalciferol in the liver. Cholecalciferol is obtained directly from diet but can also be manufactured in the skin from 7-dehydrocholesterol. This conversion occurs in the epidermis and requires ultraviolet light. The mice were treated once daily, five days per week with 1.00 mg, 0.20 mg, or 0.05 mg of 1, 25-dihydroxyvitamin D3. Skin sagging was assessed using a scale described by Bisset et al. [13] four grades were used, with the fourth being the most severe. After six weeks of treatment with 1.0 mg/day of 1,25-dihydroxyvitamin D3, skin appearance deteriorated, as there were coarse, deep wrinkles across the backs of the mice. 1,25-dihydroxyvitamin D3 also had an effect on the mechanical properties of the skin. Ue or immediate distention, a factor measuring elasticity, did not change after topical administration. However, Uv or delayed distention, a parameter of skin viscosity, increased remarkably after 1,25-dihydroxyvitamin D3 application at all doses. Note that Ue is largely a dermal component, whereas Uv is considered epidermal. This study demonstrated the effects of high levels of 1,25-dihydroxyvitamin D3 on the viscosity of the skin and suggested that changes in the mechanical properties after topical 1,25(OH)2 VD3, treatment are due to physical changes in the epidermis [12]. The experiment also measured two other parameters, Uf and Ur. Ur represents immediate retraction, whereas Uf indicates final distention. The ratio of the two, Ur/Uf, may be used as a general parameter of aging. Usually, the ratio decreases with age-related changes due to a decrease in Ur. However, in the study by Fujimara et al. the decrease in the ratio was due to an increase in Uf, which suggests a distinction from normal age-related changes in the skin.
Further studies may later reveal reasons behind these discrepancies. In addition, higher dosages of vitamin D have been recommended for prevention of osteoporosis and increased calcium absorption. However, this study suggests that high concentrations of vitamin D may contribute to symptoms in other areas of the body. Future experiments may want to explore the balance between benefits of taking vitamin D and the risks of accelerated degradation in other systems. The degradation of collagen and other components of the dermal extracellular matrix can partially be explained by the upregulation of matrix metalloproteinases (MMPs). Matrix metalloproteinases, a family of nine or more zinc-dependent endopeptidases, cleave the constituents of the extracellular matrix in connective tissues [14]. The enzymes have also been implicated in other pathologies such as atherosclerosis and emphysema. Their association with human aging has been revealed in studies showing the upregulation of matrix metalloproteinases in cultured fibroblasts after irradiation [15]. Ultraviolet (UV) B induces expression of MMP-1, MMP-3, and MMP-9, whereas UVA induces expression of MMP-1, MMP-2 and MMP-3 [16, 17]. Saarialho-Kere et al. ascertained changes in matrix metalloelastase (MME) in hairless mice skin using immunohistology. Anti-MME antibodies detected presence of the enzyme in samples taken after repeated irradiation. The first sample, taken after 12 days, indicated the dermis as the location of most MME. The last samples at 8 and 11 weeks showed a strong signal for the subsistence of MME. In every specimen, stromal cells in the dermis resembling macrophages or fibroblasts contained the enzyme, but no epidermal cells showed MME signals. The application of medications to the reversal of skin aging has also been studied using the hairless mouse. Lorraine Kligman was one of the first to illustrate the restorative capabilities of topical all-trans-retinoic acid (RA). Skh hairless-1 albino mice were irradiated dorsally thrice weekly for 10 weeks to produce the damage. She then used three groups of mice receiving RA in various percentages or different vehicles to analyze the effects. The skin was studied histologically and with electron microscopy [18]. Via these methods, she found evidence of repair to UV-damaged dermis of mouse skin and illustrated the repair was a direct result of increased collagen synthesis by hyperactive fibroblasts stimulated by RA. These experiments helped to establish the use of topical retinoids for repair of photodamaged skin. An innovative model for future regenerative processes is described by Byung-Soon Park et al. Their purpose was to assess the potential for using
Animal Models
adipose-derived stem cells (ADSCs) in treatments for aged skin. ADSCs have previously been studied for use in wound repair, and experiments have illustrated their ability to stimulate collagen synthesis and migration of fibroblasts [19]. Since the damage resulting from UV exposure includes degradation of collagen and deceleration of collagen synthesis, ADSCs were postulated a potential cosmetic treatment for photodamage. To find out, ADSCs and conditioned media of ADSCs (ADSC-CM) were injected intradermally on the backs of three micropigs, twice in a 14-day interval. One month after the second injection, skin samples from the injection site were evaluated histologically and by Western blot. Western blot study showed a remarkable increase in collagen, supporting the possibility for future use of ADSCs and their secretory factors in the treatment of skin aging [19]. UV radiation is included in a category of skin insults known as oxidative species. Alcohol consumption, ozone exposure, and cigarette smoking are included in this group, and tobacco smoke has an especially deleterious effect on the extracellular matrix of the skin. This is demonstrated in in vitro studies harvesting fibroblasts treated with tobacco smoke extract [20]. There is evidence from in vivo studies that tobacco smoke induces premature skin aging [21]. The first study examining changes in the connective tissue matrix of hairless mice due to cigarette smoke was performed in 2007 by Tanaka et al. Aqueous smoke solution was prepared using the smoke from cigarettes dissolved in phosphate buffered saline. This extract was topically or intracutaneously administered to the backs of male hairless mice thrice weekly for six months. After six months, skin specimens were taken and stained with hematoxylin and eosin (H&E). A monoclonal anti-collagen type I antibody was added to better identify collagen bundles. In the treated sample, there was a loss of discernable collagen bundles in the dermis when compared with the control. This study provided the first direct evidence that tobacco smoke induces premature skin aging using an in vivo method [21]. Leow and Maibach summarized the studies that had used Laser Doppler Flowmetry (LDF) and other methods to measure the skin’s blood flow. Despite differing methods, instruments, and test populations, there was consistent decrease in cutaneous blood flow during the first 2 minutes of smoking cigarettes [22]. No changes were found in nonsmokers. A later study by Manfrecola et al. observed a 38.1% reduction in smokers and 28.1% reduction in nonsmokers, but the recovery time was less for nonsmokers than for smokers [23]. Both studies suggest that decreased blood flow may be a contributing factor to
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visible signs of skin aging. However, additional studies on the effects of cigarette smoke in animal models would provide further molecular evidence and corroboration.
Intrinsic Aging The literature addressing innate aging is not as cohesive as that for actinic. This is because the study of intrinsic aging is more difficult for a few reasons. First, the consequences due to one measurable factor, such as UV radiation, can be observed more quickly than those from biological or physiological influences. Actinic aging describes an accelerated process. The skin’s degeneration is related to the amount and length of exposure, and the results are visible. Intrinsic aging in humans occurs over years, and it becomes more difficult to assess the effects that biological or physiological components have on the skin, free of environmental influences. In addition, scientists have yet to prove the exact reasons for the different rates of aging among individuals. Therefore, the experiments mentioned below are the beginning to the further understanding some of the processes involved with intrinsic aging using animals. Hiromi Kimoto-Nira et al. discussed the use of senescence-accelerated mice (SAM) to observe various physical changes associated with aging, including those of the skin. Senescence-accelerated mice develop normally, but then show an early onset of aging and allow scientists to observe processes that may be similar in humans over a shorter length of time. This particular experiment described the process in terms of bone density loss, incidence of skin ulcers and hair loss. Lactococcus lactic subsp. cremoris H61(strain H61), a probiotic, was administered to a specific substrain of senescence-accelerated mice, SAMP6, orally for 5–9 months [24]. The result was decreased incidence of skin ulcers and a lesser degree of hair loss, both assessed using a grading score system developed by Hosokawa et al. in 1984 [25]. In 1975, a study addressed changes in connective tissue on a more molecular level and helped to illuminate the relationship between hydroxylysine-linked carbohydrate units in collagen molecules and age. Murai et al. used the fact that glycosylation of collagen renders the molecule more insoluble to separate collagens obtained from young and old rats into soluble and insoluble fractions. They then sampled the insoluble fraction and determined the extent of glycosylation. They observed a decrease in glycosylation of hydroxylysine in collagen during maturation followed by a gradual increase in proportion to age [24]. Note that at the time of the experiment, the role of glycosylation in the formation of collagen molecules was
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not understood. Yet, the experiment utilized an animal model to observe molecular differences in collagen due to age. The study also suggested that the changes observed in skin collagens could be extended to collagens in other connective tissues. Though rats have not been examined as closely as mice genetically, they are frequently used in studies concentrated on the skin. The Ishibashi (IS) rat, a cross between Wistar and wild rats, has a unique skin appearance characterized by wrinkles and furrows appearing at an age of 12 weeks. Although the rats demonstrate the physical symptoms associated with actinic aging, the changes occur independently from the influence of UV radiation. Sakuraoka et al. explained the wrinkles by analyzing elastin and collagen content in young and old IS rats [26]. As a control, they assessed the same factors in young and old Sprague–Dawley (SD) rats. Collagen content was determined by measuring hydroxyproline content in back skins according to the method of Prockop [27]. The dermis was blended with 0.5 M acetic acid, digested with pepsin (a proteinase) at 4 C for several hours, and centrifuged. The resulting supernatant was dialyzed against 0.02 M dibasic sodium phosphate in order to precipitate collagen. This precipitate was then subjected to 5% SDS-PAGE containing 3.6 M urea under non-reducing conditions. Coommasie brilliant blue (CBB)-R250 was used to stain the gels, and the bands were examined using a densitometer. The elastin content in IS rat skin was estimated using isodesmosine, an isomer of cross-linking structures composed of four elastin molecules. The isodesmosine content was obtained by hydrolyzing the skin samples with 6 M HCl for 18 h at 110 C and by using high performance liquid chromatography. No significant differences in skin collagen content were discovered between matured and aged tissues in either IS rats or SD rats, which suggested collagen may not be related to the signs of aging seen in the IS rat. The findings were different with respect to elastin content, and the aged IS rat skins had significantly less isodesmosine than the skins from their younger counterparts. The notable reduction of isodesmosine in the aged IS rat skins and lack of isodesmosine changes in SD rat skins suggest that isodesmosine content may be related to the observable cutaneous aging of IS rat skin [26]. The authors proposed that the wrinkling occurring in IS rats is due to the decrease in elastin content and may be a good model for the study of skin aging in humans. Although methodologies for experiments on skin aging have been dominated by the use of rodentia, there have been other mammals that have also been effective. Hairless dogs have been used to investigate the improvement of aged skin after topical treatment with kinetin.
The dogs were descendants of Mexican hairless dogs that showed age-related changes in their skin structure. Their epidermis had spotty pigmentation, and lesions had heavy deposition of melanin granules. The left dorsum of each dog was treated with kinetin (KN) solutions while the right side served as the control. After about 50 days of topical application, the KN-treated sites showed normalization of hyperpigmentation and skin rejuvenation. In addition, there were no harmful effects on the skin of the dogs, which suggests this may be a safe long-term treatment for humans [28].
FGF23 and Klotho as Future Models Fibroblast growth factor 23 (FGF-23) null mice and Klotho mice are two transgenic strains with phenotypes resembling human premature aging, such as short lifespan, arteriosclerosis, osteopenia, ectopic calcification in various soft tissues, pulmonary emphysema, impaired maturation of sexual organs, senile atrophy of the skin, and defective hearing [29]. They have been used as models in experiments studying the effects of aging, particularly those correlating the levels of vitamin D with aging processes. As suggested by the experiment of Fujimara et al., high levels of vitamin D3 are correlated with aging skin. The premature aging symptoms in Klotho and FGF-23 null mice are related to high serum levels of phosphate and increases in vitamin D activity [30]. The similarities observed between the two strains have led to the discovery that the proteins encoded by the mutant genes are linked via a common pathway; in fact, the FGF-23 protein is unable to induce renal phosphate wasting in the absence of the Klotho protein [30].
Conclusion The examples presented are but a few of the existing investigations of skin aging. The representation of all related studies would require an entire textbook. In addition, these studies only address animal models, and no discussion of those involving human models, xenografting, or bioengineering methods has been included. It is hoped that this text will serve as a beginning to a more complete acquaintance with the methods used for the study of human skin aging. Though many animal models have been described, there is room for development. As the understanding of the aging mechanism becomes more intricate, new models with processes resembling those in man will be needed
Animal Models
to further knowledge of human risk. Further, many of the experiments described above observe aging processes in the skin outside of any influence from age-related pathologies. The Klotho and FGF-23 mice are good models for illustrating problems simultaneously associated with systemic and skin aging. Perhaps in the future, similar models will identify the relationships between systemic processes and physical appearance. Future cosmetic advances may then lie in treatment of the entire body instead of localized skin corruption. Hopefully, as the readily accessible skin permits aging amelioration, the information will lead to advances with other organs.
Cross-references > Basophilic
(Actinic) Degeneration of the Dermis: An Easy Histological Scoring Approach in Dermal Photo-aging
Acknowledgment We would like to thank Dr. John Epstein for his generous assistance.
References 1. Hedrick H (ed). The Laboratory Mouse. San Diego: Elsevier Academic Press, 2004. 2. Sams WM, Smith JG, Burke PG. The experimental production of elastosis with ultraviolet light. J Invest Dermatol. 1964;43:467–471. 3. Winkelmann RK, Blades EJ, Zollman PE. Squamous cell tumors induced in hairless mice with ultraviolet light. J Invest Dermatol. 1960;34:131–138. 4. Benavides F, Oberyszyn T, VanBuskirk A, Reeve V. The hairless mouse in skin research. J Dermatol Sci. 2009;53:10–18. 5. Maibach HI, Lowe NJ (eds). Models in Dermatology, Vol. 1. Switzerland: Karger, 1985. 6. Cole CA, Davies RE, Forbes PD, D’Aloisio LC. Comparison of action spectra for acute cutaneous responses to ultraviolet radiation: man and albino hairless mouse. Photochem Photobiol. 1983;37: 623–631. 7. Kligman LH, Akin FJ, Kligman AM. Prevention of ultraviolet damage to the dermis of hairless mice by sunscreens. J Invest Dermatol. 1982;78:181–189. 8. Kligman L. The Hairless Mouse Model for Photoaging. Clin Dermatol. 1996;14:183–195. 9. Berger H, Tsambaos D, Mahrle G. Experimental elastosis induced by chronic ultraviolet expotal elastosis induced by chronic ultraviolet exposure. Arch Dermatol Res. 1980;269:39–49. 10. Johnston KH, Oikarinen AI, Lowe NJ, Clark JG, Uitto J. Ultraviolet radiation-induced connective tissue changes in the skin of hairless mice. J Invest Dermatol. 1984;82:587–590.
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11. Hirose R, Kligman LH. An ultrastructural study of ultravioletinduced elastic fiber damage in hairless mouse skin. J Invest Dermatol. 1988;90:697–702. 12. Fujimura T, Moriwaki S, Takema Y, Imokawa G. Epidermal change can alter mechanical properties of hairless mouse skin topically treated with 1α, 25-dihydroxyvitamin D3. J Dermatol Sci. 2000;24: 105–111. 13. Bisset DL, Hannon DP, Orr TV. An animal model of solar-aged skin: histological, physical, and visible changes in UV-irradiated hairless mouse skin. Photochem Photobiol. 1987;46:367–378. 14. Birkedal-Hansen H, Moore WGI, Bodden MK, et al. Matrix Metalloproteinases: A Review. Crit Rev Oral Biol Med. 1993;4: 197–250. 15. Herrman G, Wlaschek M, Lange TS, Prenzel T, Goerz G, ScharffetterKochanek K. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMP) in cultured human fibroblasts. Exp Dermatol. 1993;2:92–97. 16. Scharfetter K, Wlaschek M, Hogg A, et al. UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res. 1991;283:506–511. 17. Koivukangas V, Kallioinen M, Autio-Harmainen H, Oikarinen AI. UV irradiation induces the expression of gelatinases in human skin in vivo. Acta Derm Venereol. 1994;74:279–282. 18. Kligman LH, Chen HD, Kligman AM. Topical retinoic acid enhances the repair of ultraviolet damaged dermal connective tissue. Connect Tissue Res. 1984;12:139–150. 19. Park B-S, Jang KA, Sung J-H, et al. Adipose-Derived Stem Cells and Their Secretory Factors as a Promising Therapy for Skin Aging. Dermatol Surg. 2008;34:1323–1326. 20. Morita A. Tobacco smoke causes premature aging. J Dermatol Sci. 2007;48:169–175. 21. Tanaka H, Ono Y, Nakata S, Shintani Y, Sakakibara N, Morita. A. Tobacco smoke extract induces premature skin aging in mouse. J Dermatol Sci. 2007;46:69–71. 22. Leow Y-H, Maibach HI. Cigarette smoking, cutaneous vasculature and tissue oxygen. Clin Dermatol. 1998;16:579–584. 23. Monfrecola G, Riccio G, Savarese C, Posteraro G, Procaccini EM. The Acute Effect of Smoking on Cutaneous Microcirculation Blood Flow in Habitual Smokers and Nonsmokers. Dermatology. 1998;197: 115–118. 24. Kimoto-Nira H, Suzuki C, Kobayashi M, Sasaki K, Kurisaki J, Mizumachi K. Anti-ageing effect of a lactococcal strain: analysis using senescence-accelerated mice. Br J Nutr. 2007;98:1178–1186. 25. Hosokawa M, Kasai R, Kiguchi K, et al. Grading score system: a method for evaluation of the degree of senescence in senescence accelerated mouse (SAM). Mech Ageing Dev. 1984;26:91–102. 26. Sakuraoka K, Tajima S, Seyama Y, Teramoto K, Ishibashi M. Analysis of connective tissue macromolecular components in Ishibashi rat skin: Role of collagen and elastin in cutaneous aging. J Dermatol Sci. 1996;12:232–237. 27. Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissue and urine. Anal Biochem. 1960;1:228–239. 28. Kimura T, Doi K. Depigmentation and rejuvenation effects of kinetin on the aged skin of hairless descendants of Mexican hairless dogs. Rejuven Res. 2004;7:32–39. 29. Nabeshima Y-I. Klotho: a fundamental regulator of aging. Age Res Rev. 2002;1:627–638. 30. Lanske B, Razzaque MS. Premature aging in klotho mutant mice: cause or consequence? Age Res Rev. 2007;6:73–79.
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Quality of Life
73 Assessing Quality of Life in Older Adult Patients with Skin Disorders Miranda A. Farage . Kenneth W. Miller . Susan N. Sherman . Joel Tsevat
Introduction What Is Health-Related Quality of Life? The constitution of the World Health Organization states that health is ‘‘a state of complete physical, mental, and social well-being, not merely the absence of disease’’ [1]. The organization also defines quality of life as ‘‘individuals’ perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns’’ [1]. An individual’s perception of his or her quality of life is influenced by the person’s physical health, psychological state, level of independence, social relationships, personal beliefs, and relationship to his or her environment [1]. Health-related quality of life (HRQoL) is the subset of quality of life pertaining to health.
Why Is It Important to Measure Health-Related Quality of Life? Measuring health-related quality of life (HRQoL) is an important part of overall patient care (> Fig. 73.1). Clinical examination and diagnostic testing provide information about patients’ health and the progression (or regression) of disease. HRQoL assessments involve patients in their care by allowing them to express their opinions about the value they place on health and how their illness and its treatment affects quality of life. For patients with chronic illness, HRQoL assessment measures the changes in their well-being throughout the course of the disease [2].
How to Measure Quality of Life? Quality of life (QoL) is at once a simple yet complex paradigm, with philosophers, sociologists, psychologists, economists, theologians, clinicians, and lay persons all having different conceptualizations [3–6]. While a consensus
exists that HRQoL is important to patient care, there is no absolute agreement among researchers on how to assess either HRQoL or quality of life in general [7, 8]. Nevertheless, two fundamentally different approaches are commonly applied [9]: (1) health status measurement, and (2) utility/value/preference assessment. Health status measures of HRQoL assess various domains of a person’s physical, physiological, or mental health. Health status measures can be either generic (applicable to any disease or health state) or disease-specific (applicable to a single condition or disease) [7]. One of the most commonly used generic health status instruments is the SF-12 Health Survey, a 12-item measure encompassing eight domains – physical functioning, social functioning, mental health, role limitations due to physical problems, role limitations due to emotional problems, vitality (energy and fatigue), pain, and general health perceptions – each of which is scored separately from 0 (worst) to 100 (best) [10]. Besides such generic measures, a wide variety of disease-specific measures have been developed. Health status measures specific to dermatology are reviewed in detail later in this chapter. Utility/value/preference measures of HRQoL, in contrast to health status measures, assess the value or desirability of a state of health against an external metric such as risk, time, or money [7, 11, 12]. The most common instruments used to measure utility/value/preference, hereafter referred to as utility measures, are: (1) the standard gamble, (2) time trade-off, and (3) the rating scale. The standard gamble determines the risk of (usually) death that one would be willing to take to improve a state of health. Scores on the standard gamble can range from 0 to 1, where 0 usually represents death and 1, excellent or perfect health. The time-trade-off technique asks how many months or years of life one would be willing to give up in exchange for a better health state. The rating scale (though not a strict measure of utility because it does not involve comparison against an external metric) asks the subject to rate his or her health on a scale, e.g., from 0 to 100, where 0 usually represents death and 100, perfect health. This instrument is the simplest of the three utility measures of HRQoL.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_73, # Springer-Verlag Berlin Heidelberg 2010
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Assessing Quality of Life in Older Adult Patients with Skin Disorders
. Figure 73.1 A beautiful lady in her golden years still enjoying playing music on the piano
utility measures can be used to supplement measures of health status. For example, if a particular therapy in a clinical trial is found to be superior in several aspects of health status, but inferior in others, utility measures (if assessed) could in theory ‘‘break the tie’’ and determine the optimal therapy.
Direct and Indirect HRQoL Measures While health status measures are generally ascertained directly (either from patients, their surrogate decision makers, or their health care providers), utility/value/preference measures can be ascertained either directly (from patients, surrogates, providers) or indirectly (by surveying the general population). The indirect assessment of utility involves first assessing the patient’s health status, then mapping a previously derived utility to that particular state of health. The indirectly derived utility measure is obtained by surveying a sample from the general population that has been given descriptions of various health states and asked to assess their value. Examples of indirect utility measures include the EQ-5D [14], the SF-6D [15], the Health Utilities Index [16], and the Quality of WellBeing Scale [17]. A less common utility measure, known as willingness to pay, assesses the amount of money, either in the form of cash or insurance premiums, one is willing to pay for a cure [13]. This instrument is particularly germane to nonlife-threatening conditions and hence, can be applied to many dermatologic diseases.
Applicability of Health Status Measures and Utility/Value/Preference Measures of HRQoL Health status or utility measures of HRQoL are applicable to different forms of health monitoring. For example, health status measures can be used (1) to interpret and monitor outcomes in clinical treatment programs, (2) as endpoints in clinical trials, (3) to monitor population health, and (4) to estimate the burden of different disease conditions. Utility measures are used primarily to guide decision making when uncertainty exists in other measures or when resources are limited. For example, utility measures are used to calculate quality-adjusted life years in decision and cost-effectiveness analyses. Although not as sensitive to changes in health as health status measures (particularly disease-specific health status measures),
Dermatologic Changes in Older People and their Impact on Health The aging process differs among individuals based on genetic variability, the toxicity of by-products of metabolic processes, and the sufficiency of physiologic resources available for somatic maintenance and repair [18]. Guinot and coauthors [18] identified four categories of factors that contribute to the skin aging process: (1) biological (genetically predetermined and unalterable); (2) environmental (e.g., damage from exposure to sunlight, pollutants, and/or nicotine); (3) mechanical (e.g., repetitive muscle movements such as squinting or frowning); and (4) miscellaneous (e.g., sleep patterns, dietary intake, comorbid conditions, and mental health and well-being). Skin changes associated with aging are readily apparent: thin, dry skin; age spots; wrinkles; prominent veins; etc. Such changes can be classified broadly as either age-related changes or as photoaging [19]. Age-related skin changes are further classified as (1) functional, or (2) structural. Functional changes include decreases in skin barrier function, mechanical protection, sensory perception, wound healing capability, immunologic responsiveness, thermoregulation, and vitamin D production [19] (> Table 73.1).
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. Table 73.1 Age-related changes in skin (The Merck Manual of Geriatrics [19]) Physiologic decrement
Clinical consequence(s)
Barrier function
Dryness, ease of irritation
Cell replacement
Rough surface, delayed healing
DNA repair
Increased photocarcinogenesis
Elasticity
Lax skin
Immunologic responsiveness
Chronic low-grade skin infections
Inflammatory responsiveness
Inapparent injuries and infections
Mechanical protection
Frequent injuries
Sensory perception
Frequent injuries
Sweating
Tendency for hypothermia
Thermoregulation (vascular)
Vulnerability to heat and cold
Vitamin D production
Suboptimal vitamin D stores, osteomalacia, muscle weakness
Wound healing
Persistent wounds, weak scars
Structural changes lead to dryness, roughness, wrinkling, skin laxity, and decreased skin elasticity [19]. Structural changes emerge as the skin becomes progressively thinner during adulthood (> Fig. 73.2) [20]. These changes include a reduction in the number of cells comprising the epidermis, changes in cellular shape, uneven pigmentation, reduced cutaneous immunity, reduced sebum production, and lower water content causing drier skin, even xerosis [20]. Older adults are more likely to experience skin irritation or dermatologic disease than younger adults. In fact, most persons over the age of 65 have two or more skin diseases/disorders that could require medical treatment [21]. Urinary and fecal incontinence, for example, can be common among older people; because aging skin is vulnerable, dermatologic complications associated with incontinence are frequent [22]. Untreated incontinence can lead to incontinence dermatitis, dermatological infections, intertrigo, vulvar folliculitis, and pruritus ani. Chronic incontinence can produce a continuing cycle of skin damage, irritation, and inflammation. Skin cancer is also more prevalent among older persons. In recent years, public education about skin cancer prevention has raised awareness about strategies such as using sunscreen and reducing sun exposure. However, the Centers for Disease Control and Prevention report that people over the age of 65 have greater morbidity and mortality from skin cancer: men over the age of 65 account for 22% of incident cases of malignant melanoma among men, and women over the age of 65 account for 14% of incident melanomas among women each year [23].
Health-Related Quality of Life Instruments for Skin Diseases HRQoL instruments that measure either patients’ health status or the utility and value that patients place on their state of health may be validated for use in particular populations, or validated for use in a wide variety of cultures, regions, and languages (globally validated instruments or measures). Constructing and validating a new HRQoL instrument entails both qualitative and quantitative methods [2]. HRQoL measures, particularly new ones, need to be assessed for their reliability and validity in the population of interest. The reliability of a questionnaire refers to its stability over time, that is, the consistency of answers given by the same individual to the same item. The reliability of survey items can be assessed using statistical methods, such as calculating mean absolute differences in scores on repeat measurement, coefficients of variation, kappa statistics, or correlation coefficients appropriate for the type of data (nominal, ordinal, or interval) [24]. Several types of reliability can be assessed. Test–retest reliability examines the correspondence between answers given by an individual when the item(s) is readministered over a brief interval (sufficiently brief so that an underlying change in health status is not anticipated to occur between administrations). Inter-rater reliability assesses how well data obtained by different interviewers are correlated. The validity of a questionnaire refers to whether the questionnaire or survey measures what it intends to measure that it focuses on how a questionnaire or assessment
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Assessing Quality of Life in Older Adult Patients with Skin Disorders
. Figure 73.2 Changes of aging skin (From Farage MA et al. [20]. With permission from Informa HealthCare)
process is used. The validity of survey research question can be assessed with regard to (1) content, (2) criterion (or predictivity), and (3) construct. Content validity refers to the extent to which a measure (item[s]) adequately describes the underlying statement or question. Content validity can be determined by literature review, expert reviews, or cognitive interviews. Literature and expert reviews analyze items that have been tested and utilized previously in other investigations. Cognitive interviews are in-depth, one-on-one interviews with people representative of the target population that determine how individuals process and understand the questions being posed. People are asked to describe and define what individual questions mean to them, as well as how they arrived at their answers [25]. Response categories are also analyzed to determine whether the range of responses provided is adequate and whether individuals clearly differentiate among the options. This qualitative technique for assessing content validity clarifies the wording of question and response choices, assesses possible sources of bias, and standardizes questions and the range of response choices provided. Criterion validity refers to the extent to which survey items predict or agree with an objective assessment of the particular criterion (e.g., in clinical evaluations, patient reports are compared to physician reports or medical records). Correlation coefficients are a quantitative statistical technique used to assess criterion validity. Construct validity examines the assumption that items measured in the survey correlate with other items
hypothesized to measure the same concept (e.g., whether two indicators of emotional well-being agree) and, conversely, whether items or measures in one domain of assessment that are hypothesized to be unrelated to items or measures in another domain of assessment in fact do not correlate (discriminant validity). Correlational and factor analysis are quantitative techniques to assess whether variables are measuring the same underlying construct [26]. To the authors’ knowledge, no HRQoL instruments have been developed specifically for older adults with skin disorders or diseases. HRQoL instruments used to assess skin diseases in younger adults, however, should be applicable to older patients as well, because older adults were included in the development and validation of most of the instruments.
Instruments Suitable for Dermatologic Conditions in General > Table
73.2 shows a list of instruments developed to measure quality of life for dermatologic conditions [27–43].
Skindex Skindex [31, 44, 45] is a widely used HRQoL instrument applicable to various skin diseases. Skindex originated as
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. Table 73.2 Dermatology-specific instruments for adults (Adapted with permission from The Merck Manual of Geriatrics [9]) Disease(s) Generic for dermatology
Acne
Alopecia
Measure
Abbreviation
Dermatology Life Quality Index
DLQI
Dermatology Quality of Life Scales
DQoLS
Author(s) Finlay, A.Y. [27] Morgan, M. [28]
Dermatology-Specific Quality of Life Instrument for Contact DSQL-CD Dermatitis
Anderson, R.T. et al. [29]
Family Dermatology Life Quality Index
FDLQI
Basra, M.K. et al. [30]
Skindex
Skindex
Chren, M.M. et al. [31]
Acne Disability Index
ADI
Motley, R.J. et al. [32]
Dermatology-Specific Quality of Life Instrument for Acne
DSQL-Acne
Anderson, R.T. et al. [29]
Kingsley Alopecia Profile
KPA
Kingsley, D.H. [33]
Atopic dermatitis
Quality of Life Index for Atopic Dermatitis
QoLIAD
Whalley, D. et al. [34]
Eczema
Dermatitis Family Impact Questionnaire
DFI
Finlay, A.Y. et al. [27]
Patient-Oriented Eczema Measure
POEM
Charman, C.R. et al. [35]
Charing Cross Venous Ulcer Questionnaire
CCVUQ
Smith, J.J. et al. [36].
Diabetic Foot Ulcer Scale
DFS
Bann, C.M. et al. [37]
Leg and Foot Ulcer Questionnaire
LFUQ
Hyland, M.E. et al. [38]
Onychomycosis Quality of Life Questionnaire
ONYCHO
Drake, L.A. et al. [39]
Leg ulcer
Onychomycosis Psoriasis
Systemic lupus erythematosus
Psoriasis Disability Index
PDI
Finlay, A.Y. et al. [40]
12-Item Psoriasis Quality of Life Questionnaire
PQOL-12
Koo, J. et al. [41]
Psoriatic Arthritis Quality of Life Instrument
PSAQoL
McKenna, S.P. et al. [42]
Systemic Lupus Erythematosus Quality of Life Questionnaire SLEQoL
a 61-item instrument [31], but over time, it has been reviewed and refined by its developers and today is also available in two shorter versions, a 29-item [44] and a 16-item questionnaire [45].
Skindex-61 The 61-item, self-administered Skindex questionnaire was first developed in the mid-1990s [31]. The original version focused on skin diseases and the frequency and severity of their impact on quality of life: cognitive effects, social effects, depression, fear, embarrassment, anger, physical discomfort, and physical limitations. For each domain, patients respond on a scale ranging from 0 (no effect) to 100 (maximum effect). In addition to scores in individual
Doward, L.C. et al. [43]
domains, the overall test score is defined as the average of responses to items on each domain. When tested in 201 patients, mean SD scale scores ranged from 14 (17) for physical limitations to 31 (22) for physical discomfort. Retesting at 72 h showed the scores to be reproducible and internally consistent. Construct validity (correlation among items thought to measure the same or different concepts) was demonstrated by finding that scores among patients with inflammatory dermatoses were higher (worse) than among patients with isolated lesions, and by exploratory factor analysis (a statistical approach for examining the internal reliability of a measure). Physicians’ opinions of the severity of disease did not correlate consistently with the patients’ Skindex scores, thus indicating that the Skindex instrument may be a useful adjunct to clinical assessment by physicians.
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Skindex-29 To improve the original Skindex instrument, the original 61-item questionnaire (201 patients) and a revised 29-item questionnaire (692 patients) were compared to evaluate the reproducibility, internal consistency reliability, and validity of the new version of the instrument [44]. Time needed to complete the questionnaire, the number of items that elicited the same response, and the number of scales that were responsive to patients’ self-reports of changes in their condition were evaluated, as well as reproducibility and construct validity. The questionnaire was completed in 5 min on average relative to 15 min for the original questionnaire. Only three items elicited the same response from 70% or more of those surveyed, compared to 17 items in the original questionnaire. The revised version was reproducible in a retest at 72 h and was internally reliable. Patients with psoriasis and eczema had higher scores (indicating worse HRQoL) than patients with isolated lesions, which supported construct validity. Hence, the 29-item instrument improved upon the original questionnaire in its discriminative and evaluative capability and in administration time [45]. Skindex29 has since been translated into nine languages.
Skindex-16 In 2001, the Skindex-29 questionnaire was reduced to a single-page instrument assessing how distressed patients were by the disease, as opposed to how frequently they experienced effects of the disease [45]. The evaluation compared the responses of 692 patients who completed the 29-item instrument with those of 541 patients who completed the newer brief instrument. Sixteen of the prior 29 items elicited responses of ‘‘never’’ from more than half of the responders. Data analysis and elimination of the less useful items yielded the new 16-item Skindex, which has subscales assessing distress related to symptoms, emotions, and functioning (> Table 73.3). Further analysis revealed good reliability and construct validity. Skindex-16 has been translated into 11 languages.
Dermatology Life Quality Index The Dermatology Life Quality Index (DLQI) is a simple, 10-question, validated questionnaire used widely in clinical settings and available in more than 40 languages [27]. The DLQI was developed based on the responses of 120 patients with various skin diseases who were asked how
their disease and its treatment affected their life. For further validation, the DLQI was subsequently administered to 200 consecutive new patients attending a dermatology clinic. Analysis of the responses revealed that atopic eczema, psoriasis, and generalized pruritus have a greater impact on HRQoL than do acne, basal cell carcinoma, and viral warts. When the instrument was administered to 100 healthy volunteers, mean scores were very low. A 1-week, test–retest reliability analysis in 53 patients found that the DLQI instrument was highly reliable [27].
Dermatology-Specific Quality of Life The Dermatology-Specific Quality of Life (DSQL) instrument was created to quantify the effect of skin disease on physical discomfort and symptoms, psychological wellbeing, social functioning, self-care activities, performance at work or school, and self-perceptions [29]. Reliability and validity were assessed in patients with contact dermatitis or acne vulgaris. The validity of the instrument was assessed by correlating DSQL scores with global ratings of bothersome symptoms and their perceived severity and by the instrument’s ability to discriminate among clinically defined severity-of-illness groups. Test–retest reliability was assessed at 3 and 7 days. The instrument’s domains had good internal consistency and test–retest reliability. The subscale scores were also moderately-to-highly correlated with globally validated ratings of symptoms of distress and with overall disease severity. As expected, patients with severe contact dermatitis or scarring from acne vulgaris had higher (worse) DSQL scores than those with milder disease.
The Family Dermatology Life Quality Index Although skin diseases clearly affect the well-being of patients, instruments that quantify the impact of skin diseases on patients’ family members were lacking. The Family Dermatology Life Quality Index (FDLQI) is a 10-item questionnaire administered to patients’ family members, to measure the indirect impact of skin disease on the family [30]. The FDLQI is responsive to changes in the patient: family members’ scores changed in association with improvement or worsening of the patient’s condition. FDLQI scores and the patients’ DLQI scores, FDLQI scores and inflammatory versus noninflammatory disease, FDLQI scores and the severity of the patient’s disease were strongly statistically associated. There was a positive relationship between FDLQI scores of the
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. Table 73.3 Skindex-16 (Reprinted from Chren MM et al. [45]. With permission. Skin-Index 16 is copyrighted and used with permission from both Dr. Chren and Springer Publishing Co.) THESE QUESTIONS CONCERN THE SKIN CONDITION WHICH HAS BOTHERED YOU THE MOST DURING THE PAST WEEK Never During the past week, how often have you been bothered by:
Always
Bothered
Bothered
↓
↓
□0 □1 □2 □3 □4 □5
□6
Your skin condition burning or stinging
□0 □1 □2 □3 □4 □5
□6
Your skin condition hurting
□0 □1 □2 □3 □4 □5
□6
4.
Your skin condition being irritated
□0 □1 □2 □3 □4 □5
□6
5.
The persistence/reoccurrence of your skin condition
□0 □1 □2 □3 □4 □5
□6
6.
Worry about your skin condition (e.g., that it will spread, get worse, scar, and be unpredictable)
□0 □1 □2 □3 □4 □5
□6
7.
The appearance of your skin condition
□0 □1 □2 □3 □4 □5
□6
8.
Frustration about your skin condition
□0 □1 □2 □3 □4 □5
□6
9.
1.
Your skin condition itching
2. 3.
Embarrassment about your skin condition
□0 □1 □2 □3 □4 □5
□6
10. Being annoyed about your skin condition
□0 □1 □2 □3 □4 □5
□6
11. Feeling depressed about your skin condition
□0 □1 □2 □3 □4 □5
□6
12. The effects of your skin condition on your interactions with others (e.g., interactions □0 □1 □2 □3 □4 □5 with family, friends, and close relationships)
□6
13. The effects of your skin condition on your desire to be with people
□0 □1 □2 □3 □4 □5
□6
14. Your skin condition making it hard to show affection
□0 □1 □2 □3 □4 □5
□6
15. The effects of your skin condition on your daily activities.
□0 □1 □2 □3 □4 □5
□6
16. Your skin condition making it hard to work or do what you enjoy
□0 □1 □2 □3 □4 □5
□6
Skindex-16 scoring SCALE
ITEMS
Symptoms
1–4
Emotion
5–11
Functioning
12–16
Item scores transformed to 0–100 scale Scale score: average of items in given scale Total score: average of all 16 items
family members and the patient’s disease severity, as measured by the DLQI. Thus, the FDLQI has been shown to be simple and practical and a potential additional outcome measure in clinical practice and evaluation research.
The Impact of Chronic Skin Disease on Daily Life The Impact of Chronic Skin Disease on Daily Life (ISDL) instrument assesses the effect of chronic skin diseases and
their treatments on both dermatology-specific and generic aspects of HRQoL [46]. The dermatology-specific questions in the ISDL assess physical functioning, itching/ scratching, pain, fatigue, and stigmatization; the generic questions assess psychological functioning, illness cognitions, and social support. The reliability and validity of the instrument was assessed in patients with psoriasis or atopic dermatitis. The ISDL is highly reliable, valid, and responsive to changes in health status resulting from ultraviolet B radiation therapy or cognitive behavioral therapy for itching.
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Measures Suitable for Specific Dermatologic Conditions Itch Severity Scale The Itch Severity Scale (ISS) is a self-administered questionnaire to measure the severity of pruritus [47]. To develop the instrument, an existing pruritus instrument was modified and administered to patients with psoriasisassociated pruritus, along with the RAND-36 Health Status Inventory [48] (a generic health status measure), and the DLQI. Components to be included in the new instrument were determined and the internal consistency reliability, test–retest reliability, and construct validity were analyzed. The resulting new instrument, ISS, contained just seven questions. The ISS is valid and reliable for assessing the severity of pruritus as well as the effectiveness of treatments for pruritus [47].
Itchy QoL ItchyQoL is another pruritus-specific HRQoL instrument [49]. It includes 22 pruritus-specific questions covering three major domains: symptoms, functional limitations, and emotions. Two versions of the instrument were first created (one with ‘‘frequency’’ questions and the other with ‘‘bother’’ questions) based on patient interviews and items from Skindex-16 and Skindex-29. Eighty-nine patients took part in the validation phase and 101 patients participated in the clinical application phase of instrument development. The final instrument contains 27 questions, 18 are measured with both frequency and bother and the remaining nine items pertained to emotion and were measured by frequency only. Although the authors cited lack of generalizability and potential selection bias as possible limitations, they found this initial pruritus-specific questionnaire to be reliable, valid, and responsive [49].
Scalpdex Scalpdex [50] is the first dermatitis-specific HRQoL instrument. Scalpdex is based on three major domains of quality of life: symptoms, functioning, and emotions. The 23-question instrument is reliable, valid, and responsive. Scalpdex can be used by physicians to determine what bothers the patient, as well as to assess the impact of treatments [50].
Quality of Life Index for Atopic Dermatitis Atopic dermatitis is a chronic inflammatory skin condition. The Quality of Life Index for Atopic Dermatitis (QoLIAD) is the first dermatology-specific instrument to assess quality of life impact of atopic dermatitis in adults [34]. QoLIAD was initially developed based on 65 interviews with patients with atopic dermatitis in the UK, Italy, and the Netherlands. Subsequent field tests involving 993 patients in six different countries resulted in a final 25-item instrument. Psychometric analyses demonstrate the QoLIAD to be a practical, reliable, and valid instrument for assessing the effects of atopic dermatitis, and its treatment in clinical practice and clinical trials [34].
Rosacea-Specific Quality of Life Instrument Acne rosacea (commonly referred to as ‘‘rosacea’’) is another chronic dermatologic condition that, until recently, lacked a specific HRQoL measure. Rosacea affects up to 10% of the general population [51]. Its clinical appearance varies, but rosacea most recognizably manifests as erythema on the central area of the face or as phymatoid changes around the nose. Pharmacological treatments are often inadequate [52]; thus, a rosacea-specific instrument could be beneficial in assessing the patient’s perspective on treatment effectiveness. Based on in-depth patient interviews [53], three domains pertinent to quality of life were identified: symptoms, functioning, and emotions. To validate the instrument, patients with rosacea were randomly selected from dermatology clinics and administered by telephone the Skindex-29 questionnaire, 21 rosaceaspecific questions, and five questions about general health. To assure that all four temperate seasons were represented, the interviews were performed over a 4-year period. The patients answered all questions at baseline, at 72 h (allowable range: 3–7 days), and at 4–6 months. Follow-up interviews allowed inconsequential or insensitive questions to be eliminated. This produced the 21-item, Rosacea-Specific Quality of Life (RosaQol) instrument [53] (> Table 73.4). The RosaQol’s reliability, validity, and responsiveness were assessed by psychometric and statistical analyses. At the time of this writing, the RosaQol is considered to be a pilot instrument requiring further validation in demographically diverse patient populations. Nonetheless, the developers believe it to be a promising, practical instrument for use both in clinical and research settings [53].
Assessing Quality of Life in Older Adult Patients with Skin Disorders
. Table 73.4 The rosacea-specific quality of life (RosaQoL) instrument (Reprinted from Nicholson K et al. [53]. With permission from Elsevier) RosaQoL item
Hypothesized construct
1. I worry that my rosacea may be serious Emotion 2. My rosacea burns or stings
Symptom
3. I worry about scars from my rosacea
Emotion
4. I worry that my rosacea may get worse Emotion 5. I worry about side effects from rosacea Emotion medications 6. My rosacea is irritated
Symptom
7. I am embarrassed by my rosacea
Emotion
8. I am frustrated by my rosacea
Emotion
9. My rosacea makes my skin sensitive
Symptom
10. I am annoyed by my rosacea
Emotion
11. I am bothered by the appearance of my skin (redness, blotchiness)
Emotion
12. My rosacea makes me feel selfconscious
Emotion
13. I try to cover up my rosacea (with makeup)
Functioning
14. I am bothered by the persistence/ recurrence of my rosacea
Emotion
15. I avoid certain foods or drinks because Functioning of my rosacea 16. My skin feels bumpy (uneven, not smooth, irregular)
Symptom
17. My skin flushes
Symptom
18. My skin gets irritated easily (cosmetics, Symptom aftershaves, cleansers) 19. My eyes bother me (feel dry or gritty)
Symptom
20. I think about my rosacea
Emotion
21. I avoid certain environments (heat, humidity, cold) because of my rosacea
Functioning
Utility/Value/Preference Measures for Dermatologic Conditions Several studies have assessed utility measures for one or more skin conditions [12, 24, 32, 40, 54–62]. Perceived utility or value varies not only from condition to condition, but also from patient to patient with a given condition, by assessment method, and by respondent type [12, 55–58]. For example, utility measures for controlled
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atopic eczema, uncontrolled atopic eczema, controlled psoriasis, and uncontrolled psoriasis have been evaluated in the general population in Germany and in German patients with atopic eczema or psoriasis [55]. For the time-tradeoff measure of utility, the median score for controlled atopic eczema was 0.97, indicating that respondents were willing to give up a median of 3% of their life expectancy (= [1.0 – 0.97] 100%) in order to have perfect health (no atopic eczema). For controlled psoriasis, the median time-trade-off utility measure was 0.93. By contrast, median utility measures were much lower for uncontrolled atopic eczema (0.64) and uncontrolled psoriasis (0.56). The study also asked people about willingness to pay. People from the general population would be willing to pay a median of €50/month for an effective treatment (with no side effects) for controlled atopic eczema, €150/month for uncontrolled atopic eczema, €75/month for controlled psoriasis, and €200/month for uncontrolled psoriasis. Another study reported that patients with psoriasis would be willing to pay on average 14% of their monthly income to get rid of their psoriasis [59]. Several researchers have modified the time-trade-off method by asking patients how many hours each day they would be willing to devote to treating their skin condition if the treatment was curative [40, 59, 60]. One study indicated that patients with psoriasis would be willing to spend a mean (SD) of 2.8 (3.7) h a day to be relieved of their psoriasis [59] and 1.2 (0.9) h a day to get rid of their port wine stains [60]. In a particularly comprehensive study of utility measures for dermatologic conditions, time-trade-off measures of utility for each skin condition was experienced by 236 patients [54]. The mean (SD) time-trade-off utility measure for all skin conditions was 0.943 (0.124), but ranged from 0.640 to 1.000 for specific skin conditions, depending on the disorder in question. For example, the mean (SD) utility for acne vulgaris was 0.938 (0.124); for dermatitis, 0.939 (0.098); for nonmelanoma skin cancer, 0.976 (0.052); and for actinic keratosis, 0.981 (0.056).
Conclusion Older adults experience a number of skin diseases and disorders that significantly affect quality of life. Although no disease-specific instruments have yet been developed for older adults, in the last decade a number of instruments have been developed for the general dermatology patient to assess the effects of treatment and disease progression, perceptions of well-being, and the value that
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patients place on their dermatologic state of health. Some instruments have been validated and further refined over time, while others are in earlier stages of development. Opportunity exists for developing and validating HRQoL specifically for dermatological conditions most pertinent to older patients. Dermatology-specific HRQoL instruments will continue to be investigated with the common goal of improving the understanding of how skin diseases and their treatment affect health-related quality of life in people of all ages.
Cross-references > Aging
Skin: Some Psychosomatic Aspects and Social Implications of Aging skin: Normal Aging and the Effects of Cutaneous Disease
> Psychological
References 1. World Health Organization. Division of Mental Health and Prevention of Substance Abuse. WHOQOL: measuring quality of life. Available at: www.who.int/entity/mental_health/media/68.pdf. Accessed June 25, 2007 2. Chwalow AJ. Cross-cultural validation of existing quality of life scales. Patient Educ Couns. 1995;26:313–318. 3. Wilson IB, et al. Linking clinical variables with health-related quality of life. JAMA. 1995;273:59–65. 4. Johnson RJ, et al. The structure of health status among older adults: disease, disability, functional limitation, and perceived health. J Health Soc Behav. 1993;34:105–121. 5. Calman KC. Quality of life in cancer patients – an hypothesis. J Med Ethics. 1984;10:124–127. 6. Michalos AC. Multiple discrepancies theory (MDT). Soc Indic Res. 1985;16:347–413. 7. Tsevat J, et al. Using health-related quality-of-life information: clinical encounters, clinical trials, and health policy. J Gen Intern Med. 1994;9(10):576–582. 8. Patrick DL, et al. Health Status and Health Policy: Quality of Life in Health Care Evaluation and Resource Allocation. New York: Oxford University Press, 1992. 9. PROQOLID: Patient Reported Outcome and Quality of Life Instruments Database. Available at: http://www.proqolid.org/. Accessed August 24, 2008. Accessed August 24, 2008. 10. Ware J Jr. A 12-item short-form Health Survey: construction of scales and preliminary tests of reliability and validity. Med Care. 1996;34:220–233. 11. Torrance GW. Measurement of health state utilities for economic appraisal: a review. J Health Econ. 1986;5:1–30. 12. McCombs K, et al. Patient preference quality of life measures in dermatology. Dermatol Ther. 2007;20:102–109. 13. Khanna D, et al. Willingness to pay for a cure in patients with chronic gout. Med Decis Making. 2008;28:606–613. 14. Rabin R, et al. EQ-5D: a measure of health status from the EuroQol Group. Ann Med. 2001;33:337–343.
15. Brazier J, et al. The estimation of a preference-based measure of health from the SF-36. J Health Econ. 2002;21:271–292. 16. Furlong W, et al. The Health Utilities Index system for assessing health-related quality of life in clinical studies. Ann Med. 2001;33:375–384. 17. Kaplan R, et al. The quality of Well-Being Scale: rationale for a single quality of life index. In: Walker SR, Rosser RM (eds) Quality of Life Assessment: Key Issues in the 1990s. Dordrecht: Kluwer, 1993, pp. 65–94. 18. Guinot CG, et al. Relative contribution of intrinsic vs. extrinsic factors to skin aging as determined by a validated skin age score. Arch Dermatol. 2002;138:1454–1460. 19 The Merck Manual of Geriatrics. Section 15, Dermatologic and sensory organ disorders. Chapter 122, Aging and the skin. Agerelated changes in skin structure and function; photoaging. Available at: http://www.merck.com/mkgr/mmg/sec15/ch122/ch122c.jsp. Accessed August 7, 2008. 20. Farage MA, et al. Structural characteristics of the aging skin: a review. Cutan Ocular Toxicol. 2007;26:343–357. 21. Kligman AM, et al. Demographics and psychological implications for the aging population. Dermatol Clin. 1997;15(4):550–553. 22. Farage MA, et al. Incontinence in the aged: contact dermatitis and other cutaneous consequences. Contact Dermatitis. 2007;57 (4):211–217. 23 Counseling to prevent skin cancer: recommendations and rationale of the U.S. Preventive Services Task Force. Available at: http:// www.cdc.gov/mmwr/preview/mmwrhtml/rr5215a2.htm. Accessed September 23, 2008. 24. Littenberg B, et al. Paper Standard Gamble: the reliability of a paper questionnaire to assess utility. Med Decis Making. 2003;23 (6):480–488. 25. Willis GB. Cognitive Interviewing: a Tool for Improving Questionnaire Design. Thousand Oaks: Sage, 2005. 26. Aday A, et al. Designing and Conducting Health Surveys: a Comprehensive Guide, 2nd ed. San Francisco: Jossey-Bass, 1996. 27. Finlay AY, et al. Dermatology Life Quality Index (DLQI) – a simple practice measure for routine clinical use. Clin Exp Dermatol. 1994;19(3):210–216. 28. Morgan M, et al. Dermatology quality of life scales – a measure of the impact of skin diseases. Br J Dermatol. 1997;136(2):202–206. 29. Anderson RT, et al. Development and validation of a quality of life instrument for cutaneous diseases. J Am Acad Dermatol. 1997;37 (1):41–50. 30. Basra MK, et al. The Family Dermatologyy Life Quality Index: measuring the secondary impact of skin disease. Br J Dermatol. 2007;156(3):528–538. 31. Chren MM, et al. Skindex, a quality-of-life measure for patients with skin disease: reliability, validity, and responsiveness. J Invest Dermatol. 1996;107(5):707–713. 32. Motley RJ, et al. How much disability is caused by acne? Clin Exp Dermatol. 1989;14(3):194–198. 33. Kingsley DH. The development and validation of a quality of life measure for the impact of androgen-dependent alopecia. Ph.D. thesis. Portsmouth University, Portsmouth, 1999. 34. Whalley D, et al. A new instrument for assessing quality of life in atopic dermatitis: international development of the Quality of Life Index for Atopic Dermatitis (QoLIAD). Br J Dermatol. 2004;150(2):274–283. 35. Charman CR, et al. The patient-oriented eczema measure: development and initial validation of a new tool for measuring atopic eczema severity from the patients’ perspective. Arch Dermatol. 2004;140(12):1513–1519.
Assessing Quality of Life in Older Adult Patients with Skin Disorders 36. Smith JJ, et al. Measuring the quality of life in patients with venous ulcers. J Vasc Surg. 2000;31(4):642–649. 37. Bann CM, et al. Development and validation of the diabetic foot ulcer scale-short form (DFS-SF). Pharmacoeconomics. 2003;21 (17):1277–1290. 38. Hyland ME, et al. Quality of life of leg ulcer patients: questionnaire and preliminary findings. J Wound Care. 1994;3(6):294–298. 39. Drake LA, et al. The impact of onychomycosis on quality of life: development of an international onychomycosis-specific questionnaire to measure patient quality of life. J Am Acad Dermatol. 1999;41(2):189–196. 40. Finlay AY, et al. The effect of severe psoriasis on the quality of life of 369 patients. Br J Dermatol. 1995;132(2):236–244. 41. Koo J. Population-based epidemiologic study of psoriasis with emphasis on quality of life assessment. Dermatol Clin. 1996;14(3): 485–496. 42. McKenna SP, et al. Development of the PSAQol: a quality of life instrument specific to psoriatic arthritis. Ann Rheum Dis. 2004; 63(2):162–169. 43. Doward LC, et al. The development of the SLE-QOL: a quality of life instrument specific to systemic lupus erythematosus. Qual Life Res. 1999;8:609. 44. Chren MM, et al. Improved discriminative and evaluative capability of a refined version of Skindex, a quality-of-life instrument for patients with skin diseases. Arch Dermatol. 1997;133(11): 1433–1440. 45. Chren MM, et al. Measurement properties of Skindex-16: a brief quality-of-life measure for patients with skin diseases. J Cutan Med Surg. 2001;5:105–110. 46. Evers AW, et al. The Impact of Chronic Skin Disease on Daily Life (ISDL): a generic and dermatology-specific health instrument. Br J Dermatol. 2008;158(1):101–108. 47. Majeski CG, et al. Itch Severity Scale: a self-report instrument for the measurement of pruritus severity. Br J Dermatol. 2007;156(4): 667–743. 48. Hays RD, et al. The RAND-36 measure of health-related quality of life. Ann Med. 2001;33(5):350–357.
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49. Desai NS, et al. A pilot quality-of-life instrument for pruritus. J Am Acad Dermatol. Aug 2008; 59(2):234–244, Epub 2008 Jun 11. 50. Chen SC, et al. Scalpdex: a quality-of-life instrument for scalp dermatitis. Arch Dermatol. 2002;138(6):803–807. 51. Rebora A. The red face: rosacea. Clin Dermatol. 1993;11:225–234. 52. Powell FC. Rosacea. N Engl J Med. 2005;352:793–803. 53. Nicholson K, et al. A pilot quality-of-life instrument for acne rosacea. J Am Acad Dermatol. 2007;57(2):213–221. 54. Chen SC, et al. A catalog of dermatology utilities: a measure of the burden of skin diseases. J Invest Dermatol Symp Proc. 2004; 9(2):160–168. 55. Schmitt J, et al. Assessment of health state utilities of controlled and uncontrolled psoriasis and atopic eczema: a population-based study. Br J Dermatol. 2008;158(2):351–359. 56. Lundberg L, et al. Quality of life, health-state utilities and willingness to pay in patients with psoriasis and atopic eczema. Br J Dermatol. 1999;141(6):1067–1075. 57. Chen S, et al. Cost-effectiveness and cost-benefit analysis of using methotrexate vs Goeckerman therapy for psoriasis: a pilot study. Arch Dermatol. 1998;134(12):1602–1608. 58. Zug KA, et al. Assessing the preferences of patients with psoriasis: a quantitative, utility approach. Arch Dermatol. 1995;131(5):561–568. 59. Schiffner R, et al. Willingness to pay and time trade-off: sensitive to changes of quality of life in psoriasis patients? Br J Dermatol. 2003;148(6):1153–1160. 60. Schiffner R, et al. Willingness to pay and time trade-off: useful utility indicators for the assessment of quality of life and patient satisfaction in patients with port wine stains. Br J Dermatol. 2002;146(3): 440–447. 61. Pitt M, et al. A cost-utility analysis of pimecrolimus vs. topical corticosteroids and emollients for the treatment of mild and moderate atopic eczema. Br J Dermatol. 2006;154(6):1137–1146. 62. Williamson D, et al. The effect of hair loss on quality of life. J Eur Acad Dermatol Venereol. 2001;15(2):137–139.
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Part 3
Techniques and Methods
Bioengineering Methods and Tools
65 Bioengineering Methods and Skin Aging Francesca Giusti . Stefania Seidenari
Introduction During the last decade, skin aging has become an area of increasing research interest, because of the prolongation of life span in modern society. Skin aging is an uneven process characterized by epidermal and dermal disorders, accompanied by many clinical signs, such as skin dryness, color changes, loss of elasticity, wrinkles, and risk of developing skin cancers. The elderly appearance of the skin depends on a combination of intrinsic or chronological aging, modulated by genetically predisposing factors, and extrinsic aging or photoaging, due to environmental factors, mainly UV exposure, and also wind, relative humidity, pollution, and so on. The effects of the UV radiations on sun-exposed sites are superimposed on the morphological, biochemical, and functional changes occurring with aging, making distinction between the two phenomena hard. Besides genetic aspects, all these environmental factors are responsible for the great interindividual and intraindividual variations and the site-dependent variations of the aging process. A precise and noninvasive quantification of aging is of utmost importance for in vivo studies in skin gerontology and for cosmetic research. Several bioengineering methods have been proposed to objectively, precisely, and noninvasively measure skin aging, and to detect early skin damage, which is rather difficult to demonstrate clinically. This chapter reviews the data that have emerged from recently introduced technologies aiming at quantitatively assessing the effects of aging on the skin. The variations in biophysical parameters such as hydration and trans-epidermal water loss, which have been evidenced in the skin in elderly subjects, will be discussed in detail in other chapters. To date, high-frequency ultrasonography has been used most extensively to visualize and quantify agerelated skin changes. This chapter focuses on ultrasound findings in intrinsic and extrinsic skin aging. In the last decade, a new noninvasive technique has been developed to examine the epidermis and the papillary dermis
at a resolution approaching histological detail: confocal scanning laser microscopy. Review literature data on the application of this promising technique for the study of skin aging has also been included at the end of this chapter.
pH Cutaneous acidity plays a role in skin barrier homeostasis, in stratum corneum desquamation, in skin defense against microbiological or chemical insults. In order to measure cutaneous pH, instruments like a glass planar electrode are primarily used. They create a potential difference between the two environments separated by the glass slide, that is, the skin surface and the reference solution contained in the electrode. This potential difference is linearly linked to the difference in H+ concentration. Limited data are available on skin pH and age. pH is relatively constant from childhood through age 70. Fluhr et al. did not find a significant difference in pH measured on volar forearm between 44 adults aged 21–44 and 44 children aged 1–6 [1]. These data were confirmed on the forehead in 500 female patients aged 20–70 [2]. In contrast, skin acidity decreases significantly in subjects older than 70 [3, 4]. Wilhelm et al. measured pH on 11 skin sites in 14 young volunteers (mean age 27 years) and in 15 aged volunteers (mean age 71 years), and noted significant differences at the ankle and thigh only [4]. Therefore, the authors attributed the higher pH to stasis and reduced oxygen supply frequently observed in the lower limbs in elderly patients.
Sebum Sebum production is controlled by the levels of circulating hormones and varies according to the anatomical distribution of sebaceous glands. It is generally measured by an instrument allowing a semiquantitative evaluation of sebum excretion (Sebumeter, Courage and Khazaka
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_65, # Springer-Verlag Berlin Heidelberg 2010
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Electronics, Ko¨ln, Germany). This method is based on photometric measurements of light transmission through a transparent plastic film, which is pressed against the skin in order to obtain adhesion of skin lipids. The recorded values are expressed in arbitrary units, which can be converted into microgram per square centimeter, according to the manufacturer’s calibration table. The sebum excretion rate has been demonstrated to decrease with age by different authors [4–7], more markedly at sites of elevated sebum production. When measuring the casual level of sebum in 63 healthy subjects aged 12–60 and in 24 older subjects at 14 body sites [5], a marked decrease in elderly volunteers was observed, with significantly lower values on the forehead and the upper back (> Fig. 65.1). It has been proposed that changes in sebum excretion during aging reflect the decrease in the endogenous production of androgens occurring in men and women. In menopausal women receiving hormonal replacement therapy, the sebum excretion rate shows a 35% increase, because of the stimulatory effect of the progestagen component [6]. Caisey et al., by measuring the amount of sebum produced over 1 h on the forehead of 20 young women, 19 premenopausal women, 21 postmenopausal women, and 20 postmenopausal women receiving hormonal replacement therapy, did not find a correlation between age and sebum excretion rate [7]. However, the values in postmenopausal women were significantly lower than in the other groups showing a 35–40% decrease compared with females receiving estrogen and
progesterone. The authors concluded that sebum production is more likely related to hormones than to aging.
Skin Color Skin color can be determined by a chromameter (Minolta, Osaka, Japan) according to a three-dimensional L* a* b* system. L* represents an attribute on the luminance scale, a* on the red-green color scale and b* on the yellow-blue one. Photoaging is clinically characterized by yellowish skin with erythematous areas, associated with teleangiectasia and heterogeneity of skin pigmentation. By examining the skin of sun-exposed and adjacent unexposed sites, Richard et al. reported significant differences in L* values and a* values with larger standard deviations at exposed skin sites, indicating a decrease in brightness, an increase in the red component, and color heterogeneity in photodamaged skin in the elderly [8]. In contrast, Warren et al. observed no change in L*a*b* values [9]. In order to study photodamaged skin, Kikuchi-Numagami et al. measured skin color of the dorsum of the hands in 12 middle-aged Japanese golfers, playing golf frequently for the past 4–25 years [10]. By comparing the right hand, exposed to sunlight many hours a day, to the left hand that is protected by a glove from the outer environment, he found that whereas L* value was significantly lower, a* and b* values were significantly higher on the right hand in comparison with the left hand protected
. Figure 65.1 Casual level of sebum in relation to age (p < 0.05 at forehead and upper back)
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65
. Figure 65.2 Confocal laser scanning microscopy images of the skin at the dermal–epidermal junction. (a) The dermal–epidermal junction in the young appears with bright, polygonal cells forming regular rings, surrounding the papillae; (b) in the elderly the papillae are smaller and are surrounded by small, roundish cells
by the glove. The differences in L* values were dependent on the length of past golf-playing history (> Fig. 65.2). A negative correlation between L* values and age was found on the lower lips in 80 postmenopausal women, whereas no correspondence was observed between age and the other two color components [7]. L* values were significantly lower in the group of postmenopausal women in comparison with two groups of younger and postmenopausal women receiving hormonal replacement therapy. The authors concluded that menopause may result in a slight darkening of the lip, which could be prevented or corrected by hormonal treatment. Also Guinot et al. reported that menopausal subjects treated with hormonal replacement therapy showed redder lips than untreated menopausal women [11].
Skin Blood Flow Skin blood perfusion can be quantified by Laser Doppler Flowmetry and Laser Doppler Velocimetry. A helium– neon laser light is transmitted to the skin via an optical fiber to an estimated depth of more than 1 mm. Light reflected from moving erythrocytes is Doppler shifted. The frequency-shifted signal is proportional to blood flow and can be extracted and measured by the instrument in arbitrary units (> Fig. 65.3). Data regarding age-related changes in blood perfusion are often conflicting, probably because of the small
sample sizes and the varying age ranges of the studies [1, 3, 12–15]. By using Laser Doppler flowmetry on the volar forearm in 44 children and 44 adults aged 21–44, Fluhr et al. found higher blood flow values in children. On the contrary, Kelly et al. did not observe significant differences in blood flow perfusion both in ventral forearm and forehead in a small study population comprising ten subjects aged 18–26 and ten subjects aged 65–88 [13]. Likewise, in a study comparing only nine elderly and ten young volunteers, the skin vascular response to heat and cold challenge measured by Laser Doppler Velocimetry was delayed in elderly subjects [14]. This may be due to a reduced vessel density in aged skin. Also in a study population of 201 people aged 10–89, the blood flow measured after immersion in water at 10 C was lower in subjects over 50 and the restorative ability poorer in subjects over 70 compared with younger ones [15]. The basal blood flow decreased with age in all the areas with high blood flow, such as the lip, finger, nasal tip, and forehead. In conclusion, trends indicate that aging is associated with a decrease in cutaneous blood perfusion, in particular in photo-exposed areas [1, 3, 12, 14, 15].
Skin Surface Roughness The morphological study of the skin surface can be performed by optical, mechanical, laser, and transparency profilometry. The first method is based on skin replicas
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. Figure 65.3 Confocal laser scanning microscopy images of the skin. (a) Short branches of reticulated, compact collagen fibers in young skin; (b) the collagen is thickened and unevenly disposed in bundles in aged skin
and evaluation of the black-and-white reflections by light irradiation depending on topography of skin furrows. The image is processed using a special image processing software by a CCD camera or a high-resolution blackand-white video and a connected computer. Mechanical profilometry offers a two-dimensional quantification of the absolute height and depth of wrinkles and furrows. Laser profilometry is an optical technique based on the principle of light amplification and reflection from a cutaneous replica. Recently, the visiometer method or transparency profilometry (Skin Visiometer SV600, Courage and Khazaka, Cologne, Germany) has been developed with some advantages, as short processing time and direct visual control of the skin surface topography on the computer monitor. This technique uses a thin silicon gel print of the skin surface, which allows parallel light to pass through and is registered as a change of transparency by a CCD video camera [16]. Aging is characterized by fine and coarse wrinkling, whose estimation is surely of great interest especially in the field of cosmetic research. By performing laser profilometry on the dorsal surface of the hands in Japanese golfers, Kikuchi-Numagami et al. observed that roughness parameters were increased on the right hand exposed to sun in comparison with the left hand protected by a glove when playing golf [10]. The differences became larger in golfers with lower handicap and longer golf history, probably due to elastogenesis in the dermis of the photoaged skin [9]. Likewise, Quan
et al. found significant differences in skin roughness between sun-exposed and sun-protected areas by using mechanical profilometry [17]. However, this difference was significant only in the group of older subjects. To study photodamage and the effect of tretinoin on it, Marks et al. performed optical profilometry on crow’s foot areas, associated with other noninvasive and invasive technique, concluding that there is no single method to quantify the degenerative changes due to photodamage [12]. In a study evaluating the crow’s feet of 95 women aged 30–50 by transparency profilometry and ultrasonography, an increment of all roughness parameters was observed, as age increases [18]. Moreover, a correlation between skin roughness and dermal density and thickness was found.
Skin Thickness In order to measure cutaneous thickness under a variety of normal and pathologic, ultrasonography has been widely used for about 30 years. Since penetration depth of the ultrasound waves is inversely related to its frequency, the optimal frequency for achieving a higher resolution for skin examination is 15–20 MHz. The ultrasonic wave (velocity 1,580 m/s) is partially reflected at the boundary between adjacent structures, generating echoes, whose amplitudes are characteristic of the nature of the media. The ultrasonographic image can be evaluated
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either manually or with computer assistance to quantify skin thickness. Two forms of ultrasonography, A and B modes, are available. The first gives an unidimensional representation of skin echogenicity and is easier and quicker than the B mode. However, by A scanning, the determination of the dermis–subcutis interface is based on the observation of a peak corresponding to the impedance jump between adjacent parts of the tissue, making determination of the dermis–subcutaneous tissue interface difficult, whereas B-scan measurement of skin thickness represents the mean of consecutive A-scan lines composing the whole bidimensional image. Thus, the reproducibility of B-mode assessment is higher, enabling the production of bidimensional images of cross sections of the skin. By means of the B-scan method, skin thickness values are approximately 15% greater than A-scan measurements [19]. Skin thickness has been a widely used parameter to evaluate the influence of different factors on skin aging, but the measurement of age-related changes in skin thickness yielded conflicting results. In fact, depending on the anatomical site, both thinning and thickening are observed. This poor consensus can be explained by differences in the age range and body site of study populations, and also in the frequency, mode, and gain curves of the ultrasound technique [20]. Thus, the adhesion to standardized measurements protocols in reproducible conditions is of utmost importance. Employing A-scan ultrasound on the volar forearm, Tan et al. found that skin thickness increased progressively up to the age of 20, and decreased subsequently [21], whereas Escoffier et al. observed that skin thickness increases up to the age of 20–30 years, remains constant until 65 years, then gets thinner, being at 90 years significantly thinner than at 5 years [22]. A similar trend was described by de Rigal et al., employing the B-scan method, in a population of 142 females aged from 0 to 90 years [19]. Skin thickness on both the volar and the dorsal aspects of the forearm thickened up to 15 years (maturation phase), did not vary until the seventh decade of life, and diminished thereafter (atrophy phase). These modifications in skin thickness seemed to be correlated with the age-dependent degree of elastosis. Site-to-site variations in skin thickness related to aging were reported by many authors [23–27]. In 162 volunteers in the age range of 27–90, skin thickness variations were assessed using both A- and B-mode high-frequency ultrasonography at six different anatomical locations (forehead, cheek, volar forearm,
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dorsal forearm, upper abdomen, and buttocks) [25]. It was found that subjects over 70 years have thinner skin when compared to young volunteers (age 27–31), but the differences were significant for sun-protected skin sites (abdomen and buttocks) only. In a study on 90 Danish subjects aged 18–94, skin becomes thicker with age at the forehead and buttocks, but decreases at the extremities (dorsal and ventral forearm, and ankle) significantly [26]. Since these data cannot be explained by differences in sun exposure, the authors concluded that the effect of aging on axial skin could differ from that on extremity skin. With regard to photoaging, Leveque et al. reported an increase in skin thickness in sun-exposed areas in a study on cyclists in the Tour de France [28]. Likewise, Adhoute et al. observed skin thickening on face and neck sites induced by solar exposure [29]. In 170 women aged from 17 to 76, Takema et al. measured skin thickness on the ventral forearm, forehead, cheeks, and corners of eyes and mouth by employing a 20-MHz A-mode ultrasound scanner [24]. Skin thickness increased with age on all sun-exposed areas of the face, whereas it seemed to decrease on the sun-protected volar forearm. On the contrary, using B-mode ultrasound on the neck of 30 elderly women (age 81 6 years) with high lifetime sun exposure, Richard et al. found that skin thickness was lower on an exposed area in comparison with an adjacent, anatomically equivalent unexposed area [8]. Especially on facial skin, thickness assessment yielded contrasting results. On the forehead, skin thickness appeared both to decrease [23, 30] and to increase [24, 26, 27] with age, in different studies. Employing A-mode ultrasound, Denda et al. measured skin thickness on the forehead and the cheek and observed a decrease with age [23], whereas Takema found an increase in the same areas and also on eye corners [24]. In contrast, using B-mode ultrasound in 95 Korean women aged 30–50, Lee et al. did not observe changes with age on the crow’s feet [18]. In a study population of 20 women aged 25–30 and 20 women aged 60–90, skin thickness was assessed at 12 different facial skin sites using a 20-MHz B scanner [27]. Higher values in skin thickness in the elderly were observed on all assessed facial sites except on the infraorbital region. In particular, the increase in skin thickness was statistically significant on the lateral regions of the forehead, the upper and lower lips, and the nose. The fact that facial skin thickness does not show a decreasing trend as at other skin sites can be explained by the observation that on sun-damaged facial skin, the reduction in collagen and ground substance
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content, which gradually takes place with aging, is counterbalanced by the overall rearrangement of the dermal collagen network and the accumulation of elastotic material [31, 32].
Skin Echogenicity By providing quantitative data of echogenicity, highfrequency ultrasound permits the noninvasive evaluation of age-dependent modifications in collagen structure, elastosis, and other ultrastructural features of the skin, together with the effects of diurnal and hormonal changes [6, 32–34]. When a B-mode ultrasonographic image of normal skin is generated, a hyperreflective band-like echo is observed between the medium and the skin, the socalled entry echo, corresponding to the epidermis, due to the impedance change from the coupling medium to the stratum corneum. Immediately below the entry echo, there is the corium, rich in collagen fibers, which are the main source of its echogenicity. In 1989, de Rigal et al. studied 142 females aged 1–90 by B-mode 25-MHz sonography on the volar and dorsal aspects of the forearm [19]. They identified a subepidermal hypoechogenic band, appearing as a relatively homogeneous, echolucent structure, located immediately below the entry echo. This subepidermal low-echogenic band (SLEB), which was invisible in the young, was present in most elderly subjects at the forearm and was located in the upper dermis, in some cases occupying the greater part of the dermis. The thickness of SLEB increased with age progressively and was higher on the dorsal forearm. Comparing two adjacent, anatomically equivalent sites, with and without sun exposure on the neck, Richard et al. noticed that SLEB thickness was greater on sun-exposed sites [8]. The presence of SLEB has been confirmed by many other investigators and was correlated to the severity of photodamage [12, 20, 26, 27, 34]. A Japanese study on 130 women aged 18–83 failed to demonstrate the presence of SLEB on facial areas (forehead, cheeks, and eye corners) probably because of the cultural tendency toward careful facial sun protection [35]. Changes in SLEB have been used to assess the efficacy of anti-aging cosmetics [36]. Since the main source of dermal echogenicity is represented by well-arranged collagen bundles, the appearance of SLEB is correlated to the structural changes that occur with age. In elderly skin, collagen bundles are replaced by a more homogeneously stained material, leading to the dissolution of the regular architecture of the collagen and elastic fibers and to the deposit
of a greater amount of hydrated proteoglycans and glycosaminoglycans and of unbound water [37, 38]. Gravitational changes in body water balance throughout the day may explain the diurnal variation in SLEB thickness described by Gniadecka et al. on the volar forearm of 23 subjects aged 75–100 [34]. In 74% of the study population a clear diurnal variation in SLEB thickness was found. People who have a thick SLEB in the morning before rising had a thinner SLEB in the afternoon; the opposite was also true. Moreover, a well-developed SLEB was present only in 53% of cases and in some volunteers this was irregular with ill-defined borders. In these instances, SLEB thickness measurements were complicated and unreliable. SLEB can be more precisely quantified by image analysis than by visual scoring or thickness measurement. Computer-assisted analysis of the ultrasound image is based on the attribution of arbitrary values by a 0–255 scale to the echoes’ amplitudes for each pixel, it segments the image by pixel range, and measures regions of echogenicity as specified by the investigators [20]. To calculate echogenicity, the number of low-echogenic pixels, being defined as those with echogenicity 0–30, can be measured and related to the total number of pixels, this ratio increasing with the decrease in echogenicity. Ultrasound evaluation of the dermis by means of image segmentation showed that age-related changes are not limited to the upper dermis, characterized by the appearance of SLEB, but also to the lower dermis, which appears more echogenic in elderly subjects at all examined sites [20, 26, 27, 39, 40]. This dermal hyperreflecting band has been reported to become thinner with increasing age on both the volar and the dorsal forearm in 142 females aged 0–90 by de Rigal et al. [19]. In a study population of 90 subjects aged 18–94, Gniadecka et al. studied echogenicity in the different layers of the dermis on regions with different levels of sun exposure (dorsal and volar forearm, forehead, and ankle) and nonexposed buttocks [26]. The echogenicity of the lower dermis increased at all examined sites, including those with little or no sun exposure, suggesting that changes in the dermal echogenic band characterize chronological aging. In contrast, SLEB was present at the sun-exposed dermis alone and, prior to formation of a discernible SLEB a progressive, age-related decrease in echogenicity of the upper dermis was found in sun-exposed areas (dorsal forearm and forehead), but not at moderately exposed sites (ventral forearm and ankle). A significant relationship between skin echogenicity of the upper skin layers, age, and degree of photodamage assessed clinically further indicates that the decrease in
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subepidermal echogenicity may provide an objective parameter to evaluate solar damage. These results were confirmed in a study on 55 adults aged 18–57, undergoing 20-MHz ultrasonography and image analysis on the volar and the dorsal side of the forearm [40]. The authors demonstrated that skin echogenicity measured as a ratio between the upper and the lower dermis may be used to objectively estimate photoaging. Evaluating echogenicity of the dermis as a whole, some investigators found that it decreases with increasing age [41]. In contrast, using a 20-MHz B-mode scanner on six skin sites on 24 volunteers aged 27–31 and on 24 volunteers over 60, an increase was found in overall echogenicity of the dermis in the elderly [20]. Depending on different skin areas, nonuniform variations in skin echogenicity from childhood to adulthood were observed [42]. A gradual increase was observed in echogenicity on the limbs with increasing age, whereas on the face and the trunk echogenicity was higher in children than in adults. In ultrasound images, facial skin shows scarce reflectivity, both in the young and the elderly, compared to other skin sites, such as the forearm, where the dermis is highly echogenic and the dermis– hypodermis boundary is well outlined. When evaluating overall skin echogenicity at different facial areas in young and elderly women, an increase with age at all examined sites except the infraorbital regions was found [27]. Moreover, age-related changes in skin echogenicity, consisting in the appearance of a subepidermal band and an enhancement of the lower dermis reflectivity were present at most facial sites. These findings of increased overall echogenicity in the elderly may be due to an enhancement of the lower dermis echoes, rather than a decreased echogenicity of the upper dermis.
visualization of the epidermis and the upper dermis with a good correlation to histologic sections [43, 44]. Sauermann et al. investigated skin aging using CSLM on the volar forearm of 13 young and 13 elderly volunteers [45]. The cells in the granular layer were significantly larger in the older subjects confirming histological findings documenting the increase of corneocytes with age as a result of a lower proliferation rate and turnover of the epidermis. Basal layer thickness decreased significantly, whereas thickness of the epidermis increased in the older volunteers compared to the younger ones. However, the most relevant age-related change in this study was the reduction in the number of dermal papillae per area with age reflecting the flattened epidermal–dermal junction in elderly skin. Histometric measurements by CSLM proved to be a sensitive tool for characterizing histological changes in the epidermis and papillary dermis due to aging and also for cosmetic research. The same authors evaluated the efficacy of a cream containing vitamin C applied twice a day for 4 months on the volar forearm in a study population of 33 women aged 45–67 by using CSLM [46]. Topical vitamin C resulted in a significant increase in the density of dermal papillae and in a reduction of granular layer cell size, indicating relevant effects in correcting the structural changes associated with the aging process. In CSLM images, Neerken at al. identified a reflecting layer of fibrous structures, whose depth strongly depends on age below the basal layer [47]. In addition, large structural changes, such as the flattening of the dermo– epidermal junction and a thinning of the epidermis, were observed with increasing age. CLSM was also employed to assess photodamaged skin to study alterations in dermal collagen fibers brought about by long-term sun exposure [48].
Confocal Scanning Laser Microscopy and Aging
Conclusion
In vivo confocal scanning laser microscopy (CSLM) is a noninvasive technique permitting optical en face sectioning of the skin with good contrast and high resolution, providing cellular and subcellular details. It seems to have a tremendous potential for research and diagnostic purposes in dermatology, since CSLM supplies an open ‘‘histological’’ window to the tissue noninvasively. The commercially available Vivascope microscopes (Lucid, Rochester, NY) use a diode laser source with a wavelength of 830 nm, an illumination power up to 20 mW on the object and water immersion. The penetration depth of imaging allows the
Despite the many tools and techniques available for the noninvasive evaluation of age-related changes in skin structure and functions, further work is needed to develop an unified understanding of skin aging. To date, results of some studies on skin aging are often conflicting and difficult to interpret. Instrument and other measurementrelated variables can partly explain the differing results. Moreover, the great interindividual and intraindividual variations of the human skin make consensus a challenging objective. Therefore, further studies which include larger study populations and standardized protocols are necessary.
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Cross-references > Corneocyte
Analysis of the Skin Surface > Transepidermal Water Loss and Aging > Hydration
References 1. Fluhr JW, Pfistener S, Gloor M. Direct comparison of skin physiology in children and adults with bioengineering methods. Pediatr Dermatol. 2000;17:436–439. 2. Diktein S, Hartzshtark A, Bercovici P. The dependence of low pressure indentation, slackness, and surface pH on age in forehead skin of women. J Soc Cosmet Chem. 1984;35:221–228. 3. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res Technol. 2005;11:221–235. 4. Wilhelm KP, Cua AB, Maibach HI. Skin aging: effect on transepidermal water loss, stratum corneum hydration, skin surface pH and casual sebum content. Arch Dermatol. 1991;127:1806–1809. 5. Conti A, Schiavi ME, Seidenari S. Capacitance, transepidermal water loss and casual level of sebum in healthy subjects in relation to site, sex and age. Int J Cosmet Sci. 1995;17:77–85. 6. Callens A, et al. Does hormonal aging exist? A study on the influence of different hormone therapy on the skin of postmenopaused women using non-invasive measurement techniques. Dermatology. 1996;193:289–294. 7. Caisey L, et al. Influence of age and hormone replacement therapy on the functional properties of the lips. Skin Res Technol. 2008;14:220–225. 8. Richard S, de Rigal J, Lacharriere O, Berardesca E, Leveque JL. Noninvasive measurement of the effect of lifetime exposure to the sun on the aged skin. Photodermatol Photoimmunol Photomed. 1994;10:164–169. 9. Warren R. Age, sunlight, and facial skin: a histologic and quantitative study. J Am Acad Dermatol. 1991;25:751–760. 10. Kikuchi-Numagami K, et al. Functional and morphological studies of photodamaged skin on the hands of middle-aged Japanese golfers. Eur J Dermatol. 2000;10(4):277–281. 11. Guinot C, et al. Effect of hormonal replacement therapy on cutaneous biophysical properties of menopausal women. Ann Dermatol Venereol. 2002;129:1129–1133. 12. Marks R, Edwards C. The measurement of photodamage. Br J Dermatol. 1992;127(41):7–13. 13. Kelly RI, et al. The effects of aging on cutaneous microvasculature. J Am Acad Dermatol. 1995;33:749–756. 14. Tolino MA, Wilkin JK. Aging and cutaneous vascular thermoregulation responses. J Invest Dermatol. 1988;90:613. 15. Ishihara M, et al. Blood flow. In: Kligman AM, Takase Y (eds) Cutaneous Aging. Tokyo: University of Tokyo press, 1988, pp. 167–181. 16. Hatzis J. The wrinkle and its measurement-a skin surface profilometric method. Micron. 2004;35:210–219. 17. Quan MB, Edwards C, Marks R. Non-invasive in vivo techniques to differentiate photodamage and ageing in human skin. Acta Dermatol Venereol. 1997;77(6):416–419. 18. Lee HK, Seo YK, Baek JH, Koh JS. Comparison between ultrasonography (Dermascan C version 3) and transparency profilometry (Skin Visiometer SV600). Skin Res Technol. 2008;14:8–12.
19. de Rigal J, et al. Assessment of aging of the human skin by in vivo ultrasonic imaging. J Invest Dermatol. 1989;93:621–625. 20. Seidenari S, Pagnoni A, Di Nardo A, Giannetti A. Echographic evaluation with image analysis of normal skin: variation according to age and sex. Skin Pharmacol. 1995;7:201–209. 21. Tan CY, Stathan B, Marks R, Payne PA. Skin thickness measurement by pulsed ultrasound: its reproducibility, validation and variability. Br J Dermatol. 1982;106:657–667. 22. Escoffier C, et al. Age-related mechanical properties of human skin: an in vivo study. J Invest Dermatol. 1989;93:353–357. 23. Denda M, Takahasi M. Measurement of facial skin thickness by ultrasound method. J Soc Cosmet Chem Jpn. 1990;23:316–319. 24. Takema Y, Yorimoto Y, Kawai M, Imokawa G. Age-related changes in the elastic properties and thickness of human facial skin. Br J Dermatol. 1994;131:641–648. 25. Lasagni C, Seidenari S. Echographic assessment of age-dependent variations of skin thickness. A study on 162 subjects. Skin Res Technol. 1995;1:81–85. 26. Gniadecka M, Jemec GBE. Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness. Br J Dermatol. 1998;139:815–821. 27. Pellacani G, Seidenari S. Variations in facial skin thickness and echogenicity with site and age. Acta Dermatol Venereol. 1999; 79:366–369. 28. Leveque JL, et al. Influence of chronic sun exposure on some biophysical parameters of the human skin: an in vivo study. J Cutan Aging Cosmet Dermatol. 1989;1:123–127. 29. Adhoute H, de Rigal J, Marchand JP, Privat Y, Leveque JL. Influence of age and sun exposure on the biophysical properties of the human skin : an in vivo study. Photodermatol Photoimmunol Photomed. 1992;9:99–103. 30. Nishimura M, Tuji T. Measurement of skin elasticity with a new suction device. Jpn J Dermatol. 1990;102:1111–1117. 31. Shuster S, Black MM, McVitie E. The influence of age and sex on skin thickness, skin collagen and density. Br J Dermatol. 1975;93:639. 32. Pellacani G, Giusti F, Seidenari S. Ultrasound assessment of skin ageing. In: Serup J, Jemec GBE, Grove GL (eds) Non-invasive Methods and the Skin. Boca Raton, FL: CRC press, 2006, pp. 511–514. 33. Altmeyer P, Hoffmann K, Stucker M, Goertz S, el-Gammal S. General phenomena of ultrasound in dermatology. In: Altmeyer P, el-Gammal S, Hoffmann K (eds.) Ultrasound in Dermatology. Berlin/Heidelberg: Springer-Verlag, 1992, pp. 55–79. 34. Gniadecka M, Serup J, Sondergaard J. Age-related diurnal changes of dermal oedema: evaluation by high frequency ultrasound. Br J Dermatol. 1994;131:849–855. 35. Tsukahara K, et al. Age-related alterations of echogenicity in Japanese skin. Dermatology. 2000;200:303–307. 36. Hoffmann K, Dirschka T, el-Gammal S, Altmeyer P. Assessment of actinic elastosis by means of high-frequency sonography. In: Marks R, Plewing G (eds.) The Environmental Threat to the Skin. London: Martin Dunitz, 1991, pp. 83–90. 37. Richard S, et al. Characterization of the skin in vivo by high resolution magnetic resonance imaging: water behaviour and age-related effects. J Invest Dermatol. 1993;100:705–709. 38. Oikarinen A. Aging of the skin connective tissue: how to measure the biochemical and mechanical properties of aging dermis. Photodermatol Photoimmunol Photomed. 1994;10:47–52. 39. Sandby-Moller J, Wulf HC. Ultrasonographic subepidermal lowechogenic band, dependence of age and body site. Skin Res Technol. 2004;10:57–63.
Bioengineering Methods and Skin Aging 40. Gniadecka M. Effects of ageing on dermal echogenicity. Skin Res Technol. 2001;7:204–207. 41. Nakahigashi N, Sugai T. Assessment of degeneration by sun exposure using ultrasonic imaging with dermascan C. Skin Res. 1996; 38:25–30. 42. Seidenari S, Giusti G, Bertoni L, Magnoni C, Pellacani G. Thickness and echogenicity of the skin in children as assessed by 20-MHz ultrasound. Dermatology. 2000;201:218–222. 43. Rajadhyaksha M, Gonzalez S, Avislan JM, Anderson RR, Webb RH. In vivo confocal laser scanning microscopy of human skin II: advances in instrumentation and comparison with histology. J Invest Dermatol. 1999;113:292–303. 44. Branzan AL, Landthaler M, Szeimies RM. In vivo confocal laser scanning microscopy in dermatology. Lasers Med Sci. 2007; 22:73–82.
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45. Sauermann K, et al. Age-related changes in human skin investigated with histometric measurements by confocal laser scanning microscopy in vivo. Skin Res Technol. 2002;8:52–56. 46. Sauermann K, Jaspers S, Koop U, Wenck H. Topically applied vitamin C increases the density of dermal papillae in aged human skin. Dermatology. 2004;4:13–18. 47. Neerken S, Lucassen GW, Bisschop MA, Lenderink E, Nuijs TA. Characterization of age-related effects in human skin: a comparative study that applies confocal laser scanning microscopy and optical coherence tomography. J Biomed Opt. 2004;9:274–281. 48. Bernstein EF, et al. Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun-protected and photoaged skin by northern analysis, immunohistochemical staining and confocal 3laser scanning microscopy. J Am Acad Dermatol. 1996;34:209–218.
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68 Corneocyte Analysis Tetsuji Hirao
Introduction The stratum corneum (SC) forms the outermost layer of the skin, and plays a principal role in maintaining the barrier function and the water-holding capacity of the skin. It consists of piled-up dead corneocytes and intercellular lipid lamellae. Corneocytes are the final product of terminal differentiation of epidermal keratinocytes, and are continuously renewed. Therefore, the superficial SC including corneocytes can be collected easily and noninvasively, as a source of information on the nature of the SC itself, as well as the skin beneath the SC. Extensive morphological observations of corneocytes were performed as long ago as the 1980s [1, 2], and the relationship between corneocyte size and epidermal turnover rate has been well established [3–6]. In the 1990s, biochemical methodologies were applied to analyses of SC components, leading to detailed understandings of SC architecture, organization, and physiological and pathological functions. In this review, analyses of corneocytes and SC collected by noninvasive procedures will be reviewed, focusing especially on studies of skin aging.
Collecting Procedures Various procedures have been proposed to collect SC samples. Those that impose the least burden on subjects are preferable, and reproducibility is also an important consideration. Broadly speaking, such collecting procedures applicable to healthy volunteers can be divided into three groups: tape stripping, polymeric resin, and scrub methods. In addition, SC samples from hyperkeratotic subjects can be spontaneously collected in the form of scales or dandruff, without any special collecting device. Many kinds of commercially available transparent adhesive tapes, such as Scotch tape and Cellophane tape, can be conveniently used to collect SC samples by tape stripping. However, the amount of SC collected may vary depending on the adhesiveness of the tape and conditions, such as pressure, when it is applied to the skin [7, 8]. To minimize these variations, D-squame has been
developed [9]. Thus, tape stripping has been widely used for morphological observation of corneocytes. In quantitative analyses with tape-stripped SC samples, normalization of the amount collected on the tape remains an important consideration. Simple optical procedures have been proposed to estimate the amount [10–13], and protein determination is also often used, since the major constituent of the SC is protein [14, 15]. Recently, an optical procedure using infrared absorption measurement to determine the amount of SC collected on a tape has been proposed [16]. These optical methods offer the advantage that protein content can be estimated nondestructively, so the samples remain available for further study. Repeated tape-stripping procedures are often used for the collection of SC samples of various depths. However, Van der Molen et al. [17] pointed out that tape stripping of SC yields material that originates from various depths because of the presence of furrows in the skin, so care is needed in evaluating parameters whose value varies depending on the depth in the SC. Cyanoacrylate resin is the most widely used polymeric resin to collect SC samples. This procedure, called skin surface biopsy (SSB), enables the collection of larger amounts of SC samples than tape stripping does, though collection imposes a somewhat greater burden on the subjects. This method has the advantages that the twodimensional distribution of the corneocytes is retained, and that it is applicable to undulating site, such as follicles, where tape stripping is difficult or ineffective. Collection of exfoliating corneocytes by scrubbing the skin surface with a physiological buffer using a rod is a gentler procedure than tape stripping or SSB with cyanoacrylate resin. Corneocytes can be collected as a suspension in the buffer, and can be concentrated by centrifugation. In some cases, a detergent such as Triton X-100 is added to the buffer to prevent aggregation of corneocytes. This method is suitable for morphological observation of isolated corneocytes, but not for examination of two-dimensional distribution on the skin surface, which can be evaluated by using tape-stripped SC or SSB. Thus, suitable conditions for collecting SC samples need to be selected case by case, depending on the nature of the analyses to be carried out.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_68, # Springer-Verlag Berlin Heidelberg 2010
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Corneocyte Analysis
Morphology Corneocytes have a polygonal, flattened shape, are approximately 1 mm thick and 50 mm diameter, and are piled up in 10–20 layers, depending on the anatomical site, though exceptionally, over 40 layers are stacked in the palm and the sole [18]. Classical microscopic observation of exfoliating corneocytes revealed that healthy corneocytes are anucleated, while nucleated corneocytes, well known as parakeratotic cells, are detected under pathological conditions such as dermatitis. In addition to the existence or absence of nuclei in corneocytes, the size is a parameter that can be determined easily by microscopy. Several studies have shown that the size of exfoliating corneocytes is well correlated with the turnover rate of the epidermis. Smaller corneocytes are detected in areas with faster epidermal turnover, including sites of dermatitis and tape strippinginduced rough skin [2, 5, 6]. It is noteworthy to mention that the size of exfoliating corneocytes increases with age in the trunk and extremities, but it is not the case in the face, suggesting that in these areas, the epidermal turnover rate decreases with aging. Thus, the corneocyte size can provide information about epidermal dynamics. Electron microscopic observation has enabled more precise morphological characterization of corneocytes. Corneocytes from the deeper layers of the SC exhibit a rough surface with villous projections, while those in the superficial layer have a polygonal shape with a flat surface [19]. However, corneocytes with villous-like projections were detected in the superfical SC of patients with inflammatory disorders [20], as well as in that of normal facial skin [21], as determined by scanning electron microscopic examination of corneocytes harvested with cyanoacrylate resin or adhesive tape, suggesting that this atypical morphology could reflect irregular maturation of corneocytes. Another approach to examine the surface of corneocytes is the use of scanning electron microscopy to observe freezefracture replicas by Simon et al. [22]. They reported a decrease in corneodesmosomal plaques on corneocytes during maturation within the SC. In addition to individual corneocyte morphology, the cellular arrangement has been studied in tape-stripped or cyanoacrylate-collected SC samples from patients with various skin conditions [23, 24]. Recently, highly sophisticated three-dimensional analyses of corneocytes were performed using atomic force microscopy by Kashibuchi et al. [25]. In addition to obtaining two-dimensional data of projected area, which has been widely used as a parameter of corneocyte size, they measured the volume, average thickness, and real surface area of corneocytes, and introduced a flatness
index, defined as the projected area divided by its thickness. They found that the flatness index decreased with faster turnover rate, for example, in the cheek and skin of patients with inflammatory disorders. The flatness index was also decreased in the deeper layers of the SC as compared with the superficial layer, suggesting morphological change with maturation within the SC. The flatness index of corneocytes isolated from the upper arm increased with increasing age of the subjects, in agreement with previous conventional measurements of the projected area of corneocytes.
Constituents Major constituents of the corneocytes are keratin intermediate filaments, which fill the inside of the corneocytes. In contrast, cornified envelope (CE) is a thin, insoluble structure surrounding corneocytes, consisting of various kinds of precursor proteins which are cross-linked to each other. The intercellular spaces between corneocytes, outside the CE, contain highly organized lipid lamellae. Free amino acids, which are major components of natural moisturizing factors, can also be analyzed in SC samples. Keratin intermediate filaments are aggregated in parallel, showing so-called keratin pattern-characteristic bundles, in the SC as observed with transmission electron microscopy [26, 27]. Since the arrangement of keratin filaments is likely to be affected by many conditions, such as water content, simple, noninvasive sampling procedures have not been employed. Rogers et al. [28] analyzed the intercellular lipid profile in tape-stripped SC samples by high-performance thin-layer chromatography, and examined the effect of aging as well as seasonal variation, finding significantly decreased levels of all major lipid species, in particular ceramides, with increasing age. This age-related alteration is not in agreement with the results of a previous study [29], in which the lipids were collected with solvent directly applied onto the skin surface. Rogers et al. [28] also examined seasonal change, and found that the stratum corneum lipid levels were dramatically depleted in winter compared with spring and summer. They suggested that there might be a relationship between alteration in lipid profile and senile xerosis. Furthermore, intercellular lipid depth profiles were also studied by applying high-performance thin-layer chromatography to sequentially tape-stripped SC, and altered profiles were found in different layers of the SC [30, 31]. The composition of free amino acids in the SC can be analyzed in the water-soluble fraction extracted from SC
Corneocyte Analysis
samples by means of a conventional amino acid analyzer [32]. Free amino acids in the SC are known to be derived from degradation of filaggrin, and some amino acids are further metabolized. For example, glutamine is converted to pyrrolidonecarboxylic acid, and histidine to urocanic acid; the conversion ratio was proposed to be a marker of skin condition [33]. Horii et al. [34] showed that quantity of free amino acids in the SC decreases with increasing severity of dry skin in elderly. Thus, free amino acids in the SC seem to be closely related with moisturizing function of the skin. To examine amino acid profiles, tape-stripped SC is not necessary; alternatively, direct extraction of amino acids with water from the skin surface can be applied [33, 34]. Recently, confocal Raman spectroscopy has been introduced as a sophisticated in vivo analysis method for constituents in the SC without the need for tape stripping, and has revealed the depth profile of free amino acids in the SC with a resolution of several micrometers [35]. Interestingly, vibrational imaging of isolated corneocytes by infrared and Raman microscopy by Zhang et al. [36] has revealed differences of amino acid concentration between the superficial layer and the deeper layers of the SC. These optical spectroscopic analyses may also be applicable to constituents other than amino acids. CE can be biochemically isolated as an insoluble envelope-like macromolecular structure by extensive boiling treatment of the epidermis or SC in the presence of sodium dodecyl sulfate (SDS) and a reducing agent, such as b-mercaptoethanol or dithiothreitol; most proteins, including keratins, are solubilized by such treatment. The insolubility of CE is a result of covalent cross-linking between precursor proteins. Typical cross-links are g-glutamyl-e-lysine isopeptide bonds, whose formation is mediated by transglutaminases (TGases). Michel et al. [37] first reported that CE from the deeper SC shows irregular and fragile morphology, while CE from the outer SC has a rigid and polygonal shape, suggesting morphological maturation within the SC. They also reported irregular morphology of CE from psoriatic SC. Reichert et al. [38] characterized CE maturity using tetramethylrhodamine isothiocyanate (TRITC) staining, and noted that rigid CE can be distinguished by strong staining with TRITC as compared with immature CE from the deeper SC. Since TRITC reacts with amino groups, their result may reflect abundant incorporation of precursor proteins into CE during maturation. Watkinson et al. [39] further characterized differences between rigid and fragile CEs based on biochemical as well as biophysical approaches. Repetitive tape stripping of the SC revealed that g-glutamyl-e-lysine isopeptide content in the CE gradually increases with migration
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toward the superficial layer in the stratum corneum. Interestingly, a significant difference in maximal compressional force between rigid and fragile CEs was observed; rigid CE was mechanically stronger than fragile CE, though the rigid CE population showed heterogeneity of maximal compressional force. A novel method has been established to evaluate CE maturity based on the biochemical profile by utilizing the combination of involucrin antigenicity and hydrophobicity recognized by an environment-sensitive fluorescent dye, Nile red [40]. Involucrin is one of the protein components of CE, being located at the exterior surface of CE, and is extensively modified by cross-linking or lipid attachment. Rigid CE from the outermost layer is stained with Nile red and less stained with anti-involucrin. In contrast, fragile CE from the deeper SC is less stained with Nile red, but strongly stained with anti-involucrin. These results suggest that immature CE has less extensively modified involucrin, whose antigenicity might be lost in mature CE as a result of modification by protein crosslinking and/or covalent attachment with lipids. At the same time, these modifications result in acquisition of hydrophobicity in mature CE, leading to intense staining with Nile red. This method of combined Nile red and involucrin staining of CE is a useful tool to study the biochemical maturation process of CE within the SC. It was first applied to examine regional differences of anatomical sites in healthy subjects, and found that immature and fragile CE is present in the outermost layer of the face SC, while the outermost layer of SC at other sites, including trunk and extremities, shows homogeneously mature phenotype [40]. Interestingly, CE from the deeper layers of the arm, obtained through repetitive tape stripping, exhibits immature phenotype, suggesting that maturation occurs within the SC. It has been demonstrated that the face exhibits particular characteristics, such as higher transepidermal water loss (TEWL) and easier penetration of certain drugs, suggesting some impairment of barrier function [41]. Therefore, a defect in maturation of CE may be one of the factors that impede barrier function of the facial skin. The ratio of immature CE in the face varies among individuals. However, no significant difference of the ratio with age, gender, or race was found, though seasonal change was observed. The ratio of immature CE in the face was significantly increased in winter, suggesting a relationship between defective CE maturation and rough skin in winter [42]. Furthermore, CE in the outermost SC of the involved areas of psoriasis vulgaris (PV) and atopic dermatitis (AD), which are typical inflammatory disorders associated
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Corneocyte Analysis
with impaired barrier function, showed striking heterogeneity, and consisted of immature and mature CEs, whereas CE of the corresponding uninvolved areas was relatively homogeneous, exhibiting mature phenotype [43]. The higher ratio of immature CE found in PV and AD suggests that defective CE maturation may, at least in part, account for the impaired barrier function in inflammatory disorders. It is of particular interest that corneocytes with immature CE do not always coincide with parakeratotic cells, indicating that the mechanisms of CE maturation and disappearance of nuclei do not fully overlap, although both of them are closely associated with corneocyte maturation. This method for the evaluation of CE maturity has been applied to characterization of SC of hypertrophic scar [44], as well as to examine the effect of chemical peeling on keratinocyte differentiation [45].
Biochemistry Corneocytes in the SC are biologically dead cells, but still retain various enzyme activities and biologically active substances. Various proteases and inhibitors play important roles in physiological functions of the SC. One of the most important events regulated by proteolysis in the SC is degradation of corneodesmosomes, which connect corneocytes. Their degradation leads to a decrease in cohesion of corneocytes in the superficial layer of the SC. In the 1990s, two serine proteases, SC tryptic enzyme (SCTE) and SC chymotryptic enzyme (SCCE), were shown to be involved in desquamation [46–48]. SCTE and SCCE are now called human kallikreins 5 (hK5) and 7 (hK7), respectively, according to the new nomenclature for human serine proteases [49]. hK7 can be activated by partial proteolysis by hK5, and both proteases coordinately degrade the extracellular portion of corneodesmosomes, consisting of desmoglein 1, desmocollin 1, and corneodesmosin [50, 51]. Proteolysis by these kallikreins is precisely regulated by protease inhibitors during desquamation at the superficial layer of the SC [50–52]. Komatsu et al. [53] used enzyme-linked immunosorbent assays (ELISAs) for global quantification of human kallikreins in tape-stripped SC samples, finding high levels of hK7, hK8, and hK11, a moderate level of hK5, and lower levels of hK10, hK14, hK6, and hK13. They also reported that levels of hK6, hK8, and hK13 were reduced in aged subjects, while hK5 and hK7 did not show any significant change with aging. Although the overall trypsin-like hKs showed a slight decrease in the aged
group, neither trypsin-like nor chymotrypsin-like activities in the SC differed across age groups, whereas Suzuki et al. [54] reported reduced activities of SCCE and SCTE in ichthyotic skin. In addition, no significant regional difference in the quantities of hKs in the SC was observed among forearm, abdomen, back, and thigh [53]. Komatsu et al. applied these procedures to pathogenic disorders, and found significantly higher levels of human kallikreins in lesional SC of psoriasis [55], atopic dermatitis [56], and peeling skin syndrome type B [57]. Voegeli et al. [58] profiled serine protease activities in the SC, including plasmin-like, urokinase-like, tryptase-like, hK5like, and hK7-like activities, using fluorogenic synthetic substrates, and found elevated serine protease activities in the outer SC as compared with deeper layers, and in the cheek compared with the arm, possibly indicating subclinical inflammation in the cheek, which is exposed to the environment. In addition to these serine proteases, aspartic proteases are involved in the degradation of corneodesmosomes. Horikoshi et al. [59] described a role of cathepsin D-like activity in desquamation, since the superficial pH is more acidic, around pH 5, than the deeper SC. While in vivo measurement of aspartic protease activities at the surface of animal skin using radio-labeled insulin B-chain as a substrate revealed an age-associated decrease of the activity [60], Horikoshi et al. [61] also measured cathepsin D-like and SCCE-like activities in tape-stripped SC. They showed that treatment with glycolic acid resulted in acute activation of cathepsin D in the lower SC, as well as long-term activation of de novo cathepsin D expression. Caspases are cysteine proteases that play a central role in apoptosis, as well as in the terminal differentiation of epidermal keratinocytes. Among several caspases, activation of caspase-14 is associated with terminal differentiation of human keratinocytes, and its activity is dominant in the SC, while procaspase-14 is detected in incompletely matured SC of parakeratotic skin from psoriasis [62, 63]. Gelatinases are members of the matrix metalloprotease family that degrade various components of the skin, and may be involved in photoaging. Takada et al. [64] detected gelatinase activity in tape-stripped SC of UV-irradiated skin, as well as in that of a sun-exposed area, the face, but not that of unexposed areas. Upregulation of gelatinases may be an etiological factor in photoaging. In addition to the proteases described above, other hydrolyzing enzymes have been detected in the SC. Beisson et al. [65] examined esterase activities of tape-stripped SC, toward triacylglycerol, and 4-methylumbelliferyl
Corneocyte Analysis
7-heptanoate and 7-oleate, suggesting that these esterase activities are involved not only in hydrolysis of endogenous substrates, such as triacylglycerol, but also in that of exogenous substrates, such as some ester-type drugs and ingredients of cosmetics. Jin et al. [66] examined the activities of enzymes related to ceramide metabolism in the SC. Activities of both b-glucocerebrosidase, which hydrolyzes glucosyl ceramide into ceramide, and ceramidase, which hydrolyzes ceramide into sphingosine and palmitic acid, were detected in the SC. In atopic dermatitis, both enzyme activities are unchanged as compared with healthy subjects. In an aged group, upregulation of ceramidase was observed, while b-glucocerebrosidase activity was unchanged, suggesting a possible involvement of ceramidase in the pathogenesis of aged dry skin. Takagi et al. [67] showed that b-glucocerebrosidase activity is localized in the lower part of the SC, and is involved in conversion of ceramide species within the SC. Phospholipase A2 (PLA2) catalyzes the release of fatty acids from phospholipids, and has been suggested to play a key role in the accumulation of free fatty acids in the SC. Mazereeuw-Hautier et al. [68] detected PLA2 activity in tape-stripped SC samples, and reported that deeper layers exhibited higher activity. Resoules et al. [69] studied five enzyme activities using tape-stripped SC, and reported that SC from atopic dermatitis showed significantly reduced trypsin-like activity, increased phosphatase activity, and no change in the activities of b-glucocerebrosidase, PLA2, and chymotrypsin. In the SC, protein synthesis no longer occurs, but enzymatic or nonenzymatic modifications of proteins still occur. Enzymes involved in protein modification in the SC include transglutaminase (TGase) and peptidylarginine deiminase (PAD). TGase mediates formation of g-glutamyl-e-lysine isopeptide bonds among CE precursor proteins, and its activity can be measured by using tape-stripped SC samples with labeled cadaverine as a substrate. In addition, maturation of CE was achieved by ex vivo incubation of tape-stripped SC under a humidified condition; this was mediated by TGase [70]. PAD catalyzes conversion of arginine residues in protein into citrulline residues. In the SC, keratins and fillagrin are substrates of PAD, and citrulline residues can be detected in the SC. This conversion seems to alter the isoelectric point of the proteins, and may result in a change in the conformation, but its physiological significance remains to be elucidated. Nachat et al. [71] described detection of PAD1 in extracts of superficial SC, where keratin 1 is deiminated.
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The SC at the outermost layer of the skin is exposed to various kinds of oxidative stimuli. Both oxidants and antioxidants have been detected in the SC [72, 73]. Measurement of peroxide in the SC using a fluorescent probe, 20 ,70 -dichlorofluorescein, have shown that the level varies with the depth [74]. This method can be applied to evaluate the efficacy of antioxidant treatment of the skin. Carbonylation is a hallmark of oxidatively modified proteins, and is introduced by either direct modification of amino acid residues in the protein or reaction with lipid peroxide-derived aldehydes. Carbonylated proteins in the SC can be easily detected using tape-stripped SC samples by reaction with a labeled hydrazide reagent [75]. Major targets of carbonyl modification are keratins [76], though proteins in the CE are also carbonylated [77]. The carbonyl protein level in the SC is increased under oxidative conditions, for example, in the SC of exposed areas. However, the level in the SC does not change much with age [78, 79], while it is significantly increased in photoaged dermis [78]. Elevated carbonyl level in sunexposed areas was confirmed by Fujita et al. using a simpler evaluation procedure [80]. Applying this evaluation method, the effects of carbonyl modification on the biophysical properties of the SC has been studied [81–83]. Various antioxidants in the SC, including alphatocopherol, ascorbate, uric acid, and glutathione, can also be assessed using tape-stripped SC samples [84, 85]. The levels of these antioxidants gradually decrease near the surface of the SC, and were shown to be depleted under ozone exposure [84, 85]. In addition to antioxidants, endogenous antioxidant enzymes play an important role in the protection of the skin from exogenous oxidative stimuli. Hellmans et al. [86] measured the activities of catalase and superoxide dismutase (SOD) in the SC, and showed that the activities decreased near the surface. Sun exposure or UVA irradiation resulted in the deactivation of catalase in the SC, which is consistent with reports of low activity of catalase in the SC in summer. Another important modification is glycation. SC keratins are nonenzymatically glycated in diabetic patients [87, 88], though the effect of glycation on SC function remains to be clarified. The SC contains bioactive proteins, expressed in epidermal keratinocytes, including cytokines and defensins. Interleukin 1 (IL-1) is a proinflammatory multifunctional cytokine, which had been detected in SC as epidermal thymocyte-activating factor produced by epidermal keratinocytes [89]. In addition to IL-1, IL-1 receptor antagonist (IL-1ra) has been detected in SC samples collected by
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Corneocyte Analysis
tape stripping. IL-1ra belongs to IL-1 family and can bind IL-1 receptor, but cannot activate the receptor, so that it functions as an antagonist of IL-1. It has been established that both IL-1a and IL-1ra are present in SC as active mature forms, and that the ratio of IL-1ra to IL-1a in the SC is strikingly elevated in a sun-exposed area, the face, as compared with an unexposed area, the inside of the upper arm [90]. The IL-1ra to IL-1a ratio in the SC is elevated not only in UV-induced inflammation, but also in inflammatory disorders, including lesional areas of atopic dermatitis and psoriasis [90, 91]. These results suggest that
the face exhibits a profile of subclinical inflammation. Interestingly, the IL-1ra to IL-1a ratio in the SC of the inside of the upper arm decreases with age, while that of the face remains constant [90]. Perkins et al. [92] applied a similar procedure, and showed that levels of IL-1ra and IL-8 were significantly increased in diaper rash, and further, that the IL-1ra to IL-1a ratio and the TNF-a level were higher in seborrheic dermatitis and dandruff-bearing scalp, while the level of IL-1a was not consistent, but depended on the inflammatory skin condition. De Jongh et al. [93, 94] reported
. Table 68.1 Parameters measured by means of non-invasive corneocyte analyses Category Morphology
Parameter
Method
Reference
Nuclear remnant Corneocyte size Villous-like projection Flatness index
TS, microscopy TS, microscopy TS, SEM TS, AFM
[2, 5, 6] [19–21] [25]
Intercellular lipids Aminoacids Cornified envelope maturity
TS, HPTLC TS, in vivo TS, microscopy
[28, 30, 31] [32–36] [37–45]
Protease and hydrolase
Kallikreins Cathepsin D Caspases Gelatinase Esterase b-glucocerebrosidase Ceramidase Phospholipase
TS, ELISA, activity TS, activity TS, ELISA TS, activity TS, activity TS, activity TS, activity TS, activity
[46–48, 53–58] [61] [62, 63] [64] [65] [66, 67, 69] [66] [68, 69]
Other enzyme
Transglutaminase Peptidylarginine deiminase
TS, activity TS, activity
[70] [71]
Oxidant and anti-oxidants
Peroxides Carbonyl proteins Vitamin C, E Uric acid Glutathione Superoxide dismutase
TS, colorimetry TS, staining TS, HPLC TS, HPLC TS, HPLC TS, activity
[74] [75–83] [84, 85] [85] [85] [86]
Modification
Carbonylation Glycation
TS, staining TS, chemical reaction
[75–83] [87, 88]
Bioactive molecules
Interleukin 1 Interleukin 1 receptor antagonist Interleukin 8 Nerve growth factor Defensin
TS, ELISA TS, ELISA TS, ELISA TS, ELISA TS, ELISA
[90–95] [90–95] [92–94] [95] [97]
Nucleic acid
mRNA
TS, RT-PCR
[98, 99]
Constituents
Biochemistry
AFM, atomic force microscopy; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; HPTLC, high-performance thin layer chromatography; SEM, scanning electron microscopy; TS, tape stripping
Corneocyte Analysis
that sodium lauryl sulfate-induced irritation resulted in altered cytokine levels in SC; IL-1 decreased, and IL-1ra and IL-8 increased with increasing depth. Recently, they carried out an interesting study on the relationship between IL-1 gene polymorphisms, IL-1A-889 (C to T) and IL-1B-31 (T to C), and SC cytokine levels, showing that the IL-1ra to IL-1a ratio was higher in IL-1A-889 C/T and T/T genotypes than that in C/C wild type [95]. This altered expression may be responsible, at least in part, for the interindividual differences in the inflammatory response of the skin. Yamaguchi et al. [96] reported that SC with atopic dermatitis contains an increased level of nerve growth factor, and the level may reflect the severity of itching and eruptions in atopic dermatitis. Detection of these cytokines in the SC was carried out with highly sensitive immunoassays. Antimicrobial peptides, including defensin and cathelicidin, have been reported to play important roles in cutaneous innate immunity to control colonization of microbes. Although expression of b-defensin 2 in the epidermis is downregulated in atopic dermatitis, Asano et al. [97] reported that the b-defensin 2 level is significantly higher in SC from patients with atopic dermatitis as compared with healthy controls, suggesting a role in defensive response against infection. Gene expression does not occur in SC, but mRNAs can be recovered from tape-stripped SC samples. Wong et al. [98, 99] showed that mRNA can be recovered from tape-stripped SC samples and successfully amplified. Expression of housekeeping gene mRNAs is uniform and reproducible, while the levels of IL-8 and tumor necrosis factor-a mRNAs vary with the anatomical sites and individuals. This technique may be a promising approach to examine gene expression noninvasively, although the origin of the mRNAs remains to be elucidated. In addition to biology-based technologies, great progress in spectroscopic technologies has been made recently, and constituents of corneocytes can now been analyzed and the distributions of amino acids and lipids imaged [36, 100].
Conclusion This chapter reviewed recent studies on corneocytes, as well as SC, collected by means of noninvasive methods. > Table 68.1 summarizes parameters measured by means of non-invasive corneocyte analyses. Evaluation of SC can provide a tremendous amount of information, not only on the nature of SC itself, but also on the properties of the underlying skin.
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The most typical alteration in corneocytes associated with physiological aging is an increase of corneocyte size, reflecting a slower turnover rate. In addition, the intercellular lipid profile and the IL-1ra to IL-1a ratio in SC decrease with aging. However, it is important to note that these age-associated alterations occur in the unexposed areas, and are not directly reflected in sun-exposed areas, such as the face. Since epidermal turnover is generally rapid, so that physiological age-associated changes are not likely to accumulate, changes in corneocyte parameters reflecting physiological aging are small as compared with those in the dermis. On the other hand, there are many phenotypes of corneocytes reflecting accelerated epidermal turnover, including parakeratic cells (nucleus-retaining corneocytes), smaller cell size, immature CE, villous-like projections, and so on. These phenotypes often appear in the skin with accelerated epidermal turnover, such as in inflammatory skin disorders, and in the face. Thus, the age-associated changes in corneocytes in sun-exposed areas are affected by many factors, including exogenous stimuli and intrinsic factors. Application of more sophisticated analytical technologies, including biochemical, biophysical, and optical procedures, to corneocytes is expected to give further insight into the mechanisms of skin aging in the future.
Cross-references > The
Stratum Corneum and Aging
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9. Miller DL. D-squame adhesive discs. In: Wilhelm KP, Elsner P, Berardesca E, Maibach HI (eds) Bioengineering of the Skin: Skin Surface Imaging and Analysis. Boca Raton: CRC Press, 1997, pp. 39–46. 10. Marttin E, et al. A critical comparison of methods to quantify stratum corneum removed by tape stripping. Skin Pharmacol. 1996;9:69–77. 11. Lindemann U, et al. Evaluation of the pseudo-absorption method to quantify human stratum corneum removed by tape stripping using protein absorption. Skin Pharmacol Appl Skin Physiol. 2003;16:228–236. 12. Jacobi U, et al. Estimation of the relative stratum corneum amount removed by tape stripping. Skin Res Technol. 2005;11:91–96. 13. Jacobi U, et al. The number of stratum corneum cell layers correlates with the pseudo-absorption of the corneocytes. Skin Pharmacol Physiol. 2005;18:175–179. 14. Dreher F, et al. Colorimetric method for quantifying human stratum corneum removed by adhesive-tape stripping. Acta Derm Venereol. 1998;78:186–189. 15. Dreher F, et al. Quantification of stratum corneum removal by adhesive tape stripping by total protein assay in 96-well microplates. Skin Res Technol. 2005;11:97–101. 16. Voegeli R, et al. Efficient and simple quantification of stratum corneum proteins on tape strippings by infrared densitometry. Skin Res Technol. 2007;13:242–251. 17. van der Molen RG, et al. Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin. Arch Dermatol Res. 1997;289:514–518. 18. Ya-Xian Z, et al. Number of cell layers of the stratum corneum in normal skin – relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291: 555–559. 19. King CS, et al. The change in properties of the stratum corneum as a function of depth. Br J Dermatol. 1979;100:165–173. 20. Shukuwa T. Scanning and transmission electron microscopic study of corneocytes: experimental formation of villus-like projections of the corneocytes of human epidermis by keratin layer stripping technique using cyanoacrylate (in Japanese). Nippon Hifuka Gakkai Zasshi. 1988;98:1467–1473. 21. Yanagi M, et al. Morphological investigation of desquamated corneocytes from subject with sensitive skin and improvement of their corneocytes by using skin care products (in Japanese). J Jpn Cosmet Sci Soc. 2001;25:203–210. 22. Simon M, et al. Persistence of both peripheral and non-peripheral corneodesmosomes in the upper stratum corneum of winter xerosis skin versus only peripheral in normal skin. J Invest Dermatol. 2001;116:23–30. 23. Kashibuchi N. Improved exfoliative cytology for morphological evaluation of skin. (in Japanese) J Soc Cosmet Chem Jpn. 1989;23:143–154. 24. Christophers E. Cellular architecture of the stratum corneum. J Invest Dermatol. 1971;56:165–169. 25. Kashibuchi N, et al. Three-dimensional analyses of individual corneocytes with atomic force microscope: morphological changes related to age, location and to the pathologic skin conditions. Skin Res Technol. 2002;8:203–211. 26. Norle´n L, Al-Amoudi A. Stratum corneum keratin structure, function, and formation: the cubic rod-packing and membrane templating model. J Invest Dermatol. 2004;123:715–732. 27. Norle´n L. Stratum corneum keratin structure, function and formation – a comprehensive review. Int J Cosmet Sci. 2006;28:397–425.
28. Denda M, et al. Age- and sex-dependent change in stratum corneum sphingolipids. Arch Dermatol Res. 1993;285:415–417. 29. Rogers J, et al. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 30. Bonte´ F, et al. Existence of a lipid gradient in the upper stratum corneum and its possible biological significance. Arch Dermatol Res. 1997;289:78–82. 31. Weerheim A, Ponec M. Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography. Arch Dermatol Res. 2001;293:191–199. 32. Denda M, et al. Stratum corneum sphingolipids and free amino acids in experimentally-induced scaly skin. Arch Dermatol Res. 1992;284:363–367. 33. Koyama J, et al. Free amino acids of stratum corneum as a biochemical marker to evaluate dry skin. J Soc Cosmet Chem. 1984;35:183–195. 34. Horii I, et al. Stratum corneum hydration and amino acid content in xerotic skin. Br J Dermatol. 1989;121:587–592. 35. Caspers PJ, et al. In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles. J Invest Dermatol. 2001;116:434–442. 36. Zhang G, et al. Vibrational microspectroscopy and imaging of molecular composition and structure during human corneocyte maturation. J Invest Dermatol. 2006;126:1088–1094. 37. Michel S, et al. Morphological and biochemical characterization of the cornified envelopes from human epidermal keratinocytes of different origin. J Invest Dermatol. 1988;91:11–15. 38. Reichert U, et al. A key structure of terminally differentiating keratinoytes. In: Darmon M, Blumenberg M (eds) Molecular Biology of the Skin. New York: Academic, 1993, pp. 107–150. 39. Watkinson A, et al. Its role in stratum corneum structure and maturation. In: Leyden JJ, Rawlings AV (eds) Skin Moisturization. New York: Marcel Decker, 2002, pp. 95–117. 40. Hirao T, et al. Identification of immature cornified envelopes in the barrier-impaired epidermis by characterization of their hydrophobicity and antigenicities of the components. Exp Dermatol. 2001;10:35–44. 41. Dupuis D, et al. In vivo percutaneous absorption and transepidermal water loss according to anatomic site in man. J Soc Cosmet Chem. 1986;37:351–357. 42. Hirao T, et al. A novel non-invasive evaluation method of cornified envelope maturation in the stratum corneum provides a new insight for skin care cosmetics. IFSCC Magazine. 2002;6:103–109. 43. Hirao T, et al. Ratio of immature cornified envelopes does not correlate with parakeratosis in inflammatory skin disorders. Exp Dermatol. 2003;12:591–601. 44. Kunii T, et al. Stratum corneum lipid profile and maturation pattern of corneocytes in the outermost layer of fresh scars: the presence of immature corneocytes plays a much more important role in the barrier dysfunction than do changes in intercellular lipids. Br J Dermatol. 2003;149:749–756. 45. Dainichi T, et al. Chemical peeling by SA-PEG remodels photodamaged skin: suppressing p53 expression and normalizing keratinocyte differentiation. J Invest Dermatol. 2006;126:416–421. 46. Lundstro¨m A, Egelrud T. Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation. Acta Derm Venereol. 1991;71:471–474. 47. Suzuki Y, et al. Detection and characterization of endogenous protease associated with desquamation of stratum corneum. Arch Dermatol Res. 1993;285:372–377.
Corneocyte Analysis 48. Suzuki Y, et al. The role of proteases in stratum corneum: involvement in stratum corneum desquamation. Arch Dermatol Res. 1994;286:249–253. 49. Diamandis EP, et al. New nomenclature for the human tissue kallikrein gene family. Clin Chem. 2000;46:1855–1858. 50. Caubet C, et al. Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7. J Invest Dermatol. 2004;122:1235–1244. 51. Brattsand M, et al. A proteolytic cascade of kallikreins in the stratum corneum. J Invest Dermatol. 2005;124:198–203. 52. Borgon˜o CA, et al. A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation. J Biol Chem. 2007;282:3640–3652. 53. Komatsu N, et al. Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender. J Invest Dermatol. 2005;125:1182–1189. 54. Suzuki Y, et al. The role of two endogenous proteases of the stratum corneum in degradation of desmoglein-1 and their reduced activity in the skin of ichthyotic patients. Br J Dermatol. 1996;134: 460–464. 55. Komatsu N, et al. Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy. Br J Dermatol. 2007;156:875–883. 56. Komatsu N, et al. Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients. Exp Dermatol. 2007;16:513–519. 57. Komatsu N, et al. Elevated human tissue kallikrein levels in the stratum corneum and serum of peeling skin syndrome-type B patients suggests an over-desquamation of corneocytes. J Invest Dermatol. 2006;126:2338–2342. 58. Voegeli R, et al. Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme. Int J Cosmet Sci. 2007;29:191–200. 59. Horikoshi T, et al. Role of endogenous cathepsin D-like and chymotrypsin-like proteolysis in human epidermal desquamation. Br J Dermatol. 1999;141:453–459. 60. Wormser U, et al. Noninvasive procedure for in situ determination of skin surface aspartic proteinase activity in animals; implications for human skin. Arch Dermatol Res. 1997;289:686–691. 61. Horikoshi T, et al. Effects of glycolic acid on desquamationregulating proteinases in human stratum corneum. Exp Dermatol. 2005;14:34–40. 62. Fischer H, et al. Stratum corneum-derived caspase-14 is catalytically active. FEBS Lett. 2004;577:446–450. 63. Raymond AA, et al. Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed. Br J Dermatol. 2007;156:420–427. 64. Takada K, et al. Non-invasive study of gelatinases in sun-exposed and unexposed healthy human skin based on measurements in stratum corneum. Arch Dermatol Res. 2006;298:237–242. 65. Beisson F, et al. Use of the tape stripping technique for directly quantifying esterase activities in human stratum corneum. Anal Biochem. 2001;290:179–185. 66. Jin K, et al. Analysis of beta-glucocerebrosidase and ceramidase activities in atopic and aged dry skin. Acta Derm Venereol. 1994;74:337–340. 67. Takagi Y, et al. Beta-glucocerebrosidase activity in mammalian stratum corneum. J Lipid Res. 1999;40:861–869. 68. Mazereeuw-Hautier J, et al. Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping. Br J Dermatol. 2000;142:424–431.
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69. Redoules D, et al. Characterisation and assay of five enzymatic activities in the stratum corneum using tape-strippings. Skin Pharmacol Appl Skin Physiol. 1999;12:182–192. 70. Hirao T. Involvement of transglutaminase in ex vivo maturation of cornified envelopes in the stratum corneum. Int J Cosmet Sci. 2003;25:245–257. 71. Nachat R, et al. Peptidylarginine deiminase isoforms 1–3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin. J Invest Dermatol. 2005;124:384–393. 72. Thiele JJ. Oxidative targets in the stratum corneum. A new basis for antioxidative strategies. Skin Pharmacol Appl Skin Physiol. 2001;14 (Suppl. 1):87–91. 73. Thiele JJ, et al. The antioxidant network of the stratum corneum. Curr Probl Dermatol. 2001;29:26–42. 74. Girard P, et al. A new method for assessing, in vivo in human subjects, the basal or UV-induced peroxidation of the stratum corneum. Application to test the efficacy of free-radical-scavenging products. Curr Probl Dermatol. 1998;26:99–107. 75. Thiele JJ, et al. Macromolecular carbonyls in human stratum corneum: a biomarker for environmental oxidant exposure? FEBS Lett. 1998;422:403–406. 76. Thiele JJ, et al. Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo. J Invest Dermatol. 1999;113: 335–339. 77. Hirao T, Takahashi M. Carbonylation of cornified envelopes in the stratum corneum. FEBS Lett. 2005;579:6870–6874. 78. Sander CS, et al. Photoaging is associated with protein oxidation in human skin in vivo. J Invest Dermatol. 2002;118:618–625. 79. Richert S, et al. Assessment of skin carbonyl content as a noninvasive measure of biological age. Arch Biochem Biophys. 2002;397: 430–432. 80. Fujita H, et al. A simple and non-invasive visualization for assessment of carbonylated protein in the stratum corneum. Skin Res Technol. 2007;13:84–90. 81. Kobayashi Y, et al. Increased carbonyl protein levels in the stratum corneum of the face during winter. Int J Cosmet Sci. 2008;30:35–40. 82. Iwai I, Hirao T. Protein carbonyls damage the water-holding capacity of the stratum corneum. Skin Pharmacol Physiol. 2008;21: 269–273. 83. Iwai I, et al. Change in optical properties of stratum corneum induced by protein carbonylation in vitro. Int J Cosmet Sci. 2008;30:41–46. 84. Thiele JJ, Packer L. Noninvasive measurement of alpha-tocopherol gradients in human stratum corneum by high-performance liquid chromatography analysis of sequential tape strippings. Methods Enzymol. 1999;300:413–419. 85. Weber SU, et al. Vitamin C, uric acid, and glutathione gradients in murine stratum corneum and their susceptibility to ozone exposure. J Invest Dermatol. 1999;113:1128–1132. 86. Hellemans L, et al. Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery. J Invest Dermatol. 2003;120:434–439. 87. Delbridge L, et al. Non-enzymatic glycosylation of keratin from the stratum corneum of the diabetic foot. Br J Dermatol. 1985;112: 547–554. 88. Ma´rova´ I, et al. Non-enzymatic glycation of epidermal proteins of the stratum corneum in diabetic patients. Acta Diabetol. 1995;32: 38–43. 89. Gahring LC, et al. Presence of epidermal-derived thymocyte activating factor/interleukin 1 in normal human stratum corneum. J Clin Invest. 1985;76:1585–1591.
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90. Hirao T, et al. Elevation of interleukin 1 receptor antagonist in the stratum corneum of sun-exposed and ultraviolet B-irradiated human skin. J Invest Dermatol. 1996;106:1102–1107. 91. Terui T, et al. An increased ratio of interleukin-1 receptor antagonist to interleukin-1alpha in inflammatory skin diseases. Exp Dermatol. 1998;7:327–334. 92. Perkins MA, et al. A noninvasive method to assess skin irritation and compromised skin conditions using simple tape adsorption of molecular markers of inflammation. Skin Res Technol. 2001;7:227–237. 93. De Jongh CM, et al. Stratum corneum cytokines and skin irritation response to sodium lauryl sulfate. Contact Dermatitis. 2006; 54:325–333. 94. de Jongh CM, et al. Cytokines at different stratum corneum levels in normal and sodium lauryl sulphate-irritated skin. Skin Res Technol. 2007;13:390–398. 95. de Jongh CM, et al. Polymorphisms in the interleukin-1 gene influence the stratum corneum interleukin-1 alpha concentration in uninvolved skin of patients with chronic irritant contact dermatitis. Contact Dermatitis. 2008;58:263–268.
96. Yamaguchi J, et al. Quantitative analysis of nerve growth factor (NGF) in the atopic dermatitis and psoriasis horny layer and effect of treatment on NGF in atopic dermatitis. J Dermatol Sci. 2009;53:48–54. 97. Asano S, et al. Microanalysis of an antimicrobial peptide, betadefensin-2, in the stratum corneum from patients with atopic dermatitis. Br J Dermatol. 2008;159:97–104. 98. Wong R, et al. Use of RT-PCR and DNA microarrays to characterize RNA recovered by non-invasive tape harvesting of normal and inflamed skin. J Invest Dermatol. 2004;123:159–167. 99. Wong R, et al. Analysis of RNA recovery and gene expression in the epidermis using non-invasive tape stripping. J Dermatol Sci. 2006;44:81–92. 100. Garidel P. Mid-FTIR-microspectoscopy of stratum corneum single cells and stratum corneum tissue. Phys Chem Chem Phys. 2002;4: 5671–5677.
66 Hydration of the Skin Surface Hachiro Tagami
Introduction The skin surface is tightly wrapped by an extremely thin but efficient biological barrier membrane, stratum corneum (SC). Since the SC interferes with the permeation of even small molecules, such as those of water, as a skin barrier, it can protect the underlying hydrated living skin tissue from desiccation in this dry atmosphere on the Earth. It is needless to say that the SC also protects the body from the invasion of various external injurious agents. This skin barrier function depends on the structural uniqueness of the SC, which consists of closely overlapped corneocytes, the flattened dead bodies of fully differentiated epidermal keratinocytes, whose narrow intercellular spaces are tightly packed with unique intercellular lipids, the components of which, called hydroxyceramides, tightly bind to the cornified envelope of each corneocyte. Except for the palmoplantar skin that is covered by more than 50 layers of the corneocytes to withstand a strong physical force such as required to support whole body weight, other bodily regions are covered by SC mostly composed of only 7–15 tightly stacked layers of corneocytes [1]. It is surprising that there exists fully hydrated epidermal tissue just beneath the thin and soft membranous structure of the SC. Then, why does the skin of the elderly tend to become dry and rough in winter?
Dry and Scaly Skin Surface It was found that more than 95% of the normal individuals over 60 years of age who live in Sendai, located in the northern part of Honshu Island, present with dry skin, senile xerosis, on their lower backs and lower legs in winter. Furthermore, about half of them complain of pruritus in such skin [2]. Senile xerosis, however, does not indicate lack of water in the whole skin, because the water content of the living skin tissue is rather high in the elderly [3]. The objectively noted dry skin reflects only a decrease in water content in the superficial portion of the SC. In other words, the SC of young individuals efficiently
plays another important role in keeping the skin surface soft and smooth. Almost 60 years ago, Blank [4] made interesting observations in vitro. He found that the isolated fragments of plantar SC became hard and brittle when dehydrated. Attempts to soften them with petrolatum or olive oil, the emollients conventionally used for the treatment of rough and scaly skin, totally failed. Only after absorption of water did they become soft and flexible; thus water can be regarded as the ultimate moisturizer that improves subjective perception of the mechanical properties of human skin. In contrast to such an in vitro situation, there is always a water supply from the underlying hydrated living tissue in vivo, even in an atmosphere with extremely low relative humidity. However, the SC of the dry skin is not efficient enough to take up water to keep the skin surface soft and supple. The soft and smooth SC such as that found in young skin is rich in water-holding substances, i.e., amino acids produced by proteolysis of filaggrin that takes place during their slow upward movement in the SC, lactate and potassium derived from sweat, and intercellular lipids, especially their major component ceramides that also play a crucial role in providing barrier function to the SC [5–7]. Moreover, in the locations rich in actively secreting sebaceous glands such as the face and scalp, sebum exerts an occlusive effect by itself [8], and glycerol that results from hydrolysis of triglycerides efficiently binds water in the skin surface [9]. With skin aging, deficiencies develop in these waterbinding substances in the superficial portion of the SC [2]. Moreover, there occurs a poor supply of water from the underlying, well-hydrated living tissue, because of an increase in the number of SC cell layers with age, which makes the passage of water through the SC more difficult [1, 2, 10]. Hence, the lesser water supply from the underlying epidermis as well as poor water-holding capacity of the superficial portion of the SC itself make the SC of the aged skin less efficient to bind water, leading to the development of dry skin in winter [2]. In the case of commonly observed scaly dermatoses such as chronic eczema, psoriasis, and ichthyosis, their
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_66, # Springer-Verlag Berlin Heidelberg 2010
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pathologic SC is too deficient in water-binding capacity although the water-barrier deficiency of such SC allows ample water supply from the underlying living tissue [11]. Lack of water in the skin surface produces a dry, rough, or scaly look. Traditionally, various occlusive agents such as petrolatum were applied that slowly exert a softening and smoothing effect on the skin surface by preventing the water loss. Nowadays, various moisturizing agents are available which can directly supply water to the skin surface and quickly change the rough skin surface into a smooth and soft one at least transiently. However, in the case of the treatment of those scaly dermatoses, there is no question about the importance of the medical treatment for the underlying pathologic skin changes. Thus, one of the most important issues in the field of dermatology and cosmetology is the establishment of the effective method to maintain sufficient skin surface hydration. For such a purpose, it is essential to develop a useful methodology to quantitatively measure the skin surface hydration state that enabled the evaluation of the effectiveness of various topical agents.
Assessment of Skin Surface Hydration with Bioengineering Methods From the in vitro observation, it has become clear that a decrease in the water concentration in the superficial layers of the SC is known to cause an observable alteration in the physical characteristics of the skin surface, which is noted objectively as dry and scaly skin [12]. Clinical assessment of such dry skin is done in a crude and qualitative fashion, i.e., by simply observing the skin surface and manually palpating it. However, for the quantitative evaluation, more objective and instrumental measurements are needed. It is not difficult to measure the water content of isolated SC sheets in vitro. Because of the equal distribution of water in such SC samples, it can be expressed as percentage. In contrast, it is difficult to express the amount of water contained in the SC in vivo, because there exists a concentration gradient of water within the SC [12–14] being lowest in the uppermost portion exposed to the dry atmosphere and highest in the lowermost layer facing the viable, moist epidermis. This is due to the fact that the SC is the rate-limiting barrier between the water-saturated viable tissue and the dry outer environment where diffusion of water takes place as a purely passive process through it. As mentioned above, the superficial portion of the SC can remain supple and flexible, as long as its water-holding capacity is intact
as noted in the young healthy skin. In contrast, the skin covered by pathologic SC with poor water-holding capacity such as that found in the skin of aged individuals or in that of various skin diseases presents a cracked, scaly appearance. Despite the urgent demands for the quick and quantitative techniques to assess such hydration state of the exposed portion of the SC, i.e., the skin surface hydration state, there had been a total lack of adequate in vivo methodologies or a practical technique to objectively evaluate the efficacy of moisturizers in vivo until 1980 when a quick and efficient electrical method to measure skin impedance by using high-frequency current was first reported [13]. Thereafter, various commercial instruments have become available. This electric technique can evaluate the water content at poorly defined portions of the skin surface SC. However, by combining with sequential tape-stripping method, an increasing water concentration gradient from the skin surface can be demonstrated, deep into the viable epidermis as shown with later developed different methodology such as electron probe analysis [14] or just recently introduced confocal Raman spectroscopy that can provide precise information not only about the distribution pattern of the water in the SC, but also about the distribution patterns of other functionally important substances in the SC [15]. The confocal Raman spectroscopy can also offer information about the detailed distribution of those substances that are crucial for the barrier as well as hydration of the SC [16].
Electrical Methods for the Evaluation of the Skin Surface At present, because of the handiness and convenience, the most widely employed techniques to evaluate the hydration state of the skin surface are those involving the measurement of skin impedance. Impedance (Z), the total electrical opposition to the flow of an alternating current, depends on two components, resistance (R) and capacitance (C), and their relationship may be formulated as follows: Z ¼ ½R 2 þ ð1=2pfCÞ2 1=2 where f stands for a frequency of an applied alternating current. In old days, many researchers studied the impedance of human skin for reasons of technical simplicity. Tregear [17] speculated that when the skin surface is not deliberately hydrated, the reciprocal of specific impedance should be a measure of the hydration of its surface
Hydration of the Skin Surface
position. However, because of the high impedance of human skin, which is chiefly due to the properties of the SC, measurements of the skin impedance had to employ damp contact using electrode paste between the electrodes and the skin [18]. Such an approach is not only cumbersome, but also has a great influence on the functional properties of the SC. In 1980, by using high-frequency electric current of 3.5 MHz, it was found that it is possible to evaluate the hydration state of the skin surface quickly and quantitatively in a noninvasive way even with dry electrodes in terms of either conductance (¼ 1/R) or capacitance [13]. At that time a circuit developed by Ichijo was used, which enabled the measurement of both conductance and capacitance for a high-frequency current separately. Leveque and de Rigal [19] also reported good sensitivity to the water content of the skin surface with a similar highfrequency instrument developed by them. With such a method, as soon as the probe is placed on the skin, both conductance and capacitance show a rapid initial increase followed by a gradual increase if the contact is maintained. The level of the initial increase represents the hydration state of the skin surface at the time of application of the probe and the later slow increase is due to accumulation of water beneath the probe resulting from transepidermal water loss. Thus, the initial value for the evaluation of the hydration of the skin surface is used.
In Vivo Water Sorption–Desorption Test Information can be easily obtained on the hygroscopic property and water-holding capacity of the SC in a few minutes by conducting an in vivo water sorption–desorption test [20]. This simple test procedure consists of electrical measurements before and after application of a droplet of water on the skin for 10 s to obtain data on the hygroscopic property of the skin surface and later serial measurements at an interval of 30 s for 2 min to evaluate the water-holding capacity. Under usual ambient conditions normal skin surface shows a high rise in conductance just after the application of water, which is followed by a rapid falloff within 30 s, thereafter by gradual return to the prehydration levels by 2 min (> Fig. 66.1). Such an initial increase measured with capacitance is remarkably smaller than that measured with conductance, indicating rather poor sensitivity of capacitance to a well-hydrated condition of the SC (> Fig. 66.2). This was also the case on the skin occluded by a sheet of polyethylene film. None of these parameters were influenced by the water
66
. Figure 66.1 Water sorption–desorption test on normal skin surface and that repeated 3 min after adhesive tape-stripping of the stratum corneum ten times (Reproduced with permission from Tagami, H. et al. [20])
accumulation of the tissue fluids beneath the epidermis, such as demonstrated on the top of a suction blister. This test demonstrates that the superficial SC of normal skin is much less hygroscopic and less capable of holding water than the corresponding deeper portions (> Fig. 66.1) and that scaly skin shows a functional defect in both hygroscopicity and water-holding capacity (> Fig. 66.2) between which the former normalizes much faster than the latter after effective treatment of the skin lesions [20]. These findings also indicate that variations in obtained values are much larger with conductance measurement than those with capacitance measurement especially measured on the normal healthy skin surface. It is particularly clear on the well-hydrated skin such as that under occlusion or at such anatomical locations as the face where the recorded values show much greater differences even between closely located sites with the measurement of conductance than with that of capacitance.
Characteristics Observed in the Measurement of Skin Conductance and that of Capacitance In general, skin conductance and capacitance show a very similar behavior (r ¼ 0.95; P < 0.01) [13]. However, conductance measured on dry scaly lesional skin or even
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. Figure 66.2 In vivo water sorption–desorption test measured with Skicon-200 and Corneometer CM420 (Reproduced with permission from Hashimoto-Kumasaka, K. et al. [22])
that on the plantar skin surface of normal individuals appears to be extremely low as compared to capacitance. Thus, it is not easy to compare the grade of the dry state of scaly lesions with conductance measurements, because the obtained values are mostly near zero. An in vivo simulation model of SC can be constructed to ascertain whether the hydration of the skin surface hydration can actually be evaluated with this electrical method [21]. For this purpose, an intact SC sheet separated from a normal skin edge from surgically obtained skin samples is used. First, an epidermal sheet is obtained, separating it by immersing it in hot water (60 C) for 2 min. Then, the covering SC is separated from the epidermal sheet by trypsin digestion. Such an isolated SC sheet is mounted on more than five overlapping filter papers saturated with phosphate-buffered saline (PBS), which simulates the well-hydrated living skin tissue, placed on a glass slide and all the free edges are sealed to a glass with a removable frame of adhesive vinyl tape. Similar to the SC in vivo, this in vitro simulation model of the SC tightly wraps a pad of fully water-saturated filter paper with its lower surface, while the upper surface directly faces the ambient atmosphere. Thus, there takes place water evaporation through the SC sheet just as in vivo. By placing this model in environments with different relative humidities, it was confirmed that the recorded conductance correlated well with the actual water content of the SC (r ¼ 0.99). In contrast, the correlation coefficient obtained with capacitance measurement was much less than that with conductance, showing r ¼ 0.79 (> Fig. 66.3) [22].
With regard to the electroconductive substances in the SC, it requires soaking of the SC fragment in distilled water for at least 2 weeks for them to be totally depleted, making it unable to measure conductance any more. Reflecting these differences noted in the in vitro as well as in vivo experiments, it was found that conductance measurement is suited for the evaluation of normal skin and that placed under the influence of topical moisturizing agents [21, 22]. In contrast, the measurement of capacitance seems to be more suited for the evaluation of hydration state in dry skin or clinically scaly skin where conductance cannot clearly detect differences among severely, moderately, and mildly scaly portions [22].
The Thickness of the Underlying Electroconductive Medium In the devised simple simulation model of an in vivo SC, a concentration gradient of water exists between the surface and the lowermost portion [23], because it consists of an isolated sheet of SC that tightly occludes the underlying water-saturated filter paper. The underlying watersaturated filter paper, like the living cutaneous tissues in vivo, is the water source of overlying SC and is also the conducting medium that allows the formation of an adequate electric field. Conductance values recorded with only one sheet of underlying filter paper were quite low. With an increase in the number of sheets of paper, the conductance value increased until five sheets of filter paper were in place.
Hydration of the Skin Surface
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. Figure 66.3 Relationship between electrical parameters and water content of the stratum corneum sheet placed in various relative humidities. (a) Water content by weight determination. (b) Conductance measurement. (c) Capacitance measurement (Reproduced with permission from Hashimoto-Kumasaka, K. et al. [22])
At this point the readings reached a plateau. The total thickness of five sheets of filter paper saturated with water was approximately 5 mm. Thus, to obtain an optimal reading, the high-frequency current should extend at least 5 mm into the wet and electrically conductive substances [23]. This condition is always attainable in vivo. A gravimetric determination of water content was perfomed, together with the high-frequency conductometry, in the simulation model of the SC. As a result, it was confirmed that the recorded conductance values correlated well with the actual water content of the SC (r ¼ 0.94). Moreover, by using a model consisting of five overlapped SC sheets simulating that of the palmoplantar SC, it was corroborated that there is a close correlation between the high-frequency conductance values and the water content in the uppermost SC sheet (r ¼ 0.98) [23].
Commercially Available Electrical Instruments There are now many different commercially available instruments for the evaluation of the skin surface hydration which are based on different modes of measurement [24, 25]. Moreover, even cosmetic counters in big stores are now equipped with their own simple instruments,
which are constructed by each cosmetic company based on the impedance principle. Thus, the following are the representative instruments that are routinely used for the experimental or clinical purposes worldwide. Skicon 100, which was manufactured by IBS Ltd., Hamamatsu, Japan, is the initial one among these electrical instruments. There is now a new version, Skicon 200EX, which has built-in computation. The principle is based on conductance method, operating at a single frequency (3.5 MHz). Comparison between the original model 100 and 200EX shows comparable data, although the Skicon-200EX displayed systematically 1.6-fold higher values than the Skicon-100. The two probes measured systematically different levels with the original probe measuring lower values [26]. Corneometer, CM820 and its newer version, CM 825, are manufactured by Courage-Kahzaka Electronic GmbH, Koeln, Germany. A resonating system in the instruments measures the shift in frequency of the oscillating system, which results from the changes in the total capacity of the skin surface. In the case of CM 825, the frequency shifts from 0.95 MHz in a hydrated medium to 1.15 MHz for a dry medium. DermaLab Moisture Unit, manufactured by Cortex Technology, Hadsun, Denmark, is based on impedance measurements operating at single frequency of 100 kHz.
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It is a combination instrument that allows measurements of elasticity, transepidermal water loss, and hydration. Nova Dermal Phase meter, Nova DPM 9003 is manufactured by Nova Technology Corporation, Portsmouth, NW, USA. It measures impedance-based capacitive reactance of the skin at preselected frequencies up to 1 MHz from the observed signal phase delays. MoistureMeter is manufactured by Delfin Technologies Ltd., Kuopio, Finland. Its SC-4 instrument operates at 1.25 MHz with concentric electrodes. D-3 has larger dimensions of the sensor probe, which increases the effective measuring depth from 0.5 to 5 mm.
conditions are greatly influenced by weathering of the skin in the dry and cold winter [28]. An exogenous supply of water on a skin surface results in a remarkable elevation in recorded value. However, this increase is not influenced by the application of either distilled water or a highly concentrated buffer solution; i.e., it is not affected by the presence of other electrolytes in the applied water due to the fact that the skin surface is already rich in various electroconductive substances. As mentioned above, to totally remove such electroconductive substances from a fragment of normal SC, it is necessary to soak it in distilled water for at least 14 days.
Point to Notice Before Conducting In Vivo Measurements
Assessment of the Efficacy of Skin Moisturizers
Because the SC is exposed to the atmosphere, its surface hydration state is greatly influenced by the ambient relative humidity. Thus, covered areas such as the trunk show much higher values than the exposed areas in the dry winter season due to the effect of thick, airtight clothes, when the measurement is performed just after removal of the clothes. At least 15 min should be allowed for the skin surface to adapt to the atmosphere. Measured values sometimes vary greatly even between sites only slightly apart from each other, particularly when the probe is applied to the sites rich in sweat glands such as the palmar surface, axilla, and in some individuals even the forehead. Generally, the highest values are found on the face and the lowest values on the distal portion of the limbs on the body surface [27]. The measurements on the skin areas such as the palmar skin for comparative study should be done carefully, because these areas are always under the influence of mental sweating. The comparison made between summer and winter in the same subjects at an out-patient clinic with 21–26 C room temperature showed that high environmental relative humidity of the summer and possibly invisible sweating induced higher conductance values in the summer time [28]. Therefore, the measurements should ideally be conducted in the identical environmental conditions, i.e., by using a climate-controlled chamber. Such a study performed in subjects consisting of different age groups using a climate-controlled chamber maintained at 21 C and 50% relative humidity, definitely demonstrated that the skin surface hydration state is significantly lower in winter than in summer whether on the exposed area, the cheek, or on the covered area of the flexor surface of the forearm. These data indicate that the skin surface
In the dry and cold winter, even normal healthy skin surface becomes much smoother and softer immediately after the application of moisturizing agents. This change can be shown as an increase in conductance depending on the efficacy of the agents. This is followed by a rapid decrease due to evaporation of excess water from the skin surface [29]. Thereafter, the obtained values are maintained at certain increased levels, for several hours, according to the efficacy of the agents, if undisturbed. In contrast, no initial increase is observed after the application of emollients such as petrolatum that does not contain water. However, there is a gradual increase in conductance until reaching a plateau after 2 h due to accumulation of water beneath it. To obtain reproducible results 20 mL of the agent is applied in a 4 4 cm2 skin area. Repeated daily applications of moisturizers for several days induce an increase in conductance or capacitance that is demonstrable even several days after the cessation of treatment depending on the efficacy of the moisturizers [30]. Moreover, such repeated applications of a moisturizer not only increase the hydration state of the skin surface, but also improve mildly impaired barrier function, as noted in atopic xerosis induced by the dry and cold winter air [31].
Conclusion Various electrical methods are now available to evaluate the skin surface hydration. Based on the studies conduced in vitro, the high-frequency electrical method is employed to measure quantitatively the skin surface hydration state
Hydration of the Skin Surface
to estimate the impairment in the water-holding capacity of pathologic SC of various skin lesions, or to evaluate the moisturizing effects of various topical agents. The measurement of high-frequency conductance is much more sensitive to the changes in hydration state of the superficial portions of the SC than that of capacitance, but the variations of the obtained values are much greater with the former than the latter reflecting the sensitivity of measurement. For the measurement of dry scaly lesions, the measurement of capacitance appears to be more sensitive to detect differences of severity than that of conductance.
Cross-references > Transepidermal
Water Loss and Aging
References 1. Ya-Xian Z, Suetake T, Tagami H. Number of cell layers of the stratum corneum in normal skin – relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291:555–559. 2. Hara M, Kikuchi K, Watanabe M, Denda M, Koyama J, Nomura J, Horii I, Tagami H. Senile xerosis: functional, morphological, and biochemical studies. J Geriatr Dermatol. 1993;1:111–120. 3. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–46. 4. Blank IH. Factors which influence the water content of the stratum corneum. J Invest Dermatol. 1952;18:433–440. 5. Horii I, Nakayama Y, Obata M, Tagami H. Stratum corneum hydration and amino acid content in xerotic skin. Br J Dermatol. 1989;121:587–592. 6. Nakagawa N, Sakai S, Matsumoto M, Yamada K, Nagano M, Yuki T, Sumida Y, Uchiwa H. Relationship between NMF (lactate and potassium) content and the physical properties of the stratum corneum in healthy subjects. J Invest Dermatol. 2004;122:755–763. 7. Imokawa G, Akasaki S, Hattori M, Yoshizuki N. Selective recovery of deranged water-holding properties by stratum corneum lipids. J Invest Dermatol. 1986;87:758–761. 8. O’goshi K, Iguchi M, Tagami H. Functional analysis of the stratum corneum of scalp skin: studies in patients with alopecia areata and androgenetic alopecia. Arch Dermatol Res. 2000;292:605–611. 9. Fluhr JW, Mao-Qiang M, Brown BE, Wertz PW, Crumrine D, Sundberg JP, Feingold KR, Elias PM. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice. J Invest Dermatol. 2003;120:728–737. 10. Kobayashi H, Tagami H. Distinct locational differences observable in biophysical functions of the facial skin: with special emphasis on the poor functional properties of the stratum corneum of the perioral region. Int J Cosmet Sci. 2004;26:91–101. 11. Tagami H, Yoshikuni K. Interrelationship between water barrier and reservoir functions of pathologic stratum corneum. Arch Dermatol. 1985;181:642–645.
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12. Blank IH, Moleney J, Emslie A, Simon I, Apt C. Diffusion of water across the stratum corneum as a function of its water content. J Invest Dermatol. 1984;82:188–194. 13. Tagami H, Ohi M, Iwatsuki K, Kanamaru Y, Yamada M, Ichijo B. Evaluation of the skin surface hydration in vivo by electrical measurement. J Invest Dermatol. 1980;75:500–507. 14. Warner RR, Myers MC, Taylor DA. Electron probe analysis of human skin: determination of the water concentration profile. J Invest Dermatol. 1988;90:218–224. 15. Egawa M, Hirao T, Takahashi M. In vivo estimation of stratum corneum thickness from water concentration profiles obtained with Raman spectroscopy. Acta Derm Venereol (Stockh). 2007;87:4–8. 16. Egawa M, Tagami H. Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration. Br J Dermatol. 2007;158:251–260. 17. Tregear RT. The interpretation of skin impedance measurements. Nature. 1965;205:600–601. 18. Clar EP, Her CP, Sturelle CG. Skin impedance and moisturization. J Cosmet Chem. 1973;26:337–353. 19. Leveque JL, de Rigal J. Impedance methods for studying skin moisturisation. J Soc Cosmet Chem. 1983;34:419–428. 20. Tagami H, Kanamaru Y, Inoue K, Suehisa S, Inoue F, Iwatsuki K, Yoshikuni K, Yamada M. Water sorption-desorption test of the skin in vivo for functional assessment of the stratum corneum. J Invest Dermatol. 1982;78:425–428. 21. Obata M, Tagami H. Electrical determination of water content and concentration profile in a simulation model of in vivo stratum corneum. J Invest Dermatol. 1989;92:854–859. 22. Hashimoto-Kumasaka K, Takahashi K, Tagami H. Electrical measurement of water content of the stratum corneum in vivo and in vitro under various conditions: comparison between skin surface hygrometer and corneometer in evaluation of the skin surface hydration state. Acta Derm Venereol. 1993;73:335–339. 23. Blichman CW, Serup J. Assessment of skin moisture. Measurement of electrical conductance, capacitance and transepidermal water loss. Acta Derm Venereol (Stockh). 1988;68:284–290. 24. Barrel AO, Clarys P. Measurement of epidermal capacitance. In: Serup J, Jemec GBE, Grove GL (eds) Handbook of Noninvasive Methods and the Skin, 2nd ed. Boca Raton: Taylor & Francis, 2006, pp. 337–344. 25. Gabard B, Clarys P, Barrel AO. Comparison of commercial electrical measurement instruments for assessing the hydration state of the stratum corneum. In: Serup J, Jemec GBE, Grove GL (ed) Handbook of noninvasive methods and the skin, 2nd ed. Boca Raton: Taylor & Francis, 2006, pp. 351–358. 26. O’goshi K, Serup J. Skin conductance; validation of Skicon-200EX compared to the original model, Skicon-100. Skin Res Technol. 2007;13:13–18. 27. O’goshi K, Okada M, Iguchi M, Tagami H. The predilection sites for chronic atopic dermatitis do not show any special functional uniqueness of the stratum corneum. Exog Dermatol. 2002;1:195–202. 28. Kikuchi K, Kobayashi H, le Fur I, Tschachler E, Tagami H. The winter season affects more severely the facial skin than the forearm skin: comparative biophysical studies conducted in the same Japanese females in later summer and winter. Exog Dermatol. 2002;1:32–38.
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29. Tagami H. Impedance measurement for evaluation of the hydration state of the skin surface. In: Leveque J-L (ed) Cutaneous Investigation in Health and Disease. Noninvasive Methods and Instrumentation. New York: Marcel Dekker, 1989, pp. 79–111. 30. Tabata N, O’Goshi K, Zhen YX, Kligman AM, Tagami H. Biophysical assessment of persistent effects of moisturizers after their daily applications: evaluation of corneotherapy. Dermatology. 2000;200:308–313.
31. Kikuchi K, Tagami H. Japanese Cosmetic Scientist Task Force for Skin Care of Atopic Dermatitis: noninvasive biophysical assessments of the efficacy of a moisturizing cosmetic cream base for patients with atopic dermatitis during different seasons. Br J Dermatol. 2008;158:969–978.
71 Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy Jonathan M. Crowther . Paul J. Matts
Introduction The uppermost layer of skin – the stratum corneum (SC) – plays a vital role in the functioning and protection of the human body. It provides mechanical protection and regulates water movement in and out. Despite the relatively small dimensions of the SC over most of the body parts (its thickness is of the order of 20 mm over a large portion of the body), it is far from a homogeneous structure both physically and chemically. Chemical concentrations change from its surface inwards, and these changes are responsible for both the properties it possess and the processes occurring within it. Furthermore, with the application of topical cosmetic products becoming more popular and widespread, especially in the anti-aging market, the monitoring of ingredients that are capable of penetrating into the skin from the outside, which may also influence the processes occurring within and properties of the SC, is now a necessity. To understand the role of all these components within the SC, it is not only necessary to ask ‘‘how much is there?,’’ but also ‘‘where is it located?’’ and ‘‘how is it distributed?’’ While many techniques have been developed to analyze concentration gradients within the SC, until recently, it has been impossible to quantitatively assess different chemical components as a function of depth, in vivo. The aim of this chapter is to present a brief review of research on understanding the role these concentration gradients play within the SC and provide an overview of the use of a relatively new technique, confocal Raman spectroscopy (CRS), for assessing these concentration gradients in vivo. Following this are some of the recent findings demonstrating how water and NMF profiles change throughout the skin using CRS and how this can help bring new understanding.
Measurement of Different Skin Components The SC itself is not a homogeneous structure and varies greatly across its thickness. From the basal layer, where keratinocytes are born, they mature and differentiate as they transit toward the surface, gradually flattening out to become the familiar flat, ‘‘squamous’’ corneocyte cells of the stratum corneum. Differentiating cells will cause transition through a variety of chemical gradients, including water, natural moisturizing factors (NMF), lipids, urea, lactic acid, and pH. This section outlines some of these aspects, why they are present, and how they are currently assessed.
Water The water content of the SC varies across its thickness – at the surface, the SC is constantly losing water to the environment under normal conditions, while at the basal layer, there is a continual replenishment from the viable epidermis. Therefore, across the SC, there exists a water gradient, which decreases toward the outside of the body. Maintenance of a correct state of hydration of the SC has a huge impact on its mechanical and optical properties, helping to maintain skin barrier function and playing an important role in the regulation and activation of both intra and extracellular enzymes, which control the desquamation process [1, 2]. Deviations in these processes fundamentally affect SC barrier function, and in healthy individuals, the most common expression of this is ‘‘dry skin’’ [3]. Water that is present within the SC can be
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_71, # Springer-Verlag Berlin Heidelberg 2010
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described as either free or bound [4]. Free water refers to the partially mobile molecules, which can be easily lost if exposed to a dry environment after exposure of skin to an environment of high water activity. Bound water is held within the corneocytes by both the polar groups within keratin protein molecules and by a blend of so-called natural moisturizing factors (NMF) that increase the hygroscopicity of these structures. The first attempt at determining true depth-resolved water profiling in SC was performed by Warner et al. in 1988 [5]. In this experiment, electron probe analysis was carried out on biopsied skin samples, which had been cryo-sectioned and freeze-dried (the local dry mass of a freeze-dried cryo-section of skin being inversely related to its water content). Further advancements were made to the technique, making it simpler to perform [6]; however, while the technique was able to provide hydration profiles, it still required skin biopsies to be collected, cryo-sectioning to be carried out, and analysis to be performed using a scanning electron microscope (SEM) and, as such, it is not a technique that would be possible to deploy easily in a clinical environment. Cryo-SEM has been used further to understand uptake and loss of water in salt solutions of different strengths within the SC [7]. This demonstrated that the SC does not take up water evenly across its entire thickness but that there are three ‘‘zones,’’ which respond differently when hydrated and dehydrated. The behavior of these zones is dependent on the osmotic potential of the hydrating solution. The concept of zones of hydration has also been examined before [8] showing that the central portion of the SC absorbed water strongly under high water activity, while the layers closest to the stratum granulosum showed no swelling under these conditions. The presence of a central zone capable of absorbing and holding water is in excellent agreement with the concentration of natural moisturizing factors (NMF), known to reach a maximum in the central portion of the SC [9]. Infrared (IR) spectroscopy has been used to examine water as a function of depth in combination with tape stripping [10], and Monte Carlo simulation [11]. However, these methods require compromises to be taken in collection and analysis of the data. For example, in the work by Brancaleon et al., the tape stripping approach used to sample incrementally into the skin was difficult to correlate with actual depth and, of course, was inherently destructive, thereby making it impossible to repeatedly reassess the same site over the course of a study. The Monte Carlo simulation by Arimoto et al. relies on the assumption that the water content varies linearly from 10% at the SC surface to 80% at the interface with the viable epidermis.
NMF and SC Lipids Two key classes of materials are present within the SC in addition to the corneocyte cellular structure – ‘‘natural moisturizing factors’’ (NMF) and the intercellular lipid bilayer structure. NMF comprise a collection of amino acids, salts, and other small hygroscopic molecules that are present within the corneocytes and are derived from the proteolysis of epidermal fillagrin, which initiated a few cell layers above the stratum basale. These hygroscopic NMF components are efficient humectants, helping to bind water and assisting in maintaining skin hydration and flexibility [12]. Confocal Raman spectroscopy has also been used to measure concentration profiles of different NMF components nondestructively and in vivo [13]. The concentration of most NMF components builds gradually from the stratum granulosum, peaking in the midportion of the SC and then showing a characteristic depletion near the surface. This seems to be associated with the water-labile nature of these components and their propensity to be washed out by, for example, daily cleansing. Lactate and urea, as sweatderived NMF components, are more prevalent at the surface of the SC. The ability of the SC to control the movement of molecules across it is mediated not only by the physical constraint of the corneocytes themselves, but also by the intercellular lipids. These are a mixture of ceramides, cholesterol, and free fatty acids, as well as a small amount of nonpolar liquids and cholesterol sulfates, and are organized along with a small amount of water, into a series of parallel lamellar membranes. However, while some water is trapped in the lipid lamellar structure, most is actually held within the corneocytes themselves [14]. It is this series of lipid bilayer structures, together with the tightly stacked corneocytes, which provide a tight barrier against TEWL, making it difficult for water to transfer across the SC structure. SC lipid content varies both seasonally and as a function of age, and it is generally accepted now that a reduction of approximately 30% is seen in the elderly [15–17].
Cosmetic Ingredients With the rapid expansion of the cosmetic product market in recent years, more attention is being focused on understanding how these products partition into and interact with the skin. Infrared (IR) spectroscopy has been used to monitor the effects of cosmetic ingredients on SC chemical
Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
composition [18] and, although this technique is capable of detecting changes in lipid packing and organization as a result of using different products, it is not a depthprofiling technique and it is not certain over which depth data is collected. Imhof et al. have used a modified version of IR spectroscopy to assess the delivery of topical components to the skin based on the principle of thermal emission delay from the surface after irradiation with an IR light source [19]. Confocal Raman spectroscopy has been used to monitor penetration of different lipid species into the SC and their corresponding effects on hydration and total skin lipid profiles [20]. Different lipid species were absorbed to different degrees, with petrolatum being most strongly absorbed, most likely due to a combination of its relatively short chain lengths and occlusive properties, resulting in destabilization of SC structure. They also demonstrated that infant and adult skin behaved similarly in relation to lipid uptake. This is in contradiction to the effect of topical application of water, where infant skin was more capable of uptake than adult skin. Chrit et al. have also used confocal Raman spectroscopy to assess the extent of skin hydration changes after using a glycerol-based moisturizing product in vivo [21] and were able to classify different hydrating products depending on their moisturizing effect on the skin. They showed that a polyphospholipid (poly [2-methacryoyloxylphosphorylcholine or pMPC]) was able to increase water levels in the skin, both in vivo and in vitro, although it should be noted that dosing of the products was not controlled, with any excess product being removed after application and before analysis [22].
pH While the extracellular fluid within the body is maintained at a pH of approximately 7.4, the surface of the SC is more acidic with a pH between 4.5 and 6 [23, 24]. There is, therefore, a pH gradient across the SC. While this change in pH has been linked with a variety of skin functions, such as barrier function, desquamation, and microbial defense, the mechanism responsible for its presence is not fully understood. However, it has been suggested that a buildup of trans-urocanic acid [25], carbon dioxide diffusion [26], the presence of free fatty acids [27], lactate and lactic acid produced as a by-product of sweating [28], or active regulation by a sodium–hydrogen anti-porter protein [29] are linked with this observed change. Even though humans are not born with this ‘‘acidic mantle,’’ the SC pH at the surface is nearly neutral. Over the first few months of life, the surface becomes more
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acidic to approach the values seen in adults [30]. This can have significant consequences for newborns as barrier function recovery after acute insult is not as efficient in neutral skin, when compared with acidic skin [31]. It is interesting to note that acidification of neonatal rat SC does not occur from the surface down (so is unlikely to be triggered by microbial colonization occurring after birth) but actually begins deeper down within the skin [32]. A variety of techniques have been used to examine pH as a function of depth. Tape stripping measurements combined with pH assessment has shown the existence of this gradient [24, 30]. Once again, though, it is by nature an inherently destructive technique, and is not capable of discriminating between the intra and intercellular components. Microscopy combined with a pH-sensitive fluorescent marker molecule has been used to determine pH [33], and the advent of two photon and confocal imaging has pushed resolution to submicron levels [34]. Confocal Raman spectroscopy has also been used to measure concentration profiles of trans-urocanic acid and pyrollidone carboxylic acid in vivo [13]. As with NMF, the concentration of these species is greatest in the middle portion of the SC, showing a gradual buildup from the stratum granulosum layers, and depletion near the surface.
Calcium Calcium concentration varies across the epidermis, from high levels within the stratum granulosum down to low levels in the basal layer [35]. The calcium concentration is linked with regulation of epidermal keratinocyte proliferation and differentiation and skin structural integrity. It is also strongly linked with rate of barrier recovery after acute insult by detergents, tape stripping, or organic solvents [36]. As with the pH gradient, calcium variation does not exist at birth – it manifests itself concurrently with increasing barrier function in fetal skin [37]. Analysis of calcium concentration as a function of depth has been carried out using scanning electron microscopy of biopsied samples [38]. With quantification of any component in the skin as a function of depth, the ideal solution is determination of concentration in a nondestructive manner, without the need for complex modeling.
Raman Spectroscopy In 1928, the Indian physicist C.V. Raman first reported the new type of light scattering phenomenon that was
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eventually to bear his name. Raman reported that when a liquid was irradiated by light of a specific wavelength, while most of the remitted photons were scattered elastically with no change in photon energy, an extremely small proportion of the reflected light had a wavelength different to the incident source. This wavelength shift was related directly to the change in vibrational and rotational energy states of the molecules in the liquid, thereby providing information on the energy levels of the molecules present. As the Raman phenomenon is very weak (occurring approximately once in a million photon interactions), requiring a well-defined monochromatic light source, practical applications were not readily exploited until the development of the laser in the 1960s. Improvements in detection equipment such as photomultiplier tubes further enhanced the appeal and applicability of Raman, and it is now a well-established material analysis technique. Raman spectroscopy is a complimentary technique to IR spectroscopy – molecular vibrations, which are IR active are not Raman active and vice versa. However, unlike IR spectroscopy, Raman is relatively insensitive to water, which makes it a more considered approach for analyzing skin (where with IR the intensity of the water signal can mask other chemical species) – and, indeed, a number of researchers in the last 15 years or so have reported varying degrees of success, principally using in vitro models [39–41]. The journey from measurement of in vitro systems to in vivo capture of Raman spectra on the surface of the SC presented many challenges [39, 42], specifically, the low signal to noise (based around the safety needs for relatively low laser power when used on live subjects). A huge leap forward in the use of in vivo Raman spectroscopy came with the instrument designs and research of Caspers et al. resulting in very high optical efficiency and enabling rapid, noninvasive collection of Raman spectra [43].
Confocal In Vivo Raman Microspectroscopy The ‘‘Holy Grail’’ of in vivo SC assessment is the measurement of different components within the skin as a function of depth – a joint measure of chemical composition and location within the skin structure. Recently, confocal Raman spectroscopy (CRS) has been developed to obtain real-time molecular concentration profiles and in vivo [13, 44–46]. Building on the success of their in vivo surface measurements, River Diagnostics designed and marketed the RD3100 in vivo confocal Raman spectrometer, capable of measuring skin chemical profiles by combining the principle of confocal microscopy with Raman
spectroscopy. In operation, incident monochromatic laser light is focused to a point on/within the skin tissue, which can be moved by changing the focal point of the microscope. When used to measure skin, this light enters the SC, most being scattered elastically without a change in energy and wavelength. A small proportion of this incident light, however, becomes Raman-scattered photons. Those scattered photons reaching the skins surface are re-emitted with some passing back through the microscope objective lens. Given the confocal nature of the microscope, only light re-entering the microscope from the focal plane will pass back through the pinhole – photons reemerging from other depths are excluded. The majority of these collected photons will be elastically scattered and have the same wavelength as the incident light source; however, the small proportion of Ramanscattered photons are isolated and analyzed, enabling the construction of molecular concentration profiles present within the SC. Using this technique, SC composition can be quantitatively measured by ‘‘optically sectioning’’ skin tissue and expressing the relative chemical content as a function of depth, in a noninvasive, rapid, and nondestructive manner, making it ideal for implementation in a clinical environment. For example, water concentration is calculated from the ratio of the water signal to the combined signal from water and protein within the skin. This method has also been used to estimate differences in SC thickness in vivo at different body sites and during aging [47, 48] and, recently, to evaluate the effects of water and moisturizing ingredients on SC hydration, after short-term treatment [49, 50], and long-term treatment [51]. The majority of current research, using Raman to look at the skin, focuses on the SC and upper layers of the viable epidermis, as light becomes increasingly scattered as it penetrates more deeply, reducing signal strength and making data collection more difficult. However, despite this turbidity, Naito et al. have recently reported using a 1,064 nm light source to probe the dermal chemical structure [52]. The ability to measure deep within the skin opens up the possibility of, for example, probing the development of acne in teenagers, and the effects of aging on dermal chemical composition. All the chemical components of the skin possess different groups with unique vibrational frequencies. Water and protein contain different functional groups in their chemical structure, which vibrate at different frequencies. It is these differences in vibrational frequency that enable the species to be differentiated using Raman spectroscopy. To simplify the analysis of skin, the Raman spectra can be split into two distinct zones: the ‘‘high-wavenumber’’ and
Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
‘‘fingerprint’’ regions, which can be probed independently depending on what is required. Information regarding water content is contained within the ‘‘high-wavenumber’’ region, while levels of natural moisturizing factors (NMF), cholesterol, ingredients penetrating into the skin etc. can be derived from the information in the ‘‘fingerprint’’ spectra. The high-wavenumber spectrum shows characteristic O–H and –CH3 stretching vibrations at 3,390 and 2,935 cm1, respectively. Scans in this region are used to calculate percentage hydration values by taking the ratio of the integrated signals of water (i.e., the O–H stretching vibration region between 3,350 and 3,550 cm1) to that of protein (i.e., the –CH3 stretching vibration from 2,910 to 2,965 cm1) [43–46]. A typical skin spectra for the ‘‘high-wavenumber’’ spectral region showing the water and protein peaks, collected at a single scan at an exposure time of 1 s are given in > Fig. 71.1. During collection of a depth profile, spectra like this are captured at regular intervals from the surface of the SC down into the skin. A correction factor, as determined by Caspers et al. [13] is used to normalize the spectral response of water and protein relative to their mass ratio. Percent hydration is then calculated using the formula (> Eq. 71.1): Percent hydration ¼ constant ðwaterÞ=½ðwaterÞ þ ðproteinÞ ð71:1Þ The normalized water-to-protein ratios obtained from each focal depth are plotted as percent hydration as a function of depth. This, therefore, leads to a direct semiquantitative
. Figure 71.1 Typical background-corrected confocal Raman spectra from a single scan of human skin in vivo, collected at 4 mm depth in the high-wavenumber region (671-nm laser excitation, exposure 1 s)
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measure of amount of the water present within the skin as a function of depth. Although the signal intensity drops as the laser penetrates deeper into the skin, the fact that both the water and the protein peaks are derived from the same scan enable quantification throughout the range of analysis.
Calculation of SC Thickness It has been understood for many years that SC thickness varies dynamically with its hydration state and in response to a variety of extrinsic factors. In order to analyze and interpret SC water concentration profiles properly, therefore, it is essential to take into account SC thickness – after all, in a situation where SC thickness is varying dynamically, absolute SC depths are of little meaning. It has recently been demonstrated by the authors that SC thickness can be determined directly from water concentration profiles [51], and this procedure is described below. While the work of Egawa et al. [47, 48] clearly demonstrates the utility of using CRS for deriving SC thickness estimates, these authors offer no formal validation data, establishing a direct relationship between their CRS-derived SC thickness data and values derived from other measurements. It should also be noted that a different (and, it is believed, a more rigorous) approach in calculating SC thickness from the water profile measurements have been used, validating it via correlation with values from a known objective measure of skin thickness (optical coherence tomography [OCT]). To generate representative Raman hydration profile data for a particular site, multiple, replicate water concentration profiles are derived for each location at each time point. Multiple profiles are collected as this technique is a point measure – a laser spot size in the order of microns – and the inherent biological variability of the skin means that a more accurate measure of thickness and hydration can only be obtained through the use of an average profile. After collection of a set of profiles, obvious outliers (arising, for example, from scanning through heterogeneous structures, such as skin appendages including hair follicles, sebaceous glands etc., or profiles recorded while the panelists were moving) are removed. Then, an average hydration profile is fitted through the remaining data, using a customized algorithm based on a four-parameter Weibull curve. The four-parameter Weibull curve is a well-accepted and widely used algorithm capable of accurately modeling a variety of profile shapes with the minimum number of parameters in the equation, producing model curves with very low RMS deviations from the mean data. The upper ‘‘leveling-off-point’’ of each
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Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
profile is determined by a gradient threshold method by calculating the location where the gradient reaches a value of 0.5 moving from the midpoint of the curve (> Fig. 71.2). This point was hypothesized to be the theoretical boundary of the SC (see CRS–OCT comparison below for the test of this hypothesis) and serves as the deeper limit of the SC hydration profile. The area-under-the curve values (AUC) are determined by integrating each hydration profile from the skin surface (x = 0 mm on the profile) to each individual SC boundary (point c in > Fig. 71.2), and used to express the total SC hydration.
Correlation Between NMF and Hydration Profiles Using the River Diagnostics RD3100 system, it has been possible to measure hydration and NMF profiles at exactly the same location on the skin, by retaining the volar forearm on the window of the spectrometer and alternating the laser being used from 681 to 785 nm, thereby switching between the ‘‘high-wavenumber’’ and ‘‘fingerprint’’ regions. As can be seen in the example shown in > Fig. 71.3, there is a strong correlation between the leveling-off point location of the hydration profile and the position where the NMF profile starts to rise (profiles presented here are single scans from a specific location on the skin, and that is why they are not smooth curves). This is to be expected, as NMF starts to be expressed a few microns above the base of the SC due to the breakdown of . Figure 71.2 Calculation of SC thickness using the hydration curve. Working from the middle of the curve (a) inward (i.e., deeper in the tissue), the algorithm calculates the point where the gradient equals 0.5 (b). The depth at this point corresponds to the base of the SC (c)
fillagrin, and this correlates well with the behavior of NMF reported by Caspers [13]. While these observations provide increased confidence in the correlation between the leveling-off point in the hydration profile and the location of the lower margin of the SC, it cannot be seen as definitive proof. For example, external environmental variation can also be responsible for changes in the exact location of filaggrin hydrolysis. It was necessary, therefore, to validate empirically the leveling-off point as the SC lower margin, using a separate objective measure of SC thickness.
Correlation Between Optical Coherence Tomography and SC Hydration Profiles Optical coherence tomography (OCT) is a well-established technique for examining skin structure and thickness [53]. It is based on the principle that photons are backscattered from different structures within the skin. Through the use of interferometry, the depth at which these backscattering events occur can be calculated, providing information on where different structures occur within the skin, for example, boundaries between different layers. In this work, sets of up to eight Raman profiles have been measured from each site and these are analyzed together, rather than studying each water profile in isolation. Outlying scans were removed in a quality control process before further analysis. Once this was done, the Weibull mathematical model was applied to the data point cloud representing each site, resulting in an ‘‘average’’ hydration profile for that site. The location of the SC boundary as a function of the CRS water concentration profiles was confirmed by comparing SC thicknesses from a number of different body sites obtained directly by OCT and CRS (> Figs. 71.4a, b). Linear regression through . Figure 71.3 Comparison of hydration and NMF profiles measured at the same point on the volar forearm
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. Figure 71.4 (a) Comparison of OCT- and CRS-derived SC thicknesses at a variety of body sites. (b) Comparison of OCT- and CRS-derived SC thicknesses on volar forearm, cheek, and outside of lower leg
the data shows a strong positive correlation between SC thickness derived from CRS and OCT (OCT thickness = 0.9603 CRS thickness, r2 = 0.9339; p < 0.0001). Expanding the area to the lower left of > Fig. 71.4a, corresponding to the thinner skin sites of the body (volar forearm, cheek, and outside of lower leg), shows how the dynamic range for OCT is compressed in this region (> Fig. 71.4b). It can be seen that all of the OCTderived SC thicknesses are between 9 and 15 mm, while the CRS-derived thicknesses vary between 12 and 30 mm. This is consistent with the expected behavior of the OCTmethod in areas where the SC is relatively thin – the sensitivity of the OCT is limited by the pixel size of the detector (approximately 5 mm for the system used here). For sites with SC thickness in the region of 10–20 mm, therefore, this corresponds to only a few pixels. For panelists who had cheek, forearm, and leg measures, CRS ranked the sites, in terms of SC thickness, as follows: cheek < forearm < leg (cheek 12.8 0.9 mm, volar forearm 18.0 3.9 mm, and leg 22.0 6.9 mm), whereas OCT gave very similar readings for these three different locations (cheek 11.1 1.8 mm, volar forearm 10.4 0.9 mm, and leg 13.7 1.4 mm). Of note, this ability to rank the sites in the order of thickness gave further confidence that the new CRS method was giving accurate estimates of SC thickness, as it matched exactly with the trends that would be expected, based on known, published values for these sites [54]. The limitations of OCT measurement of thinner skin sites has also been noted recently using in vivo laser scanning fluorescence microscopy [55, 56]. It is believed, therefore, that the results of this work demonstrate convincingly the capability of CRS in providing a new rapid, accurate, and sensitive means of measuring SC thickness in vivo.
Effects of Acute Hydration on SC Water Content and Thickness A simple study employing forced occlusion to drive maximal short-term acute hydration of the volar forearm was used to demonstrate the ability of the CRS system to measure dynamic, rapid change in SC water profiles. A set of hydration profiles were taken from the volar forearm after equilibration in a standardized environment (> Fig. 71.5a). The forearm was then wrapped in a wet towel soaked in deionized water. The towel was wrapped in Parafilm™ to help ensure complete saturation of the SC by occlusive hydration, and the arm left for 90 min. After 90 min, the wrap and towel were removed and any excess surface water removed by gentle patting with a dry towel. Sets of hydration profiles were then measured again using CRS over a time-course (> Fig. 71.5b). Given the complex nature of the shape of the hydration curve after this extreme treatment, the profiles here are represented as simple averages of the individual sets of scans rather than Weibull curve-modeled fits. Between measurements, the arm was removed from the CRS optical window and allowed to acclimatize within the measurement room, and the window of the CRS cleaned with methanol to remove any residue left behind from the skin. The hydration profiles in > Fig. 71.5b show the changes in SC hydration across its entire thickness, showing significant water uptake over the 90 min. Importantly, the point at which the hydration profile begins to level off after enforced hydration is further from the surface of the SC. From the OCT validation study described above, it can now be confidently said that this is because of SC swelling in the vertical axis, driven by hydration. As observation only, it is interesting to note that the magnitude of this swelling is in
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Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
. Figure 71.5 (a) Baseline volar forearm hydration profile. (b) Hydration profiles of the volar forearm measured as a function of time after 90 min occlusion with a wet towel
the region of 25%, highly consistent with that noted by Norlen [57] in ex vivo models. It is also interesting that there appears to be a central portion of the SC, which takes up more water than the upper or lower margins. As the post-occlusion time-course is followed and hydration profiles are measured over time, it should also be noted that surface hydration values fall fastest, while the ‘‘hump’’ of hydration in the central portion of the SC falls the slowest. These observations are wholly consistent with presence of higher concentrations of hygroscopic NMF components in the central portion of the SC (the lower layers containing less because of the programmed hydrolysis of filaggrin and the upper layers containing less because of an insidious wash-out of these highly waterlabile components by, e.g., daily cleansing). It is also possible that the corneocytes within the central portion of the SC are less physically constrained compared with those closer to the SC-stratum granulosum boundary and, therefore, are potentially more readily capable of swelling and increasing in thickness than those deeper down. This variance in swelling ability of the SC as a function of depth correlates with the work of Bouwstra et al. [58]. Remarkably, baseline conditions are only reestablished after a period of some 4 h, demonstrating the efficient water-binding capacity of native, untreated SC. Further in vitro validation of CRS has been reported by Wu and Polefka, where they correlated water content as measured using CRS with Karl Fischer assessment, and water content increase for a moisturizing lotion, and decrease in water content after using bar soap [59]. While SC thickness changes as a result of the treatment regimes were not taken into account, and the experiments
were carried out on excised pig skin, this does demonstrate further the capability of the technique.
Effect of Long-Term Application of Moisturizers on SC Hydration Profiles It might be expected that long-term application of moisturizers to the skin would increase SC water content and/or change the shape of the SC hydration profile. A comparison of the effect of long-term application of three moisturizers on SC hydration gradients has recently been reported by Crowther et al. [51]. To examine the effects of moisturizers on SC thickness, water gradients and total SC hydration CRS were used to compare the effects of a formulation containing niacinamide (A), which is known to improve SC barrier function and desquamation better than two other commercially available moisturizers (formulations B and C) [60]. For illustration, average hydration profiles from each treatment from this work are given in > Fig. 71.6. All hydration profiles start at 20–30% hydration at 0 mm depth (i.e., the SC surface) and rise in a ‘‘sigmoidal’’ type curve to 65–70% hydration, where they plateau. While all hydration profiles at baseline and 1-day treatment show the same shape, differences in shape begin to appear after 1 week of treatment. After 2 weeks, notable differences are observed for formulation A, where a laterally ‘‘stretched’’ profile is evident, which is still present after 1 week of regression. As a result of this stretching, the leveling-off point of the profile has moved deeper in the skin (which indicates an increase in SC thickness).
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. Figure 71.6 Average hydration profiles over the course of the study
After 2 weeks of treatment, the increase in SC thickness induced by formulation A was significantly different from the other two products being tested and the untreated control site (p = 0.0121), and this difference remained at the 1 week regression timepoint (p = 0.0162). The observed change corresponded to an approximate 10% increase in SC thickness.
Total hydration in the SC can be calculated from the area under the profile (AUC; integration between x = 0 mm and the calculated SC leveling-off point). Concomitant with the increase in SC thickness, total skin hydration increased significantly following treatment with formulation A after 2 weeks of product usage and the 1-week regression (p = 0.0275 and 0.0435, respectively).
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Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
While all moisturizers have the effect of alleviating dry skin when formulated appropriately, it has become apparent in recent years that different moisturizer formulations can have different effects on the SC and the epidermis (for review see Loden [61]). Naturally, in the short term, moisturizers will increase SC hydration [46, 49, 50, 60–62], and in the medium term improve desquamation [63, 64], however, in the longer term, it has become apparent that some can actually compromise SC barrier function [65–69], while others can strengthen it [60, 62, 70–72]. In vitro [73–77] and in vivo [78, 79] studies have also demonstrated the ability of some moisturizing ingredients to influence SC thickness. Therefore, it is becoming increasingly apparent that not only is there a need for longer term studies to evaluate the effect of moisturizers, but also that the introduction of new measurements in addition to the more ‘‘traditional’’ electrical parameter-based devices is needed to understand their effects more completely [68]. In the moisturizer study presented here, on the first day after starting product application, little difference in CRS-derived hydration profiles is observed between any of the treatments or the untreated control site. After the first week of treatment though, there was a numerical diminution in SC thickness. While not statistically significant, it could have been due to the osmotic effects of glycerol (which was present in all the three formulations). This behavior has been reported before – Caussin et al. [77] reported that changes in SC swelling can occur, when examining the effects of moisturizers on SC hydration and swelling. Lipophilic moisturizers increased SC thickness whereas hydrophilic moisturizers tended to reduce SC thickness. This apparent SC thinning may, therefore, be due to osmotic effects of the moisturizing ingredients (used at high concentration) and the work of Fluhr et al. [80] describing the effect of glycerol on reducing corneocyte surface area would tend to support this. However, inconsistencies remain where in vitro [77] and in vivo [78, 79] increased corneocyte swelling has been reported with glycerol solutions. Another possible explanation would be that during the first week of treatment, there was some activation of SC protease activity (simply by elevated water activity), resulting in more efficient desquamation and an ensuing reduction in SC thickness. This effect prompts further investigation. After the second week of the study, formulation A induced a statistically significant increase in SC thickness (2 mm average increase, corresponding to an approximate 10% increase in thickness). As already discussed, water content measurements at absolute depths are simply not comparable between time points in a study where SC thickness may change or vary. It is, therefore, more meaningful to extract information from the profiles regarding
total SC thickness and express water measurement derivatives as a function of this (e.g., the use of total SC water content). Considering SC thickness first of all, after 2 weeks of treatment, formulation A produced a significantly greater increase in this parameter than the other two treatments and the untreated site (p = 0.0121), and this difference remained at the 1-week regression time point (p = 0.0162). Of note, increases in SC thickness have also been reported by Jacobson et al. [81] using a lipophilic niacin derivative. Concomitant with this increase in SC thickness, total SC hydration as measured by CRS increased significantly with use of formulation A after 2 weeks of treatment. This increase remained at the 1-week regression time point. However, no such effect was observed for treatment with formulations B and C. This data did not, however, correspond with Corneometer measurements taken at the same time points. Significantly, increased Corneometer values were observed for all products even after 1 day of application and, indeed, values remained elevated throughout the 2-week treatment phase. Corneometer values also remained elevated for all treatments at the 1-week regression (although all values were significantly lower than those at the 2-week treatment time point – an effect observed in other regression studies [71, 82]). Considering the ingredients present in all the three formulations, the capacitance effects noted may be attributable partially to the high dielectric constant of glycerol [83, 84]. It therefore appears from the CRS hydration profiles and their relative difference to corresponding Corneometer values, however, that measured changes in capacitance do not directly reflect total SC hydration. This raises the question as to where the capacitance signal is coming from within the skin and what moieties are driving changes in this parameter in the context of treatment with a moisturizer. While it is not possible here to go into a detailed examination of the role of different ingredients on skin properties (a more complete discussion is given in [51], it is believed that niacinamide (nicotinamide, vitamin B3), present only in Formulation A, is probably the agent responsible for these SC effects. Recent work has been undertaken to further examine the role of niacinamide in SC swelling using freshly prepared biopsy cross sections in a vehicle controlled study [85].
Changes in Raman Profiles as a Function of Age The ability of confocal Raman spectroscopy to rapidly and nondestructively examine large numbers of people in a clinical environment has opened up the possibility to
Molecular Concentration Profiling in Skin Using Confocal Raman Spectroscopy
assess changes in skin properties in wide-ranging age groups – from infants to the elderly. Egawa et al. have reported significant differences in NMF levels, lactate, trans-urocanic acid, ceramide, and cholesterol, along with differences in water gradient, and apparent SC thickness, when assessing volar forearm and cheek sites of young (aged 22–40) and older (aged 59–76) female subjects [48]. It should be noted that only two or three Raman spectra were collected for a particular subject, a low number of replicates given the structural heterogeneity of the skin and laser spot size. However, they were able to derive similar forced hydration profiles to the ones described here in > Fig. 71.5b. Raman profiling of very young skin (3–12 months in age) has shown decreased NMF content and increased water at all depths throughout the SC when compared to adult skin [86]. In their work Nikolovski et al. also showed that infant skin was more capable of quickly absorbing and desorbing exogenous water when compared to adult skin, which might be expected from the higher TEWL values seen in infants.
Conclusion While a number of techniques are currently available to provide information regarding distribution of different components within the skin, until recently none have been capable of determining these, nondestructively, rapidly, and in vivo. As such, it has been difficult to incorporate them into routine clinical test protocols. The recent development of confocal Raman spectroscopy has enabled the assessment of changes in molecular concentration gradients in vivo for the first time for a variety of different chemical species. It is clear that CRS represents a powerful new class of measurement device with significant advantage over traditional measurement techniques through its ability to assess these changes, both rapidly and in a nondestructive manner. The advent of this new technology seems timely as it is considered the development of moisturizers that truly augment SC barrier function. The capability of in vivo confocal Raman has only been touched upon as the technique is still in its infancy. Even considering its relative youth, Raman spectroscopy of the skin has already provided a valuable new insight into SC behavior and function, and demonstrated its potential as a valuable tool for determining chemical concentration gradients in a clinical environment, both in relation to the use of cosmetic products and with regards to the changes associated with the aging process. As the technique becomes more established its ability
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to measure these profiles within the skin will provide a deeper understanding of the interaction between chemical composition and location and skin health and function.
Cross-references > Bioengineering
Methods and Skin Aging
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on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291:555–559. Gambichler T, Boms S, Stacker M, et al. Epidermal thickness assessed by optical coherence tomography and routine histology: Preliminary results of method comparison. J Eur Acad Dermatol Venereol. 2006;20(7):791–795. Lademann J, Otberg N, Richter H, et al. Application of optical noninvasive methods in skin physiology: a comparison of laser scanning microscopy and optical coherent tomography with histological analysis. Skin Res Technol. 2007;13(2):119–132. Norlen L. Stratum corneum keratin structure, function and formation – a comprehensive review. Int J Cosmet Sci. 2006; 28(6):397–425. Bouwstra JA, de Graff A, Gooris GS, et al. Water distribution and related morphology in human stratum corneum at different hydration levels. J Invest Dermatol. 2003;120(5):750–758. Wu J, Polefka TG. Confocal Raman microspectroscopy of stratum corneum: a pre-clinical validation study. Int J Cosmet Sci. 2008;30:47–56. Matts PJ, Gray J, Rawlings AV. The ‘‘dry skin cycle’’ – a new model of dry skin and mechanisms for intervention. International Congress and Symposium Series, 256. London: The Royal Society of Medicine Press Ltd, 2005, pp. 1–38. Loden M. The clinical benefit of moisturizers. JEADV. 2005;19: 672–688. Breternitz M, Kowatski D, Langenauer, M, et al. Placebo controlled, double blind, randomized prospective study of a glycerol-based emollient on eczematous skin in atopic dermatitis: biophysical and clinical evaluation. Skin Pharmacol Physiol. 2008;21:39–45. Summers RS, Summers B, Chandar P, et al. The effect of lipids with and without humectants on skin xerosis. J Soc Cosmet Chem. 1996;47:27–39. Rawlings AV, Watkinson A, Hope J, et al. The effect of glycerol and humidity on desmosome degradation in stratum corneum. Arch Dermatol Res. 1995;287:457–464. Held E, Sveinsdottir S, Agner T. Effect of long term use of moisturizer on skin hydration, barrier function and susceptibility to irritants. Acta Derm Venereol. 1999;79:49–51. Zachariae C, Held E, Johansen JD, et al. Effect of a moisturizer on skin susceptibility to NiCl2. Acta Derm Venereol. 2003;83:93–7. Berardesca E, Distante F, Vignoli GP, et al. Alpha hydroxyacids modulate stratum corneum barrier function. Br J Dermatol. 1997;137:934–938. Buraczewska I, Berne B, Lindberg M, et al. Changes in skin barrier function following long-term treatment with moisturizers, a randomized controlled trial. Br J Dermatol. 2007;156:492–498. Barany E, Lindberg M, Loden M. Unexpected skin barrier influence from non-ionic emulsifiers. Int J Pharm. 2000;195:189–195. Fluhr JW, Gloor M, Lehmann L, et al. Glycerol accelerates recovery of barrier function in vivo. Acta Dermatol Venereol. 1999;79: 418–421.
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71. Loden M, Andersson AC, Andersson C, et al. Instrumental and dermatologist evaluation of the effect of glycerine and urea on dry skin in atopic dermatitis. Skin Res Technol. 2001;7:209–213. 72. Rawlings AV, Conti A, Verdejo P, et al. The effect of lactic acid isomers on epidermal lipid biosynthesis and stratum corneum barrier function. Arch Dermatol Res. 1996;288:383–390. 73. Norlen L, Emilson A, Forslind B. Stratum corneum swelling. Biophysical and computer assisted quantitative assessments. Arch Dermatol Res. 1997;289:506–513. 74. Richter T, Muller JH, Schwarz UD, et al. Investigation of the swelling of human skin cells in liquid media by tapping mode scanning force microscopy. Appl Phys A. 2001;A72:S125–S128. 75. Bouwstra JA, de Graaff A, Gooris GS, et al. Water distribution and related morphology in human stratum corneum at different hydration levels. J Invest Dermatol. 2003;120:750–758. 76. Richter T, Peuckert C, Sattler M, et al. Dead but highly dynamic – the stratum corneum is divided into three hydration zones. Skin Pharmacol Physiol. 2004;17:246–257. 77. Caussin J, Groenink HWW, Graaff de AM, et al. Lipophilic and hydrophilic moisturizers show different actions on human skin as revealed by cryo-scanning electron microscopy. Exp Dermatol. 2007;16:891–898. 78 Orth DS, Appa Y, Contard P, et al. Effect of high glycerin moisturizers on the ultrastructure of the stratum corneum. Poster at the 53rd Annual meeting of the American Academy of Dermatology, February 1995. 79. Orth DS, Appa Y. Glycerine: a natural ingredient for moisturizing skin. In: Loden M, Maibach HI (eds) Dry Skin and Moisturizers: Chemistry & Function. Boca Raton: CRC Press, 2000, pp. 213–228. 80. Fluhr JW, Bornkessel A, Berardesca E. Glycerol-just a moisturizer? Biological and biophysical effects. In: Loden M, Maibach HI (eds) Dry Skin and Moisturizers, 2nd ed. London: Taylor & Francis, 2006, pp. 227–244. 81. Jacobson EL, Kim H, Kim M, et al. A topical lipophilic niacin derivative increases NAD, epidermal differentiation and barrier function in photodamaged skin. Exp Dermatol. 2007;16(6):490–499. 82. Loden M, Wessman C. The influence of a cream containing 20% glycerin and its vehicle on skin barrier properties. Int J Cosmet Sci. 2001;23:115–119. 83. Fluhr JW, Mao-Qiang M, Brown BE, et al. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice. J Invest Dermatol. 2003;120:728–737. 84. Choi EH, Man MQ, Wang, F, et al. Is endogenous glycerol a determinant of stratum corneum hydration in humans? J Invest Dermatol. 2005;125:288–293. 85 Crowther JM, Matts PJ. Publication in preparation. 86. Nikolovski J, Stamatas GN, Kollias N, Wiegand C. Barrier function and water-holding and transport properties of infant stratum corneum are different from adult and continue to develop though the first year of life. J Invest Dermatol. 2008;128:1728–1736.
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74 Percutaneous Penetration of Chemicals and Aging Skin Michael F. Hughes
Introduction The human body undergoes changes, for better or worse, from infancy to the elderly stages of life. The change is observed from the biochemical level within cells to the morphology and function of whole organs. The skin is an organ that changes with age due to both intrinsic and extrinsic factors. Intrinsic aging of the skin is primarily determined by genetics. Extrinsic aging, also termed photoaging, is principally caused by environmental exposure to ultraviolet light. In areas of the skin that are sun-exposed, these two processes of aging are superimposed upon one another [1]. In the industrialized world, humans live longer because of advancements in agriculture, medicine, and public health. Because of the increasing age of the population, it is important to know how age-related changes in organs, such as skin, impact overall health. Changes occur in the epidermis and dermis with increasing age [2–4]. Within the stratum corneum, the outermost layer of the epidermis, alterations with increasing age include decreased content of moisture [5] and skin surface lipids [6]. The epidermis becomes thinner and more flattened, although the thickness of the stratum corneum is not altered [7, 8]. The area of contact and adherence between the epidermis and the underlying dermis is decreased. Thus in the elderly, simple trauma to the skin may remove the epidermis more easily than in younger adults. Within the dermis, the vasculature, principally the capillaries that supply blood to the viable epidermis, is diminished. The decreased blood flow in the skin of the aged appears to be affected by autonomic influences and may be site-dependent [9]. This reduced blood supply can result in a longer time for wounds to heal, decreases in inflammatory response, and altered thermal regulation. Humans are exposed to chemicals throughout their lives. Exposure to chemicals, including drugs, environmental contaminants, industrial and household chemicals, agrochemicals, personal products, and others, can be intentional or accidental. Exposure to chemicals occurs by the oral, pulmonary, or percutaneous routes and can result in their systemic absorption. The amount of
chemical absorbed and the potential effects that may result are dependent on the route of exposure as well as other factors. In order for percutaneous or dermal absorption of a chemical to occur, it must undergo a series of partitioning and passive diffusion steps [10]. Active and facilitated transport mechanisms, such as those that occur in oral absorption, are not involved in dermal absorption. First, the chemical on the skin surface partitions into the stratum corneum. This may require partitioning of the chemical from a vehicle or formulation. The chemical then diffuses across the stratum corneum and partitions into the underlying viable epidermis. Diffusion of the chemical across the viable layers of the epidermis occurs, followed by partitioning into the dermis. The final steps are diffusion across the dermis and partitioning into the circulatory or lymphatic system. The main driver in dermal absorption is the concentration of the chemical on the surface of the stratum corneum. Upon dermal exposure to a chemical, a concentration gradient is produced and results in a mass transfer of the chemical from the surface of the skin, through the epidermis, and into the dermis. There are three proposed routes of dermal absorption [10]. These include the intercellular, transcellular, and appendegeal routes. It is generally thought that the intercellular route predominates in dermal absorption, while the other two routes have a minor role. The mechanism by the intercellular route involves partitioning of the chemical into the lipid-rich extracellular regions of the stratum corneum cells, the corneocytes. The chemical then diffuses around the corneocytes. Lipophilic chemicals diffuse through the lamellar acyl chains of the lipid, while hydrophilic chemicals diffuse through the polar head groups of the lipid. For the transcellular route, the chemical goes through the keratinfilled corneocytes by partitioning into and out of the extracellular lipid. For the appendageal route, the chemical bypasses the stratum corneum by entering the shunts of the hair follicles and sebaceous and sweat glands. Chemicals are absorbed through the skin and into the systemic circulation, but not to the same extent or rate. The main barrier is the stratum corneum, which is lipid-rich
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_74, # Springer-Verlag Berlin Heidelberg 2010
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and impedes the absorption of many compounds. Because of its lipid-rich nature, the stratum corneum can be a reservoir for lipophilic chemicals [11]. They partition into the stratum corneum, but diffuse no further, or to a limited extent because of their physical and chemical properties. The more aqueous environment of the lower layers of the epidermis and dermis than the stratum corneum also has an impact on the penetration of lipophilic compounds, adding to this reservoir effect. Over time, these chemicals may eventually be systemically absorbed or lost when the stratum corneum is sloughed off. Dermal absorption of chemicals can be affected by exposure-, skin-, and chemical-related factors (> Table 74.1). These include the anatomical site of exposure, species, vehicle, and age. A classic example of the effect of age on dermal absorption is the toxicity that occurred following the use of hexachlorophene as a disinfectant on the skin of preterm infants [12]. Preterm infants are susceptible to the effects of chemicals and drugs applied to the skin, because their stratum corneum is not fully developed at birth [13]. These preterm infants later developed neurological deficiencies because the hexachlorophene, a neurotoxicant, was absorbed through their skin that had not fully matured. Skin that has fully developed can retard the absorption of many chemicals. Whether aging skin maintains its barrier properties to the absorption of chemicals has been questioned and is
. Table 74.1 Exposure-, skin-, and chemical-related factors that can affect dermal absorption of chemicalsa Exposure-related
Skin-related
Chemical concentration
Age
Duration
Thickness
Lipid solubility
Use of protective equipment
Blood flow
Water solubility
Climate (temperature and humidity)
Damage
Vehicle
Matrix (e.g., soil)
Metabolism
Irritancy
Occlusion
Other chemicals (e.g., enhancers)
Anatomical site of exposure Hair and pore density a
Chemical-related
Adapted from Semple [30]
Molecular weight
still being investigated. From microscopic examination, the barrier of the stratum corneum does not appear to be compromised in aged skin [8]. Nevertheless, this alone does not indicate that the barrier properties of aged skin are intact. Studies in the 1960s by Christophers and Kligman [14] suggested that the permeability of skin from the elderly (>66 years old) was different from that of younger adults ( Fig. 74.1). There was a significantly greater percent of the dose of these two compounds in the receptor fluid of the 27-month-old
mice than those of younger age groups. Conversely, there was a significantly greater percent of the dose of these two compounds in skin of the two younger age groups. Also, there was a significantly greater percent of the dose of phenol in the receptor fluid of the 15-monthold than of the 3-month-old mice. Overall, these agedependent differences were < 5% for phenol detected in the receptor fluid, but were > 40% for heptyloxyphenol. There was no difference in the absorption of cyanophenol among the three age groups. For acetamidophenol, there was a significantly greater percent of the dose in the skin of the 15- and 27-month-old mice than in the 3-monthold mice. Although not significantly different, the skin wash removed about twice as much acetamidophenol from the 3-month-old mice than from the skin of the two older groups. The phenols tested differed in lipophilicity, with acetamidophenol the least lipid soluble (octanol/water partition coefficient (Log P) 0.32) and heptyloxyphenol the most lipid soluble (Log P 4.75). The thickness of the dermis in this strain of mouse decreases with age [23]. Thus, the chemicals have a shorter distance to completely diffuse through the skin of the older than the younger mice in the in vitro setting. This change in mouse skin thickness may result in increased in vitro absorption of lipophilic compounds that can penetrate into skin but are unable to efficiently partition out of it into the receptor fluid as observed with heptyloxyphenol in the 3- and 15-month-old mice. In this study, the mouse skin was a reservoir for heptyloxyphenol. However, the composition of the lipids in the skin of the 28-month-old mice may have changed that increased the ability of heptyloxyphenol to partition from the skin into the receptor fluid. This study indicates that in addition to aging skin, the physical and chemical properties of a chemical can alter the extent of dermal absorption. Banks et al. [24] examined the in vivo dermal absorption of radiolabeled 2,3,7,8-tetrachlorodibenzo-r-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) in male Fischer 344 rats of various ages (10, 36, 96 weeks for TCDD; 10, 36, 64, 96, and 120 weeks for 4PeCDF). The lifespan of this rat strain ranges from 120 to 160 weeks. The chemicals were applied in acetone at a dose of 0.1 mmole/kg over 1.8 cm2 on the back of a shaved animal. The dose site was then covered with a non-occluding stainless-steel cap. The animals were housed in individual metabolism cages for 3 days following exposure. Urine and feces were collected over the 3-day period. At the end of the experiment, the animals were sacrificed and tissues and the skin dosing site were removed. The excreta, tissues, and skin were analyzed for chemical-derived radioactivity.
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. Figure 74.1 Distribution of acetamidophenol-, phenol-, cyanophenol-, and heptyloxyphenol-derived radioactivity in receptor fluid ( ), skin (▒ ), and skin wash (□) 72 h after exposure to mouse skin of different age groups (Hughes M.F. et al.) [22]. Data represent mean standard deviation, N = 4–9. aSignificantly greater than 3-month old mice; bsignificantly greater than 27-month-old mice; csignificantly greater than 3- and 15-month-old mice; dSignificantly greater than 15- and 27-month-old mice
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Chemical-derived radioactivity detected in excreta and tissues was considered absorbed. The treated skin was not washed to remove unabsorbed chemical. A previous study from this laboratory [25] reported that 80% or more of the dose of TCDD and 4-PeCDF was removed by an acetone swab wash 3 days after application. Thus, the authors considered chemical-derived radioactivity detected in skin not absorbed. In the treated skin, 80–94% and 66–87% of the dose of TCDD and 4PeCDF, respectively, was detected. Absorption of TCDD and 4PeCDF decreased with increasing age of the animals (> Fig. 74.2). The changes were primarily between the 10-week and 36-week-old rats. After 36 weeks, there were no significant age-related changes in the absorption of TCDD. There was a significant difference in the
tissue levels of 4PeCDF between the 64- and 96-week-old rats, but the authors suggested that this difference was due to experimental variability. Thus, this age-related decrease in the dermal absorption of these two lipophilic compounds occurred at an age when the rats would not be considered elderly, but approaching mid-life. Lehman and Franz [26] examined the effect of age and caloric-restricted diet on the in vitro dermal absorption of water, lidocaine, and hydrocortisone in female Fischer 344 rat skin. Rodents on a caloric-restricted diet live longer and the age-related changes in the body are delayed compared to rodents fed ad libitum. The ages of the rats tested were from 11 to 144 weeks old. They used fullthickness skin and the finite dosing technique with Franz static diffusion cells. The hair on the dorsal surface of the
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. Figure 74.2 Percent of the administered dose of TCDD- and 4PeCDFderived radioactivity in tissues (▒ ), feces (□), and urine ( ) of rats of different ages following a 3-day dermal exposure (Banks YB et al.) [24]. Data represent mean standard deviation, N = 3–4. aSignificantly different (p < 0.05) versus 36- and 96-week-old TCDD-treated rats; bsignificantly different (p < 0.05) versus next age group of 4PeCDFtreated rats; csignificantly different (p < 0.05) versus 36, 64, 96, and 120-week-old 4PeCDF-treated rats
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at 44 weeks and continued up to the last age group tested. In the caloric-restricted animals, lidocaine permeability increased at 44 weeks, but then decreased with increasing age. For hydrocortisone, the penetration followed the same course as lidocaine, increasing at 44 weeks for both groups, and continuing to increase in the ad libitum group, but decreasing in the caloric-restricted group. Thus the penetration of the lipophilic compounds lidocaine and hydrocortisone increased with age in both dietary groups, relative to the ad libitum 11-week-old animals. This suggested that the innate aging of the skin was independent of dietary factors. The difference between the dietary groups was that in the ad libitum animals, the total penetration of lidocaine and hydrocortisone kept increasing with age, whereas it decreased for both from the peak at 44 weeks in the caloric-restricted animals (but still remained higher than the ad libitum 11-week-old animals). The caloric-restricted animals were most likely not deficient in essential fatty acids, because they received 10% corn oil in their diet. For some unknown reason, the skin of the caloric-restricted animals became more impervious to the lipophilic chemicals relative to the age-matched ad libitum animals. The agerelated changes in the skin of the rat result in increased in vitro dermal absorption of lidocaine and hydrocortisone. This effect on the dermal absorption can be modified with diet. The different result from the Banks et al. [24] study could be from the techniques used (in vitro vs. in vivo) and the chemical and physical properties of the chemicals used in these two studies.
Studies in Humans
rats was carefully clipped before placing the skin on the cells. In static cell studies, the receptor fluid remains below the skin and is directly sampled over time. The aliquots are then analyzed for the presence of chemicals. In the case of water, with the exception of the 144week-old group of the ad libitum animals, neither age nor diet had an effect on its penetration (> Fig. 74.3). (Note: there were only two surviving ad libitum animals in the last age group, out of the original 50.) With lidocaine in the ad libitum group, its permeability increased starting
Roskos et al. [27] examined the in vivo percutaneous absorption of a select group of chemicals in humans of two age groups. A young group consisted of males and females of ages 22–40 years. An old group consisted of males and females of ages 65–86 years. All subjects were Caucasian and had no history of dermatologic disease. The test compounds were radiolabeled and included testosterone, estradiol, hydrocortisone, benzoic acid, acetylsalicylic acid, and caffeine. The chemicals were applied (4 mg/cm2) in acetone onto the ventral surface of the forearm. Following evaporation of the acetone, a nonoccluding patch was placed over the dosing site. Urine was collected at several time points up to 7 days postexposure. At 24 h postexposure, the patch was removed and the dosing site was washed to remove unabsorbed chemical. Following this wash, the dosing site was covered with a non-occluding patch. At 7 days postexposure, the patch
Percutaneous Penetration of Chemicals and Aging Skin
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. Figure 74.3 Total in vitro penetration of water, lidocaine, and hydrocortisone through rat skin of different ages and diets (□, caloricrestricted; , ad libitum) (Lehman PA) [26]. Data represent mean standard deviation, N = 2–6. aSignificantly different from 11-week-old ad libitum animals; bsignificantly different from diet match cohort; csignificantly from 144-week-old ad libitum animals
▪
was removed and the dosing site was washed again. To account for incomplete urinary excretion of the chemical, each subject was administered the chemical intravenously. Urine was collected for 7 days after intravenous administration of the chemical. The washes, patches, and urine were analyzed for chemical-derived radioactivity. The percent of the dose in the urine following dermal administration was corrected for incomplete urinary excretion by dividing this value by the percent of the dose in urine collected after intravenous administration. The adjusted value for percent of the dose in urine following dermal exposure to the chemicals was considered the absorbed dose. The chemicals tested were divergent with respect to their water and lipid solubility. Testosterone is insoluble
in water and the most lipophilic of the chemicals tested (Log P 3.32). Caffeine is the most water soluble (21.7 g/L) and the least lipid soluble (Log P 0.01). Roskos et al. [26] reported that there were no differences between the young and old groups in the urinary excretion of chemicalderived radioactivity of the six chemicals administered intravenously. This suggests that once any of these chemicals is absorbed, for the two age groups, the chemicals are excreted similarly. The urine was not analyzed for metabolites of these chemicals, so the effect of age on their metabolism could not be determined. Nevertheless, if there were age differences in the metabolism, it did not affect the urinary elimination of the chemicals or their metabolites.
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For the two most lipid-soluble compounds (and least water soluble), testosterone and estradiol, there was no difference in the cumulative percent dose absorbed between the two age groups (> Fig. 74.4). However, an age difference was observed in the dermal absorption for the more hydrophilic compounds. Significant differences were noted in the absorption of benzoic acid, acetylsalicylic acid, and caffeine, with a greater cumulative percent of dose absorbed in the young than in the old age group. For hydrocortisone, the cumulative percent of dose absorbed in the young age group was about three times greater than in the old age group. However, no statistical difference was detected. The reason may have been a low sample size (n ¼ 3 for young; n ¼ 7 for old), or that the overall absorption was low (60 years) patients who had undergone lower abdominal surgery. The fentanyl was administered to relieve pain from the surgery. The patients were treated with a transdermal patch of fentanyl for 72 h following surgery. Blood was withdrawn from the patients over time following application of the patch and after its removal. The blood was centrifuged and the resulting plasma was analyzed for fentanyl. Between the two age groups, there was no difference in the maximal plasma concentration of fentanyl, the time these occurred, the plasma area under the curve, and the elimination half-life after the patch was removed. The only significant difference was in the half-time for the plasma concentration of fentanyl to double. In the older group, this half-time was 11.1 h and in the younger group,
. Figure 74.4 Cumulative % dose absorbed of 14C-labeled testosterone, estradiol, hydrocortisone, benzoic acid, acetylsalicylic acid, and caffeine in vivo in young (18–40 years, ) and old (65–86 years, □) humans following dermal exposure (Roskos KV et al.) [27]. Absorption was determined by collecting urine of treated subjects for 7 days and analyzing for chemical-derived radioactivity. The absorption data was adjusted for incomplete elimination of radioactivity following intravenous administration of radiolabeled chemical and collection of urine for 7 days. Data represent mean standard deviation, N = 3–8. aSignificantly different from young group, p < 0.05. bsignificantly different from young group, p < 0.01
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Percutaneous Penetration of Chemicals and Aging Skin
it was 4.2 h. This lag in fentanyl absorption could be due to changes in the lipid composition of skin as it ages. It could also be due to the alteration of the microcirculation in the dermis. Fentanyl is a very lipophilic compound (Log P 9550) and may be retained in the skin because of the decreased circulation. Also, the elderly may react differently than the younger patients to the surgery. This could impact cardiac output, circulatory volume, and body temperature.
Conclusion Age-related changes occur in the biochemistry, physiology, and morphology of the skin. Experimental studies suggest there is an age-related effect on the dermal absorption of chemicals. This could be important as the age of the population increases in developed countries. Knowledge of this potential effect on the dermal absorption would be beneficial for the elderly who use transdermal drug delivery of drugs or are exposed residentially or environmentally to chemicals. The results from the animal studies are somewhat conflicting. In one in vitro study [21], the age-related change in absorption occurred when the animals were young and appeared to be related to their hair-growth cycle. So the relevance of this study to human exposure can be questioned. In the two other in vitro studies [22, 26], there was an increase in the absorption of lipophilic compounds as the animals aged beyond young adulthood. In an in vivo rat study [24], the absorption of the lipophilic compounds decreased. The differences in results could potentially be due to technique (in vitro vs. in vivo) as well as disparities in the physical and chemical properties of the chemicals studied. It has not been adequately established whether animal skin, particularly that of rodents, can adequately predict permeability of chemicals in humans. There are also questions about the in vitro technique, because it only tests the barrier property of the skin, not the removal of absorbed chemicals by the vasculature in the dermis. The limited number of rigorous studies in humans suggests that age affects the dermal absorption of hydrophilic compounds, but not lipophilic compounds to the same extent. The data from Thompson et al. [29] are consistent with the Roskos et al. [27] study. The extent of the lipophilic compound fentanyl [29] was not affected by age, as observed with testosterone and estradiol [27]. What is needed for a better understanding of the agerelated changes in dermal absorption are more complete studies, with a greater number of subjects and chemicals
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that vary in physical and chemical properties. Certainly, Roskos et al. [27] did this with the chemicals that varied in water and lipid solubility. One of the limitations of this study was the low number of subjects. However, it is difficult to conduct laboratory studies in humans for ethical, monetary, and logistical reasons. The animals studies presented looked at a wide range of ages and included very old mice and rats. However, the results were not consistent with the human data [27, 29], although only a few of the same chemicals were tested. An approach that could be taken is for a laboratory to test the same set of chemicals, in animals and humans, using in vitro and in vivo techniques. The chemicals should be carefully selected, so that their physical and chemical properties are well known. Because the age of the population is increasing in many parts of the world, it is important to understand their risk for potential dermal absorption of the chemicals that surround everyone. Understanding whether the agerelated changes on the skin alter the dermal absorption of chemicals will ultimately lead to reduced risk for the development of adverse health effects in the elderly population. Disclaimer: This article has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Cross-references > Susceptibility
to Irritation in the Elderly: New
Techniques
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7. Fenske NA, Lober CW. Structural and functional changes of normal aging skin. J Am Acad Dermatol. 1987;14:571–585. 8. Lavker RM, Zheng P, Dong G. Aged skin: a study by light, transmission electron, and scanning electron microscopy. J Invest Dermatol. 1987;88:44S–51S. 9. Harvell JD, Maibach HI. Percutaneous absorption and inflammation in aged skin: a review. J Am Acad Dermatol. 1994;31: 1015–1021. 10. Hotchkiss SAM. Cutaneous toxicity: kinetic and metabolic determinants. Toxicol Ecotoxicol News. 1995;2:10–18. 11. Poet TS, McDougal JN. Skin absorption and human risk assessment. Chem Biol Interact. 2002;140:19–34. 12. Anderson JM, Kilshaw BH, Harkness RA, et al. Spongioform myelinopathy in premature infants. Br Med J. 1975;2:175–176. 13. Harpin VA, Rutter N. Barrier properties of the newborn infant’s skin. J Pediatr. 1983;102:419–425. 14. Christophers E, Kligman AM. Percutaneous absorption in aged skin. In: Montagna W (ed) Advances in biology of the skin. Vol. 6:Aging. Long Island City:Pergaman Press, 1965, pp. 163–175. 15. DeSalva SJ, Thompson G. Na22Cl skin clearance in humans and its relation to skin age. J Invest Dermatol. 1965;45:315–318. 16. Tagami H. Functional characteristics of aged skin. Acta Dermatol (Kyoto). 1972;67:131–138. 17. Kaestli L-Z, Wasilewski-Rasca A-F, Bonnabry P, et al. Use of transdermal drug formulations in the elderly. Drugs Aging. 2008; 25:269–280. 18. Jeter KF, Lutz JB. Skin care in the frail, elderly, dependent, incontinent patient. Adv Wound Care. 1996;9:29–34. 19. Bronaugh RL, Stewart RF, Congdon ER. Methods for in vitro percutaneous absorption studies. II. Animal models for human skin. Toxicol Appl Pharmacol. 1982;62:481–488. 20. Behl CR, Flynn GL, Kurihara T, et al. Age and anatomical site influences on alkanol permeation of skin of the male hairless mouse. J Soc Cosmet Chem. 1984a;35:237–252.
21. Behl CR, Flynn GL, Linn EE, et al. Percutaneous absorption of corticosteroids: age, site, and skin-sectioning influences on rates of permeation of hairless mouse skin by hydrocortisone. J Pharm Sci. 1984b;73:1287–1290. 22. Hughes MF, Fisher HL, Birnbaum LS, et al. Effect of age on the in vitro percutaneous absorption of phenols in mice. Toxicol In Vitro. 1994;8:221–227. 23. Monteiro-Riviere NA, Banks YB, Birnbaum LS. Laser Doppler measurements of cutaneous blood flow in ageing mice and rats. Toxicol Lett. 1991;57:329–338. 24. Banks YB, Brewster DW, Birnbaum LS. Age-related changes in dermal absorption of 2,3,7,8-tetrachlorodibenzo-r-dioxin and 2,3,4,7,8-pentachlorodibenzofuran. Fundam Appl Toxicol. 1990; 15:163–173. 25. Brewster DW, Banks YB, Clark A-M, et al. Comparative dermal absorption of 2,3,7,8-tetrachloridibenzo-r-dioxin and three polychlorinated dibenzofurans. Toxicol Appl Pharmacol. 1989;90: 243–252. 26. Lehman PA, Franz TJ. Effect of age and diet on stratum corneum barrier function in the Fischer 344 female rat. J Invest Dermatol. 1993;100:200–204. 27. Roskos KV, Maibach HI, Guy RH. The effect of aging on percutaneous absorption in man. J Pharmacokinet Biopharm. 1989;17: 617–630. 28. Pochi PE, Strauss JS, Downing DT. Age-related changes in sebaceous gland activity. J Invest Dermatol. 1979;73:103–111 29. Thompson JP, Bower S, Liddle AM, et al. Perioperative pharmacokinetics of transdermal fentanyl in elderly and young adult patients. Br J Anaesth. 1998;81:152–154. 30. Semple S. Dermal exposure to chemicals in the workplace: just how important is skin absorption? Occup Environ Med. 2008;61:376–382.
78 Probiotics in Aging Skin Benedetta Cinque . Paola Palumbo . Cristina La Torre . Esterina Melchiorre . Daniele Corridoni . Gianfranca Miconi . Luisa Di Marzio . Maria Grazia Cifone . Maurizio Giuliani
Introduction Health benefits of probiotics have been established by several studies in animals and humans and the scientific literature shows that the clinical uses of probiotics are broad and are open to continuing evaluation. The most common microorganisms used as probiotics are strains of lactic acid bacteria (LAB), which are gram-positive, nonsporing, catalase-negative organisms that are devoid of cytochromes and of nonaerobic habit, but are aerotolerant, acid-tolerant, and strictly fermentative; lactic acid is the major end product of sugar fermentation. Particular attention is paid to specific species of lactic acid bacteria (LAB), including Lactobacilli and Bifidobacteria, that are part of the intestinal microbiota. Most probiotics are included in foods or dietary supplements and are aimed at functioning in the intestine. However, even if gastrointestinal tract has been the primary target, it is becoming evident that other conditions not initially associated with the gut microbiota might also be affected by probiotics. It was speculated that the skin status could benefit from reinforced gut homeostasis. Nutritional intervention, particularly with dietary antioxidants have been proposed to protect against UV-induced skin damage and an increasing interest has been shown for new nutritional approaches using live microorganisms as probiotics. Moreover, the capacity of probiotics to modulate the systemic immune status, including the release of regulatory cytokines, might influence skin homeostasis. In addition, reports showing the efficacy of a selected probiotic extract in increasing ceramide levels in vivo, on stratum corneum (SC) of healthy young and old subjects as well as in atopic dermatitis patients thus reducing dryness, loss of tone, fullness, and water loss, opened new potential probiotic-based strategies against those pathophysiological skin alterations, including aging, associated with a reduced amount of the ceramide, major water-holding molecule in the extracellular space of the horny layer. Overall, even if the potential use of probiotics for the skin has been hardly considered in the past, more recent experimental studies have suggested interesting, potential,
new applications. The aim of the present review is to outline the main challenges associated with accumulating evidence in support of skin health claims for probiotics and to give a perspective of the scientific gaps that need to be addressed to advance the probiotic-based preventive or therapeutic approaches in aging skin, which is one of the most common dermatologic concerns.
Probiotic Microorganisms and Health Benefits The history and evolution of the definition of ‘‘probiotic microorganism’’ has been extensively reviewed by Fioramonti et al. [1]. The concept of probiotics was most likely derived from a theory first proposed by Nobel Prize-winning Russian scientist Elia Metchnikoff, who suggested in 1908 that long life of Bulgarian peasants resulted from their consumption of fermented milk products. The term ‘‘probiotic,’’ which literally means ‘‘for life,’’ was first used by Lilly and Stillwell (1965) to describe ‘‘substances secreted by one microorganism, which stimulate the growth of another.’’ A powerful evolution of this definition was coined by Parker (1974), who proposed that probiotics are ‘‘organisms and substances which contribute to intestinal microbial balance.’’ Fuller (5. R. Fuller, Probiotics in man and animals. Journal of Applied Bacteriology 66 (1989), pp. 365–378. View Record in Scopus | Cited By in Scopus (1099) 1989) then modified the definition in 1989 to ‘‘a live microbial feed supplement, which beneficially affects the host animal by improving its microbial balance.’’ Afterwards, Salminen et al. in 1998, defined probiotics as ‘‘foods which contain live bacteria, which are beneficial to health,’’ whereas Marteau et al. in 2002 defined them as ‘‘microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being.’’ In these definitions the concept of an action on the gut microflora, and even that of live microorganisms disappeared. In 2001, a new and complete definition of probiotic has been presented by the Food and Agriculture Organization of the United
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_78, # Springer-Verlag Berlin Heidelberg 2010
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Nations–World Health Organization (FAO–WHO) and approved by the International Scientific Association for Probiotics and Prebiotics and best exemplifies the breadth and scope of probiotics as they are known today: ‘‘Live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.’’ This definition retains the historical elements of the use of living organisms for health purposes, but does not restrict the application of the term only to oral probiotics with intestinal outcomes [2]. Probiotics represent a large variety of bacterial genera, species, and strains. Several criteria have been proposed for considering a given microorganism as probiotic. These include the ability to adhere to cells, exclude or reduce pathogenic adherence, persist and multiply, produce acids, hydrogen peroxide, and bacteriocins antagonistic to pathogen growth, be safe, noninvasive, noncarcinogenic, and nonpathogenic, and coaggregate to form a normal balanced flora. Different strains have different actions in different clinical situations and moreover, it is important to stress that each probiotic microrganism displays its own properties and so data obtained from one strain cannot be extrapolated to another. The most common microorganisms used as probiotics are strains of lactic acid bacteria such as Lactobacillus, Bifidobacterium genera, other bacterial genera including Enterococcus and Streptococcus. Moreover, VSL#3, a patented bacterial preparation including four strains of Lactobacilli, three strains of Bifidobacteria, and one strain of Streptococcus salivarius subsp. thermophilus, also possesses properties that make it a probiotic agent. With the first publication in 1987 on the general properties of the Lactobacillus GG and its antimicrobial substance [3], a new era was initiated in which research laboratories from many countries began serious investigations on a variety of probiotic strains. Probiotics provide an attractive alternative to antibiotics in the treatment of inflammatory bowel disease (IBD) [4]. In addition, there is considerable evidence that the highly concentrated cocktail of probiotics, VSL#3 is efficacious in preventing onset and relapse of pouchitis, a nonspecific inflammation of the ileal reservoir after ileoanal anastomosis, which appears to be associated with bacterial overgrowth and dysbiosis [5]. Probiotics have also been implicated in the prevention and decreased recurrence of colon and bladder cancer [6, 7]. Antitumoral effects of selected strains of probiotics in vitro and in vivo have also been reported [8–10]. Probiotics have been demonstrated to have an adjuvant effect on immunological responses; their interaction with mesenteric lymph nodes can result in an
up-regulation of pIgA against intestinal pathogens and food antigens [11]. Promising applications include the prevention of respiratory infections in children, prevention of dental caries, elimination of nasal pathogen carriage, prevention of relapsing Clostridium difficile-induced gastroenteritis. Proposed future applications include the treatment of rheumatoid arthritis, treatment of irritable bowel syndrome, prevention of ethanol-induced liver disease, treatment of diabetes, and prevention or treatment of graft versus host disease [10].
Probiotics in Aging Skin Aging has been defined as the accumulation of molecular modifications, which manifest as macroscopic clinical changes. Human skin, unique among mammalians insofar as it is deprived of fur, is particularly sensitive to environmental stress. Major environmental factors have been recognized to induce modifications of the morphological and biophysical properties of the skin. Factors as diverse as ultraviolet radiation, atmospheric pollution, wounds, infections, traumatisms, anoxya, cigarette smoke, and hormonal status have a role in increasing the rate of accumulation of molecular modifications and have, therefore, been termed ‘‘factors of aging.’’ Aging of the skin is commonly associated with increased wrinkling, sagging, and increased laxity, but when considering the underlying reasons for these changes, it is important to distinguish between the effects of true biological aging (intrinsic aging) and environmental factors, such as exposure to the sun (extrinsic aging). Generally, the molecular changes of photoaging are considered to be as augmentation and amplification of the molecular changes associated with chronological skin aging [12]. In terms of biochemical and molecular mechanisms, skin aging is a really complex process, which involves a variety of changes and a lot of molecules. This section highlights certain aspects of the properties of probiotics that could have interesting implications in the skin aging treatment, even if future investigations will be indispensable. In > Fig. 78.1, a scheme is reported, which summarizes the main biochemical, molecular, and cellular changes underlying skin aging process that include alterations of skin-associated microflora, skin pH increase, reduced stratum corneum lipid levels, abnormal oxidative stress, collagen level reduction, and altered immune responsiveness. The possible sites of action of probiotics useful to slow down or inhibit the process of cutaneous aging are also highlighted.
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. Figure 78.1 Skin aging-associated biochemical, molecular, and cellular changes and possible sites of action of probiotics
Probiotics and Skin Aging-associated Microflora Changes The probiotic principle is likely to be applicable to any environment where a normal microflora exists. The skin also has a normal microflora [13], albeit less complex than the intestinal microflora because of the harsh environment provided by the human skin. The normal microflora of the skin is composed of a limited number of microbial types, mainly gram-positive species. A number of physiological conditions such as hydration, pH, O2, and growth substrates are the major factors in determining the limited number of microbial species that colonize human skin. Cutaneous microflora defends the skin against premature aging, inflammation, and dehydration and is involved in competitive exclusion of pathogens and increases the acidic nature of the skin, thereby making it even more inhospitable to many pathogens [14]. Some microflora are able to breakdown the fatty acid molecules (from the natural oils) in the skin and thereby increase its acidity. Skin microflora is different depending on the site
of the body. The most common genera found in the microflora of the skin are Propionibacteria, Staphylococcus, Micrococcus, Corynebacterium, and the yeast Malassezia [13]. Based on the proposed probiotic therapy to positively modulate the intestinal microflora, the use of probiotics is postulated to also change the composition of the skin microflora from a potentially harmful composition towards a microflora that would be beneficial for the host [15]. Due to competition for adhesion sites and nutrients, and possibly the production of antimicrobial substances, levels of certain less desirable genera can decrease. However, because the skin has an entirely different environment than the intestine, some different selection criteria for probiotics would be applied. Acid and bile resistance are prime selection criteria for intestinal probiotics, obviously these are not relevant for application to the skin. On the other hand, adhesion is important for the skin as well, to improve transient colonization and colonization resistance toward potential pathogens. Also, production of antimicrobial substances is important for an application on the skin, which, together with
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inhibition of pathogen adhesion, provides colonization resistance. Of interest, Ouwehand et al. (2003) have investigated the possibility of applying probiotics to the skin [15]. Propionic acid bacteria (PAB) were chosen as potential probiotics because they are members of the normal microbiota [16] and have been observed to exhibit antifungal activity [17]. All tested probiotic strains were found to inhibit the growth of some of the target strains, the Candida albicans strains being mainly sensitive. All of the tested potential probiotic strains were found to exhibit some adhesion to keratin, the main protein of the skin. Two of the tested strains were, in fact, found to adhere well; 16% and 20% of the applied cells, Propionibacterium freudenreichii ssp. freudenreichii 20271 and Lactobacillus rhamnosus 5.5a, respectively. The results of this study strongly encourage the use of skin probiotics even if further studies are needed and should focus on the identification and assessment of strains that also exhibit activity in vivo against potential skin pathogens and will indeed persist on the skin in vivo and be active there.
Probiotics and Skin Aging-associated pH Changes Normal skin pH is somewhat acidic and in the range of 4.2–5.6 and has been attributed largely to endogenous agents including the Na+/H+ antiporter, NHE1, and one or more secretory phospholipase/s A2 (sPLA2) enzymes, which hydrolyses membrane phospholipids, thereby generating free fatty acids (FFAs) that contribute to the acidification of the stratum corneum [18, 19]. The acid mantle, the combination of sebum (oil) and perspiration on the skin’s surface protects and renders the skin less vulnerable to damage. It also protects from attack by environmental factors such as the sun and wind and leaves it less prone to dehydration. The acid skin pH keeps the resident bacteria flora (see above) attached to the skin, whereas an alkaline pH promotes the dispersal from the skin [20]. The natural pH varies from one part of the body to the other and, in general, the pH of a man’s skin is lower than a woman’s skin. This acidic environment is very important, as it discourages bacterial colonization and provides a moisture barrier through absorption of moisture by amino acids, salts, and other substances in the acid mantle and in addition regulates the activity of many of the enzymes in the stratum corneum [21]. For example, the activities of both b-glucocerebrosidase and acidic sphingomyelinase are optimal at or below pH 5.5. If the pH of the stratum corneum is increased, the activities of b-glucocerebrosidase and acidic sphingomyelinase are
reduced and the extracellular processing of glucosylceramides and sphingomyelins to ceramides is impaired, leading to abnormalities in the structure of the extracellular lipid membranes and decreased permeability barrier function [22–24]. On the other hand, many of the proteases in the stratum corneum have, instead, an optimum pH of 7 or higher; therefore, their activity is low at the usual stratum corneum pH. Thus, increases in stratum corneum pH stimulate protease activity, resulting in increased corneocyte desquamation [22–24]. Skin pH is relatively constant from childhood to approximately age 70, then rises significantly, the increase being especially pronounced in lower limbs, possibly related to impaired circulation and, consequently to stasis, and reduced oxygen supply [25]. Recently, a decreased NHE1 expression that accounts for the pH abnormality in moderately aged epidermis in mice and human has been reported [26]. The reduced NHE1 expression could account the impairment of lipid processing and epidermal barrier homeostasis in aged skin even if further studies will be required to delineate whether altered sPLA2 activity also contributes to the functional abnormalities in moderately aged epidermis. An interesting property of probiotics is the fermentative metabolism that involves the production of acid molecule, thus acidifying the surrounding environment. Moreover, Yadav and Sinha (2007) have recently reported the ability of probiotic Lactobacilli to increase the production of free fatty acids (FFAs) by lipolysis of milk fat and to produce conjugated linoleic acid (CLA) by using internal linoleic acid, which may confer nutritional and therapeutical value to probiotic treatment [27]. These evidences suggest that the oral assumption and/ or the topical application of probiotic preparation on the aged skin could cause a pH decrease thus coming back near the physiological acid pH. Consequently, the most important cutaneous enzymes that have been impaired by aging, could again function.
Probiotics and Skin Aging-associated Altered Stratum Corneum Lipid Composition Several studies have demonstrated that ceramides play an essential role in both the barrier and water-holding functions of healthy stratum corneum (SC), suggesting that the dysfunction of the stratum corneum associated with aging as well as that observed in patients with several skin diseases could result from a ceramide deficiency [28]. A previous study reported a significant increase in skin ceramide levels in healthy subjects, after a treatment in vivo with a cream containing sonicated S. salivarium ssp.
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thermophilus [29]. The presence of high levels of neutral sphingomyelinase activity in this organism was responsible for the observed increase of stratum corneum ceramide levels, thus leading to an improvement in barrier function and maintenance of stratum corneum flexibility. There is also evidence that the treatment with a sonicated preparation of a S. salivarium ssp. thermophilus S244 was able to induce an increasing ceramide levels in vivo, on stratum corneum of atopic dermatitis patients [30]. Considering the role of the ceramides in regulating the waterholding capacity and in maintaining skin integrity, the possibility that the topic application of a probiotic formulation, representing a source of exogenous SMase able to hydrolyze skin SM and consequently to generate ceramides, may lead to reduce dryness, loss of tone, fullness, and water loss, thus slowing the process of skin aging [31], has been recently investigated. The skin barrier and the water-holding capacity are the other most important functions of the SC and these functions are related to the composition and structure of SC intercellular lipids [32, 33], including cholesterol, ceramides, and fatty acid. Therefore, the capacitance and ceramide levels as markers of epidermal hydration were determined. The findings indicated that the barrier improvement, resulting in a prompt increase in the water-holding capacity, was observed when the aged subjects was applied S. thermophilus-containing cream. In fact at the end of the treatment a statistically significant increase in hydration values was showed when compared with the values observed at the beginning. An amelioration in hydration skin could be attributed to the increase of the stratum corneum ceramides levels. Topical application of a sonicated S. salivarium spp. thermophilus preparation lead to increased non-hydroxy and hydroxy fatty acid ceramides levels in stratum corneum. These results could be again explained with the presence of high levels of neutral SMase in S. thermophilus. Altogether, the findings suggest that there are two eventual possibilities by which topical application of a sonicated S. thermophilus preparation may contribute to the improvement of lipid barrier and a more effective resistance against aging-associated skin xerosis. One possibility would be that the presence of high levels of neutral SMase in S. thermophilus hydrolyses skin SM thus generating ceramides, with structural function in the stratum corneum lipid bilayers. The other eventuality is that S. thermophilus SMase-produced ceramides are involved in epidermal differentiation and proliferation signaling pathway as important second messenger, as previously described [34]. Thus, although the mechanism of action of topical application of a sonicated S. thermophilus preparation needs to be further elucidated, the results
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obtained with this experimental cream consist in a relevant increase of skin ceramide levels, which was associated to a more effective resistance against aging-associated skin xerosis.
Probiotics and Skin Aging-associated Oxidative Stress The epidermis of skin possesses an extremely efficient antioxidant activity that is superior to most tissues [35], and it has been proposed that the reduction in efficiency of this system during aging is an important factor in skin aging. There are many reports describing the reduction of antioxidant enzymes in skin with age, while others suggest that skin aging is not due to a general decline in antioxidant capacity. However, all agree that the accumulation of free radicals throughout life most likely promotes cellular aging. Generation of reactive oxide species (ROS) is thought to play a major role in skin aging. All the biological structures, as human skin, undergo the detrimental action of ROS. The free radical theory of aging proposes that aging results from accumulation of oxidative damage over a lifetime due to excess ROS, which result from aerobic metabolism [36]. ROS generation is increased in aged skin and represents a key step in molecular pathways, which eventually lead to increased collagen breakdown. ROS cause damage to lipids, proteins, and DNA and also influence cellular senescence [37]. In addition, free radicals also cause damage to connective tissue components of the dermis, particularly collagen [38], which again is likely to influence cell behavior via cell– matrix interactions. Indeed, poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NF-kB and AP-1, which are involved in several aging-associated degenerating processes, including extracellular matrix degradation [39]. Probiotics have been demonstrated extracellularly to produce effective bioactive molecules exerting several beneficial effects as antioxidative effects by different mechanisms, including the release of exopolysaccharides (EPSs), a class of such effective biomolecules that probiotic bacteria release into the surroundings to protect themselves under starvation conditions and also at extreme pH and temperature conditions [40]. These EPSs are long-chain, high-molecular-mass polymers, which are used in food and dairy industries as texturizers, viscosifiers, and syneresis-lowering agents [41, 42]. They have also been reported to show antiulcer, immunomodulatory, antiviral, antioxidant, and various other biological activities. Recently, studies have demonstrated that microbial EPS has significant
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antioxidant and free radical scavenging activities, and also have numerous potential applications as pharmaceutical formulations [43]. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzyme superoxide dismutase (SOD). Bruno-Ba`rcena et al. (2004) showed that heterologous expression of an SOD gene in intestinal Lactobacilli provides protection against peroxide toxicity [44]. Indeed, the authors suggest that it may be possible to use these SOD-rich species in biotherapy for treatment of peptic ulcers or ulcerative colitis. Using a similar approach the cumulative oxidative damage could also be reduced in the aged skin.
Probiotics and Skin Aging-associated Collagen Level Reduction The processes associated with intrinsic skin aging are thought to result from a combination of events including decreased proliferative capacity of skin-derived cells, decreased matrix synthesis in the dermis, and increased expression of enzymes that degrade the collagenous matrix. Collagen is one of the main building blocks of human skin, providing much of the skin’s strength. Dermal fibroblasts make precursor molecules called procollagen, which is converted into collagen. There are two important regulators of collagen production: transforming growth factor (TGF)-b, a cytokine that promotes collagen production, and activator protein (AP)-1, a transcription factor that inhibits collagen production and up-regulates collagen breakdown by up-regulating enzymes called matrix metalloproteinases (MMPs) [45, 46]. In aged skin, there is elevation of AP-1 as compared to young skin [47]. MMP activity is increased in aged human skin, and is associated with dramatic increased levels of degraded collagen [48]. In addition, synthesis of types I and III procollagen is reduced in aged human skin [49]. The combination of increased breakdown of collagen and decreased synthesis of new collagen results in an overall decrease in collagen levels in the dermis. The MMPs are a large family of degradative enzymes and four in particular are thought to be important in matrix degradation in the skin. The combined actions of collagenase (MMP1), 92 kDa gelatinase (MMP2), 72 kDa gelatinase (MMP9), and stromelysin 1 (MMP3) can fully degrade skin collagen and components of the elastic network. Coupled with these changes, elastin gene expression is markedly reduced after the age of 40–50, as determined by mRNA steady state levels in cultured fibroblasts, and there is a progressive disappearance of elastic tissue in the dermis. In aged
skin there is an increase of MMP activity and reduced collagen I expression. Moreover, an irradiation of human skin with just a single dose of UV light has been shown to increase the activities of MMPs, and this has been associated with significant degradation of collagen fibers. In presenescent dermal fibroblasts, metalloproteinase activity is relatively low with MMP1 and MMP3 shown to be expressed at very low levels. In contrast, levels of matrix metalloproteinase inhibitors TIMP1 and TIMP3 are high, further reducing degradative capacity. In senescent fibroblasts, however, this is reversed with an increase in matrix metalloproteinase expression and a reduction in the expression of tissue inhibitors of metalloproteinase (as a review see [50]). Ulisse et al. (2001) demonstrated the capacity of an oral VSL#3 treatment to decrease MMP activity tissue levels in the maintenance treatment of patients with pouchitis [51]. Further insights into the molecular basis of periodontitis have identified the potential clinical significance, giving the experimental ground for a new innovative, simple, and efficacious therapeutical approach of periodontal disease [52]. In particular, the anti-inflammatory effects of L. brevis extracts on periodontitis patients, which were associated to a significant decrease of MMP levels in saliva samples, was assayed by zymogram and Western blotting. Moorthy et al. (2007) evaluated the effect of L. rhamnosus and L. acidophilus on neutrophil infiltration and lipid peroxidation during Shigella dysenteriae 1-induced diarrhea in rats demonstrating a reduction of levels of myeloperoxidase, lipid peroxidation, alkaline phosphatase, and the expression of MMP2 and MMP9 [53]. Together these data suggest that probiotic treatment could decrease skin aging-associated MMP activity and may represent a new, promising, and inexpensive approach to treat the cutaneous laxity.
Probiotics and Skin Aging-associated Altered Immune Response Aging is accompanied by a reduction in the functional capacity of all the organs in the body and accordingly the activity of the immune system also declines with age (as a review see [54]). The senescence of the immune system especially affects cell-mediated as well humoral immunity. A decrease has also been observed in the ratio of mature to immature T lymphocytes and an increase in proinflammatory cytokine and ROS production [55]. Age-related alterations in immune function also affect the skin, and may account for the increased susceptibility in the elderly to cutaneous infections and malignancies, and decreased or variable contact hypersensitivity reactions [56]. Perhaps
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associated with these immunological changes, and certainly with other physiological and environmental factors, the bifidobacteria numbers in the gut decrease markedly after 55–60 years of age [57]. The immune system of the elderly is a potential target for probiotics, as it is known to be affected adversely by the aging process, leading to decreasing resistance to diseases [57]. Several studies have reported the capacity of probiotics to counteract the immunosenescence process and to protect against infection [58, 59]. Probiotics have been demonstrated to induce an adjuvant effect on immunological responses and this evidence suggests that the use of probiotics could be also effective in enhancing the skin barrier function, even if not all probiotics have the same immunologic properties (as a review see [60]). Immune regulation by probiotics is thought to be mediated through the balancing control of pro- and anti-inflammatory cytokines. Some strains of the genus Bifidobacterium exhibit powerful anti-inflammatory properties, and thus may be able to restore an unbalanced cytokine production [61]. The efficacy of probiotic organisms in the treatment of pouchitis is also reported; an effect that could be in part attributed to nitric oxide synthase (NOS)-II activity decrease [51]. Nitric oxide (NO) is a paracrine regulator of various biological functions and is known to be involved in the physiology and pathophysiology of many systems, including skin. It is synthesized from L-arginine by NOS. Of interest, the expression of NOS-II is also strongly implicated in several inflammatory skin conditions [62]. A recent study aimed to investigate the beneficial effects of L. brevis extracts on periodontitis patients reported that the relevant anti-inflammatory effects of L. brevis extracts could be attributed to the presence of high levels of arginine deiminase, which, also in this inflammatory model, metabolizing arginine to citrulline and ammonia, indirectly leads to nitric oxide (NO) generation inhibition, by competing with NOS for the same substrate, arginine [52]. The association between the composition of the Bifidobacterium microbiota and the different level of proinflammatory cytokine TNF-a as well as anti-inflammatory cytokine TGF-b and regulatory cytokine IL-10 has been recently investigated [63]. The results showed that Bifidobacterium microbiota of the elderly may be modified through a probiotic intervention, and that even modest changes in the levels of specific Bifidobacterium species may be associated with changes in the cytokine levels, indicating that modulation of the intestinal Bifidobacterium microbiota may provide a means of influencing the inflammatory responses in the elderly. Nutritional intervention, particularly with dietary antioxidants have been proposed to protect against UV-induced skin damage and
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during recent years an increasing interest has been shown for new nutritional approaches using live microorganisms as probiotics. UV radiation is known to alter the cutaneous and systemic immune systems implicated in the development of skin tumors. Of note, findings suggest that ingested probiotic bacteria (L. johnsonii La1) can maintain in a mouse model as well as in humans a normal cutaneous immune capacity after UV exposure have been reported [64, 65]. The presented evidence would suggest that La1, via priming the immune system in the gut, may be considered an immunoprotector against the predicted immunosuppressive effect of UV on the skin immune system. In particular, probiotic ingestion was able to allow a protective cutaneous hypersensitivity reaction, a normal epidermal Langerhans cell density, as well as to maintain or restore the systemic IL-10 production to levels equivalent to non-UV-exposed conditions, thus confirming the ability of La1 to preserve the capacity of the organism to respond to immunological changes. Of note, the ingestion of probiotics has been associated with a diminution in the severity of autoimmune, particularly intestinal inflammatory, diseases as well as allergic disorders [66, 67]. In conclusion, the scientific literature strongly supports the ability of probiotics to modulate the immune response as well their capacity to counteract the immunosenescence process and to protect against infection [58, 59].
New Frontiers in Probiotic Research and Concluding Remarks As the elderly population increases, the prevalence of aging-related diseases will increase, and functional foods that provide health benefits to control aging and prolong health span will become more desirable. The experimental evidences summarized in the present review strongly encourage the treatment with selected probiotic strains as sun protector, in ameliorating the aging skin condition, in improving dermocosmetic treatments, in recovering skin properties after an injury, as well in preparing skin to cutaneous laser resurfacing. Laser procedures for the aging face are numerous and emerging rapidly. Ablative laser resurfacing is considered to be the gold standard to improve clinical features of the aging face and generally refers to treatment with a carbon dioxide laser (10,600 nm) [68]. It improves fine and some coarse wrinkles and overall dyspigmentation, lightens dark under-eye circles, and generally improves the texture of skin; it can also be used to ameliorate old acne scarring. Side effects include increased erythema immediately following the treatment,
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slight discomfort, swelling, and potential bruising. Pretreatment clinical assessment and consultation are critical before prescribing or performing the treatments and procedures to review the risks and complications [39]. The potent anti-inflammatory effects exerted by selected strains of probiotics could render them good candidates to prevent or reduce, at least partially, the side effects associated to ablative laser resurfacing. Moreover, considering that most of the topically applied cosmetic products have only a short-term effect on superficial structures, the oral supplementation can be integrated with topical products to obtain an even more effective result. Since probiotics are intrinsically benign bacteria and they appear to implant, at least temporarily, in the gastrointestinal tract of nearly everyone who consumes them, they can be used as vehicles for transporting genes of medical importance to the host [69]. This approach is also helpful to slow the process of skin aging. Advances in the field of probiotic research provide new delivery systems, creation of disease-targeted recombinant strains, and isolation and characterization of signaling molecules that can modulate microbial biofilms and infectious processes [2] (> Fig. 78.2). Moreover, the development of new delivery mechanisms could provide encapsulating probiotics, such that they rehydrate at specific sites, and encasing prebiotics in nanoaggragates that could protect against stomach acid and deliver their payload when the pH reaches 7.4 [70]. Potentially, such nanoencapsulation will also allow probiotic delivery in foods, such as biscuits [2]. At the macromolecular level, it will be possible to coat capsules with biosensors that detect the optimal conditions for release of probiotic contents as suggested by Hopper [71]. An alternative approach to improving probiotic efficacy is to enhance a strain’s ability to cope with stress at the genetic level. This approach has been successfully employed to increase the stress tolerance profile of two probiotic strains: L. salivarius UCC118 and Bifidobacterium breve UCC [72, 73]. Cloning the betL gene into L. salivarius resulted in a significant increase in the ability of the transformed strain to accumulate . Figure 78.2 New frontiers in probiotic research
betaine, which confer increased salt tolerance and osmotolerance, thus improving the clinical efficacy of the probiotic. The growing rates of antibiotic resistance and the realization that biofilm formation makes it more difficult for antibiotics to eradicate infections, have led to studies of new approaches for managing infectious biofilms using probiotics. These include disruption or penetration of biofilms by beneficial microbes or alteration of the environment to restore a noninfectious biofilm. An in vitro study has shown that Gardnella vaginalis species biofilms can be penetrated by L. rhamnosus GR-1, leading to rapid disruption and death of the pathogens [74]. Other studies have shown that this Lactobacillus strain can also prevent the formation of C. albicans biofilms, and it can kill the yeast in vitro [2]. Moreover, fluorescent in situ hybridization and confocal laser microscopy, used successfully to study complex oral biofilms, could be used to better understand how gram-negative and gram-positive bacteria interact in biofilms and are affected by different nutrients, as suggested by Thurnheer [75]. In summary, biotechnology holds the key to future advances in the clinical application of probiotic product. An additional message, supported by scientific evidence, strongly emerges that probiotics can enhance health in a more holistic manner, by improving the balance of the intestinal flora, preventing disease, reducing allergic events, interdicting the introduction of harmful microorganisms, and suppressing intestinal enzyme activity that could have detrimental effects. Probiotics must be viewed as healthy additions to everyone’s diet. The discovery of new probiotics has been based on a calculated strategy of considering the characteristics of an ideal strain and then asking nature to provide it within the diversity of the microbial world. Now the objective should be to define the appropriate uses of probiotics and to discover new applications, which will bring benefit to human health including ameliorating the aging skin condition.
Cross-references > The
Potential of Probiotics and Prebiotics for Skin Health
References 1. Fioramonti J, et al. Probiotics: what are they? What are their effects on gut physiology? Best Pract Res Clin Gastroenterol. 2003;17 (5):711–724. 2. Reid G. How science will help shape future clinical applications of probiotics. Clin Infect Dis. 2008;46(Suppl. 2):S62–S66.
Probiotics in Aging Skin 3. Silva M, et al. Antimicrobial substance from a human Lactobacillus strain. Antimicrob Agents Chemother. 1987;31:1231–1233. 4. Heczko PB, et al. Critical evaluation of probiotic activity of lactic acid bacteria and their effects. J Physiol Pharmacol. 2006;57(Suppl 9):S5–S12. 5. Gionchetti P, et al. Antibiotics and probiotics in treatment of inflammatory bowel disease. World J Gastroenterol. 2006;12:3306–3313. 6. Farnworth ER. The evidence to support health claims for probiotics. J Nutr. 2008;138(6):1250S–1254S. 7. Sleator RD, et al. New frontiers in probiotic research. Lett Appl Microbiol. 2008;46(2):143–147. 8. Di Marzio L, et al. Apoptotic effects of selected strains of lactic acid bacteria on a human T leukemia cell line are associated with bacterial arginine deiminase and/or sphingomyelinase activities. Nutr Cancer. 2001;40(2):185–196. 9. de Moreno de LeBlanc A, et al. The application of probiotics in cancer. Br J Nutr. 2007;98(Suppl 1):S105–S110. 10. Goldin BR. Clinical indications for probiotics: an overview. Clin Infect Dis. 2008;46(Suppl 2):S96–S100. 11. Macpherson AJ, et al. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science. 2004;303:1662–1665. 12. Fisher GJ, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138(11):1462–1470. 13. Bojar RA, et al. Review: the human cutaneous microflora and factors controlling colonisation. World J Microbiol Biotechnol. 2002;18 (9):889–903. 14. Cogen AL, et al. Skin microbiota: a source of disease or defence? Br J Dermatol. 2008;158(3):442–455. 15. Ouwehand AC, et al. Probiotics for the skin: a new area of potential application? Lett. Appl Microbiol. 2003;36(5):327–331. 16. Tannock GW. Normal Microflora. An Introduction to Microbes Inhabiting the Human Body. London: Chapman & Hall, 1995. 17. Suomalainen R, et al. Propionic acid bacteria as protective cultures in fermented milks and breads. Lait. 1999;79:165–174. 18. Fluhr JW, et al. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity. J Invest Dermatol. 2001;117:44–51. 19. Fluhr JW, et al. Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum. J Invest Dermatol. 2004;122:320–329. 20. Lambers H, et al. Natural skin surface pH is on average below 5, which is beneficial for its resident flora. Int J Cosmet Sci. 2006;28 (5):359–370. 21. Mauro T. SC pH: measurement, origins, and functions. In: Elias P, Feingold K (eds) Skin Barrier. New York: Taylor & Francis, 2006, pp. 223–229. 22. Fluhr JW, et al. Functional consequences of a neutral pH in neonatal rat stratum corneum. J Invest Dermatol. 2004;123:140–151. 23. Hachem JP, et al. pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol. 2003;121:345–353. 24. Hachem JP, et al. Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity. J Invest Dermatol. 2005;125:510–520. 25. Waller JM, et al. Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res Technol. 2005;11(4):221–235. 26. Choi EH, et al. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol. 2007;127 (12):2847–2856.
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27. Yadav H, et al. Production of free fatty acids and conjugated linoleic acid in probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei during fermentation and storage. Intern. Dairy J. 2007;17(8):1006–1010. 28. Holleran WM, et al. Epidermal sphingolipids: metabolism, function, and roles in skin disorders. FEBS Lett. 2006;580(23):5456–5466. 29. Di Marzio L, et al. Effect of the lactic acid bacterium Streptococcus thermophilus on ceramide levels in human keratinocytes in vitro and stratum corneum in vivo. J Invest Dermatol. 1999;113(1): 98–106. 30. Di Marzio L, et al. Effect of the lactic acid bacterium Streptococcus thermophilus on stratum corneum ceramide levels and signs and symptoms of atopic dermatitis patients. Exp Dermatol. 2003;12 (5):615–620. 31. Di Marzio L, et al. Increase of skin-ceramide levels in aged subjects following a short-term topical application of bacterial sphingomyelinase from Streptococcus thermophilus. Int J Immunopathol Pharmacol. 2008;21(1):137–143. 32. Denda M, et al. Age- and sex-dependent change in stratum corneum sphingolipids. Arch Dermatol Res. 1993;285(7):415–417. 33. Motta S, et al. Abnormality of water barrier function in psoriasis. Role of ceramide fractions. Arch Dermatol. 1994;130(4):452–456. 34. Jensen JM, et al. Acid and neutral sphingomyelinase, ceramide synthase, and acid ceramidase activities in cutaneous aging. Exp Dermatol. 2005;14(8):609–618. 35. Kohen R, et al. Skin low molecular weight antioxidants and their role in aging and in oxidative stress. Toxicology. 2000;148(2–3):149–157. 36. Hensley K, et al. Reactive oxygen species and protein oxidation in aging: a look back, a look ahead. Arch Biochem Biophys. 2002;397 (2):377–383. 37. Tzaphlidou M. The role of collagen and elastin in aged skin: an image processing approach. Micron. 2004;35(3):73–177. 38. Dalle Carbonare M, et al. Skin photosensitizing agents and the role of reactive oxygen species in photoaging. J Photochem Photobiol. 1992;14(1–2):105–124. 39. Helfrich YR, et al. Overview of skin aging and photoaging. Dermatol Nurs. 2008;20(3):177–183. 40. Kodali VP, et al. Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium. Biotechnol J. 2008;3(2):245–251. 41. Cerning J. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol Rev. 1990;7(1–2):113–130. 42. Welman AD, et al. Exopolysaccharides from lactic acid bacteria: perspectives and challenges. Trends Biotechnol. 2003;21(6):269–274. 43. Kishk YFM, et al. Free-radical scavenging and antioxidative activities of some polysaccharides in emulsions. LWT Food Sci Technol. 2007;40(2):270–277. 44. Bruno-Ba´rcena JM, et al. Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity. Appl Environ Microbiol. 2004;70(8):4702–4710. 45. Kang S, et al. Photoaging and topical tretinoin: therapy, pathogenesis, and prevention. Arch Dermatol. 1997;133(10):1280–1284. 46. Massague´ J. TGF-b signal transduction. Annu Rev Biochem. 1998;67:753–791. 47. Chung JH, et al. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J Invest Dermatol. 2000;115(2):177–182. 48. Fisher GJ, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138(11):1462–1470. 49. Varani J, et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and
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64. Gue´niche A, et al. Supplementation with oral probiotic bacteria maintains cutaneous immune homeostasis after UV exposure. Eur J Dermatol. 2006;16:511–517. 65. Peguet-Navarro J, et al. Supplementation with oral probiotic bacteria protects human cutaneous immune homeostasis after UV exposuredouble blind, randomized, placebo controlled clinical trial. Eur J Dermatol. 2008;18:504–511. 66. Canche-Pool EB, et al. Probiotics and autoimmunity: an evolutionary perspective. Med Hypotheses. 2008;70:657–660. 67. Hsu CJ, et al. Emerging treatment of atopic dermatitis. Clin Rev Allergy Immunol. 2007;33:199–203. 68. Railan D, et al. Ablative treatment of photoaging. Dermatol Ther. 2005;18:227–241. 69. Gorbach SL. Probiotics in the Third Millennium. Digest Liver Dis. 2002;34(Suppl 2):2–7. 70. Fan YF, et al. Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-by-layer adsorption. Int J Pharm. 2006;324:158–167. 71. Hooper LV, et al. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proc Natl Acad Sci USA 1999;17(96):9833–9838. 72. Sheehan VM, et al. Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118. Appl Environ Microbiology. 2006;72(3):2170–2177. 73. Sheehan VM, et al. Improving gastric transit, gastrointestinal persistence and therapeutic efficacy of the probiotic strain Bifidobacterium breve UCC2003. Microbiol. 2007;153(10):3563–3571. 74. Saunders S, et al. Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms. Colloids Surf B Biointerfaces. 2007;55(2): 138–142. 75. Thurnheer T, et al. Multiplex FISH analysis of a six-species bacterial biofilm. J Microbiol Methods. 2004;56:37–47.
Modeling
76 Skin Aging: A Generalization of the Micro-inflammatory Hypothesis Paolo U. Giacomoni . Glen Rein
Introduction This chapter describes the phenomenology of skin aging. It analyzes the clinical, biophysical, histological, electronmicroscopy, cellular, and macromolecular aspects of skin aging. It also identifies environmental and lifestyle factors that accelerate the rate of aging. In addition, it explores metabolic and genetic factors. Factors of aging provoke physiological responses that share common features. For example, consider the pathways to the onset of solar elastosis and varicose vein. Ultraviolet (UV) radiation damages epidermal cells. Damaged cells trigger the arachidionic acid cascade and the release of prostaglandins and leukotriens. Upon binding these molecules, resident mast cells release histamine and tumor necrosis factor a (TNF-a), which promote synthesis and mobilization of intercellular adhesion molecule 1 (ICAM-1) in nearby endothelial cells. Circulating monocytes and macrophages bind ICAM-1, roll over, enter the dermis, and chemotactically migrate to reach the UV-damaged cell. Evidence for this phenomenon is provided by the histology documented inflammatory infiltration [1]. In so doing, they release proteases and damage the extracellular matrix (ECM), thus accelerating the aging process, which has been defined as accumulation of damage versus time [2–4]. Damaged elastic fibers are slowly replaced by new, disorganized ones [5, 6] and, with chronic exposure to solar radiation, the elastic properties of the skin are lost. When constrained to a static position, a person develops anoxia in the veins of the lower legs. Anoxia provokes the synthesis and the mobilization of ICAM-1 in the endothelial cells lining the vein walls [7], monocytes and macrophage bind ICAM-1, roll over, perform diapedesis, infiltrate the surrounding extracellular matrix (ECM) and, not having chemotactical signals to follow, exert their destructive action on the smooth muscle cells surrounding the vein. With chronic exposure to anoxia, the smooth muscle cells are heavily damaged, the vein walls collapse, and varicose veins appear as a sign of accelerated vascular aging.
It appears that two totally unrelated phenomena such as solar elastosis and varicose vein are the consequences of a common physiological response (the synthesis and mobilization of ICAM-1) to causes as different as ultraviolet radiation and anoxia. The comparison of the onset of solar elastosis and of varicose vein leads to the proposal of the micro-inflammatory model for skin aging [4]. Due to accumulating evidence that environmental, lifestyle, and metabolic factors can also trigger its onset, the micro-inflammatory model can be now considered more of a testable hypothesis than a simple mechanistic model.
The Micro-inflammatory Hypothesis of Skin Aging The aging of skin is the consequence of three oxidative steps subsequent to the synthesis and mobilization of intercellular adhesion molecule 1 within the endothelium of cutaneous vessels. Agents able to provoke this synthesis and mobilization contribute to skin aging [4]. First oxidative step. Vascular cell-adhesion molecule-1 (VCAM-1) activates endothelial cell NADPH oxidase, which catalyzes production of reactive oxygen species (ROS). This activity is required for VCAM-1-dependent lymphocyte migration [8]. Upon binding ICAM-1 and rolling over, circulating inflammatory cells release hydrogen peroxide [9], endothelial cells lose intercellular contact, round up, and allow monocytes and macrophages to perform diapedesis across the vascular wall. Second oxidative step. In the extracellular matrix, inflammatory cells can either follow chemotactical signals to reach damaged somatic cells or agents of infection, or just exert their lytic functions randomly. In both cases, reactive oxygen species are released, together with specific proteases. Third oxidative step. In the presence of cells to destroy, engulf, and remove (such as damaged somatic cells, foreign bacteria, or molds), inflammatory cells release H2O2. By-standing resident cells can be damaged by this oxidative burst, and trigger the arachidonic acid cascade, the release of prostaglandins and leukotriens
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_76, # Springer-Verlag Berlin Heidelberg 2010
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which will be relayed by the secretion of histamine and TNF-a from resident mast cells, and these cytokines and autacoids will induce synthesis and mobilization of ICAM-1, thus perpetuating the inflammatory process. The accumulation of oxidative and proteolytic damage experienced over time by the extracellular matrix, together with the remodeling of fibers in a disorganized mode, leads to skin aging. The micro-inflammatory hypothesis emphasizes the aging of the cutaneous connective tissue and of the extracellular matrix. Macroscopic consequences of this hypothesis are verified by experiment. The recognition that post-UV-repair and wound-healing share ECM remodeling as a common feature has allowed one to understand why blood vessels are deeper down in aged skin than in young skin. The sagging of the dermis is the consequence of a modified ECM and is accompanied by an overall increase in the surface area of the skin, particularly of the face. With time, under the action of gravitational pull, the surface of the skin increases. It can be surmised that in order to keep skin around the skull, nerves and muscles act and pull the skin. Facial wrinkles form along the sites of attachment of skin to muscles. Wrinkles have a neuro-muscular cause and that is evidenced by their disappearance in hemiplegics, or when the individual is under general anesthesia or when treated with botox. The increase of skin surface area and the reduction of total body volume can be invoked to explain the observation that, notwithstanding a nearly constant rate of turnover of the keratinocytes through the life span, the thickness of the epidermis is diminished with aging. This is more the consequence of the stretching of the skin than the consequence of a modification of the turnover rate of the keratinocytes. Indeed, the turnover of the keratinocytes does not change with aging and this is confirmed by the fact that the thickness of the stratum corneum is, in fact, constant with aging [10].
Early Justification of the Micro-Inflammatory Hypothesis Environmental and lifestyle factors capable of accelerating skin aging were recognized by biomedical investigations in the course of the twentieth century and, as a consequence of the work of the European Network for the Biology of Aging, it was pointed out in 1996 that several factors of skin aging share as a common feature, the capability of inducing the synthesis and the mobilization of ICAM-1 in the endothelium [4]. The factors first recognized as having this capability were ultraviolet radiation, tractions, wounds, infections, trauma, anoxia, cigarette smoke, and specific
hormonal imbalances. A cause-effect relationship can be inferred between ICAM-1 synthesis and mobilization and protein glycation, stretching, electromagnetic fields, psychological stressors, and neuropeptides [11]. Some of the factors of skin aging are also direct cell- or tissue-damaging factors. When damage to cells or tissue is generated (e.g., by UV radiation, smoke-related free radicals, infectious agents, or wounds and traumas), a number of modifications are provoked to the ECM, to resident cells, and to vessel walls by the free radicals and lytic enzymes which are released in the course of the inflammatory response, consequent to the diffusion of cytokines produced via the arachidonic acid cascade. What about other factors of aging which are not directly damaging agents? Traction and gravitational forces provoke the activation of phospholipase A2, an enzyme involved in the arachidonic acid cascade. Anoxia induces ICAM-1 synthesis and diapedesis of macrophages, which start digesting the ECM around veins or other blood vessels. Glucose binds to proteins in a nonenzymatic glycation process and glycated proteins are inducers of ICAM-1 synthesis. Electromagnetic fields associated with computers provoke the release of histamine, IL-1, and IL-6. Neuropeptides regulate the expression of cell adhesion molecules on both leukocytes and endothelial cells in a coordinated effort to control neurogenic inflammation. These phenomena trigger a cycle of self-maintained inflammatory responses, which comprises the induction of mobilization and neosynthesis of ICAM-1, and are summarized in [4, 5, 11].
Extension of the Validity of the Micro-inflammatory Hypothesis Recent investigations have generated further results indicating that other factors of skin aging, the mode of action of which was not previously understood, induce physiological responses, which are in keeping with the microinflammatory hypothesis. Exposure to low temperatures, consumption of specific nutritional elements, neuromediators, physical exercise, and sleep deprivation are discussed.
Cold Epidemiological evidence indicates that exposure to low temperatures is associated with visible signs of skin aging, from type I and type II rosacea to the appearance of spider veins. Exposure to low temperatures provokes a vasoconstriction, which is mediated by endothelin-1. Levels of
Skin Aging: A Generalization of the Micro-inflammatory Hypothesis
circulating endothelin-1 increase sevenfold in venous plasma from a hand immersed in ice water, and threefold in venous plasma from the nonimmersed, controlateral hand [12]. Another study [13] reported that plasma endothelin-1 increased nearly two-fold in borderline hypertensive volunteers subjected to standard cold pressor test. In a field study, it was observed that exposure to high altitude (2,500 and 5,000 m above sea level), moderate cold (4 C), or freezing temperatures ( 18 C) increased endothelin-1 production in 25 healthy volunteers [14]. The levels of circulating adhesion soluble molecules, such as sI-CAM 1, sV-CAM 1, and sE-selectin, which are indicators of an existing inflammatory reaction, were found to increase within an hour after healthy individuals were subjected to a cold pressor test [15]. Levels of circulating adhesion molecules were not increased in 12 young healthy individuals infused with 0.4 pmol/kg of endothelin-1 for 6 h [16], whereas hypertensive patients have nearly threefold higher levels of endothelin-1 than normotensive individuals, and display 20–30% higher levels of circulating soluble ICAM-1 and VCAM-1 [17]. On the other hand, nanomolar to micromolar concentrations of endothelin-1 was observed to enhance the adhesion of human neutrophils to lipopolysaccharides and to endothelin-1-activated human coronary artery endothelial cells (HCAEC). This adhesion was inhibited by PAF antagonists as well as antagonists to endothelin receptor A. Remarkably enough, endothelin-1 increased the expression of E-selectin and ICAM-1 on HCAEC [18]. Endothelin-1 was also reported to mediate the induction of ICAM-1 and VCAM-1 by C-reactive protein in human saphenous vein endothelial cells [19]. Recent in vivo studies with diabetes mellitus patients and healthy controls pointed out that levels of endothelin-1 and ICAM-1 were positively correlated [20]. All these results are consistent with the conclusion that endothelin-1 regulates cell surface adhesion molecules including ICAM-1, which is key to cell–cell and cell–matrix adhesion and leukocyte infiltration [21]. From these studies one can expect that, upon chronic exposure to cold, the vasoconstriction provoked by endothelin-1 will be associated with a moderate increase in the levels of adhesion molecules with consequent diapedesis of circulating inflammatory cells. These could damage the smooth muscle cells surrounding the cutaneous blood vessels, thus maintaining a mild vasodilation, which can provoke a persistent erythema sometimes diagnosed as type I or type II rosacea. When the damage to smooth muscle cells causes the collapse of the capillary walls, the erythema can become permanent and be accompanied by broken capillaries, visible as spider veins.
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Ethanol The physiological effects of ethanol consumption are known to differ according to the dose and the frequency of ingestion. Serum levels of ICAM-1 and E-selectin were higher in a group of 30 chronic alcoholics than in 30 teetotaler controls [22]. Moderate drinkers, with a daily intake of 20–40 g ethanol/day, have lower serum ICAM-1 and VCAM-1 levels than teetotalers, whereas heavy drinkers display much higher level of adhesion molecules than moderate drinkers or abstinent controls [23]. Furthermore, moderate consumption of sparkling wine, red wine, or white wine in healthy individuals promotes the decrease of serum level of circulating VCAM-1, E-selectin, and P-selectin [24, 25]. These results suggest that moderate alcohol intake has antiinflammatory effects on the cardiovascular system, but at higher doses ethanol can exert a pro-inflammatory effect. Indeed, chronic alcoholics exhibit significantly higher serum levels of endothelial adhesion molecules than abstainers or moderate drinkers [26]. One hour after consumption of 4 mL/kg (about three glasses) of wine the serum level of alcohol was about 6 mM and 6 h after ingestion of either red or white wine the serum level of IL-6 was about 60% higher than in controls having ingested a nonalcoholic beverage [27]. These results were confirmed by a recent study on the relative effects of consumption of water, ethanol, red wine, or beer [28].
Neuro-mediators The skin is innervated by peripheral sensory nerves, which can form direct synapses with epidermal and dermal cells. These sensory neurons contain and release a variety of neuropeptides and neurohormones which regulate a wide variety of biochemical processes and cell functions of keratinocytes, Langerhans cells, mast cells, dermal microvascular endothelial cells, and infiltrating leukocytes under physiological and pathological conditions. Furthermore, many of these cell types can act as a source of neuropeptides and in turn affect the survival, regeneration, and functional capacity of sensory neurons. Expression and regulation of neuropeptide receptors that are synthesized on a variety of skin cells determine the cellular responses mediated by these peptides. A majority of studies address diseased human skin, but in most cases these phenomena are universal across most species and tissues and the results from these studies can be extrapolated to normal skin. Of particular interest here are the vasoactive effects of cutaneous neuropeptides
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resulting in inflammatory processes, previously proposed as a hallmark of skin aging [11]. Substance P and CGRP are co-localized in sensory cutaneous neurons and are released locally in response to injury and induce neurogenic inflammation [29]. These neuropeptides increase leukocyte adhesion to endothelia cells [30], increase microvascular permeability, enhance leukocyte extravasation, and increase cellular migration [31] resulting in vasodilation [32], erythema [33], wheal and flare [34], and pain [35]. There is strong evidence of the role of cell adhesion molecules in neurogenic inflammation [11]. The role of substance P and CGRP in increasing the expression of ICAM-1, VCAM-1, and P- and E-selectins is well established. This has been shown at the protein and mRNA levels both in vitro and in vivo using immunofluorescent staining. These effects are mediated by neurokinin receptors on leukocytes [36] and cell adhesion molecule receptors on human dermal microvascular endothelial cells [37]. Other neuropeptides present in human skin also play a role in the modulation of neurogenic inflammation. Several studies have demonstrated a role for vasointestinal peptide (VIP), a neurotransmitter found in cutaneous sensory neurons. VIP is a neuropeptide belonging to the secretin/glucagon family of peptides, well known for its immune-modulating effects. It is particularly interesting because of its ability to regulate the production of both pro- and anti-inflammatory mediators. In the skin, its net effect is pro-inflammatory, whereas in other tissues it has anti-inflammatory properties and is therefore used to treat a variety of acute and chronic inflammatory diseases. Inflammatory skin conditions like psoriasis show increased plasma levels of VIP (66.9 vs. 60.1 pg/mL in healthy subjects) [38]. In patients with atopic eczema, a single intracutaneous injection of VIP increased local blood flow in a dose-dependent manner, induced a wheal-and-flare reaction, and increased pruritus [39]. These results have been corroborated in in vivo animal studies where subcutaneous injections of VIP caused concentration-dependent plasma extravasation in rat skin, although this was significantly less effective than identical concentrations of substance P [40]. Using another animal model, it was demonstrated that substance P induced plasma extravasation when exogenously perfused over a blister induced on the rat hind foot pad [41]. This effect of substance P was enhanced in the presence of VIP in a dose-dependent manner. In vitro, VIP increases lymphocyte binding to fibronectin (a cell-free model for binding to endothelial cells) via up-regulation of ICAM-1 [42]. Furthermore, using a Boyden chamber to measure migration, VIP has been
shown to act as a chemoattractant for human eosinophils [43], T cells, and lymphocytes [44]. These effects are mediated via VIP receptor type I. The pro-inflammatory activity of VIP in human skin is also mediated by a direct action on inflammatory mediators: the addition of VIP to human keratinocytes in culture increases in the intracellular expressions of IL-1a, IL-8, and TNF-a mRNA [45]. IL-1 and IL-8 protein levels were also increased in culture medium of VIPtreated cells. A similar effect of VIP was observed on human mast cells where it induced the release of IL-8, TNF-a, and monocyte chemoattractant protein-1 [46].
Physical Exercise Psychological stress triggers the release of pro-inflammatory mediators [11]. In at least one study, similar phenomena were observed with both psychological stress and physical exercise [47], suggesting that both factors mediate ageaccelerating inflammatory processes. Several studies indicate that moderate-intensity exercise is associated with a reduction in inflammatory mediators (perhaps by reducing the risk of anoxia) [48–50], and public health authorities recommend aerobic exercise for 30 min/day for 5 days a week as having beneficial effects on health and protection from chronic diseases [51, 52]. However, the body’s response to moderate or extensive, acute exercise appears to be more complex, as several studies report the co-release of anti-inflammatory IL-10 [53, 54] along with pro-inflammatory IL-6 [55]. It has therefore been suggested that the health benefits associated with physical exercise is due to this anti-inflammatory response. This conclusion is supported by a recent study measuring the expression of hundreds of neutrophil genes before and after a single 30-min cycling exercise at 80% peak oxygen uptake. Both pro-inflammatory and anti-inflammatory genes showed increased expression immediately following this exercise regime [56]. However, the beneficial anti-inflammatory effects of exercise are critically dependent on the type of exercise, its duration and intensity, the level of fitness, and the time when the markers of inflammation are measured after the exercise regime. Since these factors vary from one study to another, results reported in the scientific literature are sometimes difficult to compare. Exercise intensity is often calculated as a percentage of the maximum VO2 level for a given individual, which is typically measured using a ramp-type cycle ergometer. Exercise intensities vary from 40% to 80% depending on the study. VO2 levels are also used as a measure of individual
Skin Aging: A Generalization of the Micro-inflammatory Hypothesis
cardiorespiratory fitness, where baseline values between 40% and 50% are considered fit, whereas values below 30% are usually found with older, less fit individuals. Typically, studies which employ more intense exercise regimes use more fit individuals. One interesting study measured the inflammatory response to 20 min of treadmill exercise (65–70% VO2 maximum) in younger/fit vs. older/unfit individuals. This regime increased leukocyte adhesion to endothelial cells in vitro only in younger and fitter subjects [57]. In another study, less fit, middle-aged men showed no changes in serum levels of IL-6 after a 30-min treadmill exercise (50% VO2 maximum) [58]. Other studies, using different exercise programs, however, have shown pro-inflammatory changes immediately after relatively intense resistive exercise. In an attempt to use real-life exercises, wrestling matches are often used as an acute, intense, resistive form of exercise for adolescents, as they are known to induce muscle injury. This form of exercise was used to study the change in the number and type of circulating leukocytes. Using FACS flow cytometry an increase in the density of lymphocytes expressing ICAM-1 and LFA-1 was observed immediately after a single 1.5-h wrestling practice session [59]. Furthermore, results from the redistribution of other lymphocyte subsets indicate an increase in memory T cells. These lymphocytes are intimately involved with inflammation since they preferentially adhere to endothelial cells, and are selectively recruited into inflammatory sites [60] and express IL-6 [61]. Other studies have shown that within 20 min of a wrestling practice session, there is an increase in the number of circulating monocytes, granulocytes, and lymphocytes [62]. After several hours, the number of leukocytes decreases. This biphasic response may explain the fact that some studies show that intense exercise produces both pro- and anti-inflammatory effects. In addition to leukocyte cell numbers, other studies have measured circulating levels of pro-inflammatory markers following physical exercise in healthy individuals using a variety of regimes. These studies report increases in ICAM-1/VCAM [63], IL-6 [64], TNF-a [65], IL-1b [66], PGE2 and substance P [67], iNOS and NF-kb [68], C-reactive protein [69], and CC-chemokine receptor-2 [70]. These effects are typically measured following a single, acute bout of intense exercise, but are also observed after repeated endurance exercises like long distance cycling over a 6-day period [71]. The acute phase inflammatory response typically occurs within the first 48 h, but can be maintained for several days [72], depending on which inflammatory marker is measured. These inflammatory responses occur in both young and old healthy individuals.
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Sleep Deprivation A life-style factor known to promote apparent skin fatigue and perhaps to accelerate aging, and which has been proven to trigger inflammatory responses in healthy individuals, is sleep deprivation. Like extensive physical exercise, sleep deprivation is associated with poor quality of life, mood changes, higher psychological stress levels, and increased susceptibility to a variety of diseases (notably cardiovascular disease). Both sleep deprivation and extensive physical exercise increase serum concentrations of pro-inflammatory cytokines, circulating leukocytes, and soluble cell adhesion molecules. In a study where volunteers were subjected to both 7 days of semicontinuous strenuous exercise and sleep deprivation (1 h/night), plasma levels of IL-6, TNF-a, and IL-1b were increased and isolated leukocytes showed enhanced release of these proinflammatory markers when stimulated with LPS [73]. Similar results were obtained by numerous other investigators examining sleep deprivation alone, although severe sleep deprivation protocols often keep subjects awake for extended periods of time with no sleep at all. In these studies, sleep is typically monitored using polysomnography and sleep quality is usually assessed by subjective reports using the Pittsburgh Sleep Quality Index (PSQI). Results from these studies reveal that in addition to the pro-inflammatory cytokines [73], increased plasma levels of ICAM-1 and E-selectin [74], endothelin-1 [75], PGE2 [76], and leukocytes counts [77] were observed in healthy individuals. Results from these studies are often complicated by the fact that circadian fluctuations in levels of pro-inflammatory cytokines are known to exist [78]. However, several studies have taken these circadian variations into account and reached the same conclusions. In the case of IL-6, for example, sleep deprivation leads to daytime oversecretion and nighttime undersecretion [79]. In general, similar pro-inflammatory changes are reported in both young and old subjects, although it is worth considering that chronic sleep impairment can contribute to age-related changes in inflammatory responses [80].
Conclusion The micro-inflammatory hypothesis of skin aging was proposed as a mechanistic model [4]. The original model showed that skin aging is accelerated by any agents or treatments able to induce the synthesis and mobilization of ICAM-1 in endothelial cells. Most currently, the analysis of other factors proved to induce the synthesis
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and the mobilization of ICAM-1 in endothelial cells and/ or in circulating leukocytes. These factors are now believed to be accelerators of skin aging. Thus, the micro-inflammatory mechanistic model of skin aging could be considered a testable hypothesis open for experimental challenge and further scientific confirmation.
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70 Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance Kouichi Nakagawa
Introduction The stratum corneum (SC) is the outermost layer of skin and the skin barrier against chemicals, surfactants, UV irradiation, and environmental stresses. The SC has a heterogeneous structure composed of corneocytes embedded in the intercellar lipid lamellae as illustrated in > Fig. 70.1. The morphology of the SC lipids is closely associated with the main epidermal barrier. Knowledge of the lipid structure is important in understanding the mechanism of irritant dermatitis and other SC diseases. The structural information of the SC lipids is obtained by an analysis of aliphatic spin probes incorporated into intercellar lamella lipids using Electron Paramagnetic Resonance (EPR) [1–6]. EPR in conjunction with the spin probe method non-distractively measures the ordering of the lipid bilayers of the SC. EPR (or ESR: electron spin resonance) utilizes spectroscopy, which measures the freedom of an unpaired electron in an atom or molecule. The principles behind magnetic resonance are common to both EPR and nuclear magnetic resonance (NMR), but there are differences in the magnitudes and signs of the magnetic interactions involved. EPR probes an unpaired electron spin, while NMR probes a nuclear spin. EPR can measure 109 M (moles per liter) concentration of the probe and is one of the most sensitive spectroscopic tools. Therefore, EPR is able to elucidate skin lipid structures as well as skin lipid dynamics. It is important to know the composition of SC lipids as well as their structure in relation to depth. The various components of SC lipids, such as ceramides, cholesterol, and free fatty acids of SC lipids have been investigated by thin-layer chromatography (TLC) [7, 8]. It was also pointed out that the levels of SC lipids in a group of women aged 41–50 years showed a decrease in SC lipid levels [9]. Structural information organized by the components is essential for knowing the detailed functions of the SC. The role of the intercellular SC lipid bilayer in
relation to barrier function has been investigated by infrared (IR) spectroscopy [10, 11] and X-ray diffraction [12, 13]. IR examination showed that the outer layers are less cohesive and the intercellular lipids are more disordered compared with the deeper membrane, based on the C-H stretching absorbance of the methylene groups of the lipid acyl chains. The X-ray approach is somewhat limited to model lipid membranes containing water or in vitro SC specimens, and it is difficult to obtain information about depth-related changes of the SC. On the other hand, the EPR probe method can provide an insight into the organization of SC lipids as well as its dynamics. The physicochemical properties of intercellar lipids of SC as a function of various surfactants [1, 2], water contents [3], various kinds of spin probes [4], and ordering (or fluidity) change of the SC lipid [1] were investigated. EPR is a reliable, sensitive, and non-distractive technique to measure the probe in SC lipids at ambient temperature. An introduction on EPR spectroscopy and its application in conjunction with slow-tumbling simulation to elucidate the organization of SC lipids are discussed next. This technique provides confirmatory and complementary information about the structure and physicochemical properties of the SC on a molecular level. The advantage of using the spin probes is that it is possible to determine not only the structure, but also the acyl chain motion in the SC lipids. These studies provided the fluidity related behaviors of the SC at the different conditions by measuring EPR signal intensities and hyperfine coupling values. EPR measurements and the simulation analysis can potentially provide further quantitative insight into the skin-lipid structures of the skin. An additional EPR spectroscopic method is introduced for elucidating the structure of the SC. The structure of the SC calculated from a slow-tumbling simulation is an appropriate index for defining the ordering. Applications of the EPR technique for understanding the various SC structures should provide fruitful information.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_70, # Springer-Verlag Berlin Heidelberg 2010
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Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
. Figure 70.1 Schematic representation of the modified ‘‘brick and mortar’’ model of the stratum corneum (SC) is shown. Also, shown is the most likely probe location in the lipid bilayer and the pathways of drug permeation through the intact stratum corneum
EPR Apparatus The EPR apparatus consists of a klystron to generate microwaves, a electromagnetic, resonant cavity, a microwave detector, an amplifier, an A/D converter, and a PC, as shown in > Fig. 70.2. The microwaves from the klystron have a constant frequency, and those microwaves reflected from the resonant cavity are detected, changed to an electronic signal, amplified and then recorded. In contrast to NMR, substances that contain unpaired spin can be observed using EPR. Paramagnetic substances including transition metal complexes, free radicals, macromolecules, and photochemical intermediates are observed. Approximately 1013 mole of a substance gives an observable signal; thus EPR has great sensitivity.
EPR of Nitroxide Spin Probes (or Spin Labels) The momentum of electron spin in a magnetic field orients only two quantum states: ms = ½ and – ½. Application of an oscillating field perpendicular to a steady magnetic field (H) induces transitions between the two states provided the frequency (n) of the oscillating field satisfies the resonance condition: DE ¼ hn ¼ gbH;
ð70:1Þ
where DE is the energy-level separation, h is Planck’s constant, g is a dimensionless constant called the gvalue, and b is the electron Bohr magneton, and H is the applied magnetic field.
The interaction of an electron spin in resonance with a neighboring nuclear spin in a molecule is called hyperfine coupling. In the case of a nitroxide spin probe, 14N of the probe has three quantum states: mI = +1, 0, and 1. Each quantum state interacts with an electron spin and further splits into two sets of energy states as shown in > Fig. 70.3. The selection rules for transitions in hyperfine coupling are Dms = 1 and DmI = 0. Thus, one can observe three transition (resonance) lines for a fast-tumbling nitroxide spin probe in a spectrum. The interval of the resonance lines is called the hyperfine coupling constant. The EPR spectra are usually recorded as the first derivative of the absorption spectrum, as shown in lower part of > Fig. 70.3.
SC Cyanoacrylate Glue Stripping The sampling method was first utilized by Marks and Dawber [14] to obtain SC sheets. Recently, Yagi, Nakagawa, and Sakamoto developed a process to study SC properties [5, 6]. The SC specimens were successively removed from the mid-volar forearm and shank of the volunteers (male, age 49; male, age 59; male, age 78), who had given informed consent to the procedure. All subjects had normal skin, as judged by visual assessment. A glass plate (7 37 mm; Matsunami Glass Ind., Ltd., Tokyo, Japan) on which a single drop (1.2 mg) of a commercially available cyanoacrylate resin had been uniformly spread was used to strip the SC sheet, as depicted in
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
. Figure 70.2 Block diagram of electron paramagnetic resonance (EPR) spectrometer
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. Figure 70.4 Chemical structures of various doxylstearic acid (DSA) spin probes
the spin probe in the attached SC sheet. This method has the advantage of avoiding prior exposure of the SC to enzymes. EPR intensity slightly depends, to a small extent, on the thickness of the sample is removed by each stripping, but it can be adjusted by the amount and areas of glue on the glass plate. . Figure 70.3 Hyperfine levels and transitions for a nitroxide nitrogen nucleus (14N) of I = 1 with positive coupling constant. An observable EPR observable spectrum is shown
Spin Probes Organic free radicals containing the nitroxide group are called spin probes or spin labels. The ordering (or fluidity) of the lipid bilayers is obtained with doxylstearic acid (DSA) which is most commonly used. The chemical structures of DSAs are depicted in > Fig. 70.5. Changes in the lipid chain ordering enable monitoring using various probes. The orientation of the spin probe reflects the local molecular environment and should serve as an indicator of conformational changes in the lipid bilayers. The ordering at different position of the lipid bilayers is obtained with 5-, 7-, 12-, and 16-DSA. The 5-DSA is usually used for extraction of information near the surface region in a lipid membrane. The 16-DSA is usually used for near the end of the lipid chain. It is notable that other spin probes are also commercially available.
Preparation of SC Sheets for EPR Measurements
> Fig. 70.4. Only approximately 1 mg of a SC sample is required for the spin probe studies. Once the glue has solidified, no significant signal arises from the cured resin or from the spin probe dissolved in the resin; the only signal observed arises from
One piece of stripped SC (7 37 mm2) was incubated in 50 mM 5-DSA aqueous solution for about 60 min at 37 C (> Fig. 70.4). The probe solution was dropped on the SC sheet. The SC sheet repels the aqueous solution but the probe goes into the lipid phase during the incubation. After rinsing with deionized water to remove excess spin probe, the SC sample was mounted on an EPR cell.
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Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
EPR Measurements A commercially available X-band (9 GHz) EPR spectrometer (> Fig. 70.2) was used to measure the SC samples. Typical spectrometer settings were as follows: microwave power, 10 mW; time constant, 1 s; sweep time, 8 min; modulation, 0.2 mT; sweep width, 15 mT. All measurements were performed at ambient temperature. The obtained EPR spectra of 5-DSA in the various SC samples were digitized using the UN-SCAN-IT for Windows, Version 6.0 software (Silk Scientific Inc., USA). The digitized EPR spectra of the SC samples were analyzed using two . Figure 70.5 Schematic representation of SC sample procedures and the EPR spectrum
methods: qualitative order parameter (S) and quantitative simulated order parameter (S0) [5, 6]. The detailed sample preparations are described elsewhere [6].
EPR Line-shapes Due to Spin Probe Motion The line-shapes and line-widths can vary under certain spin probe environments. When line broadening arises from incomplete averaging of the g-value and the hyperfine coupling interactions within the limit of rapid tumbling in a medium, the EPR line-shape starts changing from the triplet pattern. EPR spectra of nitroxide radicals for different tumbling times as well as different order parameters are presented in > Fig. 70.6. Schematic illustration of lipid bilayer structures and the corresponding EPR spectra is shown in > Fig. 70.7. If a spin probe is oriented (immobilized) in a lipid membrane, the EPR spectrum is an anisotropic pattern which clearly shows parallel (2A||) and perpendicular (2A⊥) hyperfine coupling structures (the top spectrum in > Fig. 70.6). The order parameter is approximately 0.7 or higher. If a spin probe tumbles relatively fast (weakly immobilized) in a lipid membrane, the EPR spectrum is a triplet pattern with unequal intensities. The order parameter is usually very small (0.1).
Qualitative Order Parameter (S) The inclination of the principal axis of the nitroxide radical to the rotational axis of the long-chain probe . Figure 70.6 Nitroxide EPR line-shape as a function of tumbling time and order parameter. The parallel and perpendicular hyperfine couplings, 2A|| and 2A⊥, are also indicated for an anisotropic (immobilized) EPR spectrum
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
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. Figure 70.7 Schematic representation of lipid bilayer structures as a function of lipid ordering. The corresponding EPR spectral patterns are also indicated
molecule represents a measure of the order-disorder of the molecular assemblies of a membrane. The order parameter indicates the membrane chain dynamics and microenvironment of the medium in which the spin probe is incorporated. The conventional order parameter (S) is determined from the hyperfine coupling of the EPR signals according to the following relations [15]: S¼
AZZ
Ak A? a 0; 1 2 ðAXX þ AYY Þ a
a0 ¼
Ak þ 2A? ; 3
ð70:2Þ
ð70:3Þ
where a is the isotropic hyperfine value, (AXX + AYY + AZZ)/3; AXX, AYY, and AZZ are the principal values of the spin probe. The following principal components were used for 5-DSA [16]. AXX ; AYY ; AZZ ¼ ð0:66; 0:55; 3:45Þ mT
of the lipid structure, its EPR spectrum represents the microscopically oriented profile as depicted in > Fig. 70.7. When the normal structure is completely destroyed by chemical and/or physical stress, the EPR spectral profile changes to three sharp lines because the probe mobility is unrestricted. Thus, the EPR spectral profile reflects the rigidity of the environment of the probe moiety. However, conventional analysis measuring 2A|| and 2A⊥ from the observed spectrum gives limited information concerning the probe moiety in the membrane, and may not reveal subtle differences in the overall spectra related to the membrane chain ordering [6].
ð70:4Þ
The experimental hyperfine couplings of 2A|| and 2A⊥ are obtained from the experimental spectrum (> Fig. 70.6). The order parameter indicates that the S value increases with increasing anisotropy of the probe site in the membrane. On the other hand, the S value becomes zero for completely isotropic motion of the nitroxide radical. As the spin probe is incorporated into the highly oriented intercellular lipid structure in normal skin, in which the probe cannot move freely due to the rigidity
Quantitative Order Parameter (S0) by Slow-Tumbling Spectral Simulation The slow-tumbling motions on the order of 107 s of the aliphatic spin probes in membranes were evaluated by using the nonlinear least-squares (NLLS) to calculate the EPR spectra based on the stochastic Liouville equation [17, 18]. The EPR spectra for spin probes incorporated into the multilamellar lipid bilayers were calculated according to the microscopically ordered but macroscopically disordered (MOMD) model introduced by Meirovitch et al. [19]. In this model, lipid molecules are preferentially oriented by the local structure of the bilayers, but the bilayer fragments are overall distributed
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Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
randomly. The spectrum of a sample can be regarded as the superposition of the spectra of all of the fragments. The lipid and 5-DSA molecules in the lipid bilayers experience ordering potentials, which restrict the amplitude of the rotational motion. The ordering potential in a lipid bilayer determines the orientational distribution of molecules with respect to the local ordering axis of the bilayer [20]. The overall orientation of the probe can be expressed by the order parameter (S0), which is defined as follows [18, 21]: 2 1 2 S0 ¼ D00 ¼ 3 cos g 1 2 R 2 dO expðU =kT ÞD00 ð70:5Þ ¼ R ; dO expðU =kT Þ which measures the angular extent of the rotational diffusion of the nitroxide probe moiety. Gamma (g) is the angle between the rotational diffusion symmetry axis and the z-axis of the nitroxide axis system, as shown in > Fig. 70.8. In addition to S0, the simulation calculates slow-tumbling motions of the probe in the bilayers, providing rotational diffusion coefficients, as described in detail elsewhere [20]. The A of Eq. 70.4 and the g of the principal components were used for the simulation of 5-DSA [16]. gX X ; gY Y ; gZ Z ¼ ð2:0086; 2:0063; 2:0025Þ
ð70:6Þ
The local or microscopic ordering of the nitroxide probe in the multilamellar lipid bilayer is characterized by the S0 . Figure 70.8 A schematic representation of a conformation of DSA spin probe in the SC membrane, where the Z-axis of the acyl chain is parallel to z-axis of the nitrogen 2Pz orbital
value. A larger S0 value indicates highly ordered structure and a smaller S0 shows less ordered structure. The modern simulation takes into account EPR intensities, linewidths, and hyperfine coupling values and provides the quantitative information regarding the probe environment. Therefore, S0 value reflects the local ordering of the lipid structure in the membrane.
Qualitative Order Parameter (S) and Quantitative Order Parameter (S0) of SC Lipids The modified ‘‘brick and mortar [22]’’ model of the SC is illustrated in > Fig. 70.1. The intercellular lipids of the SC arrange themselves into bilayer and pack into lamellae. The single-chain 5-DSA normally dissolves into lipids and fat phases. The most likely location of the single-chain probe in the SC is shown in > Fig. 70.1. The aliphatic probe will be located in the lipid phase and fat-like sebaceous secretion of the SC. > Figure 70.9 shows the experimental and simulated EPR spectra of 5-DSA in the SC. The reasonable agreement of the experimental and simulated spectra suggests that simulation analysis can provide detailed information . Figure 70.9 Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA probe. Stripping numbers show consecutively stripped SC from the surface downward. The arrow of stripping number 1 indicates the characteristic peak. The EPR spectra were obtained with the single scan
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
regarding SC lipids. The S0 value changes from 0.61 to 0.96, while the S value is in the range of 0.56–0.59. The conventional S value was obtained by Eq. 70.2 measuring the hyperfine values from the observed spectrum. There are significant differences between the conventional and simulated order parameters. Because the slowtumbling simulation calculates the total line-shape of the spectrum, it is able to extract more detailed information about the SC structure than the conventional analysis, which is normally ambiguous in distinguishing the two hyperfine components (parallel and perpendicular) from the experimental spectrum due to the presence of weak and broad signals [5]. Thus, the S0 values (0.2 0.9) obtained by the simulation suggest that the outermost SC layers are less rigid (or more mobile), while the deeper lipid layers (S0 0.9) have more rigid and oriented structures. The arrow in the spectrum indicates the characteristic peak, which is prominent only for the first strip (> Fig. 70.9). This peak diminishes in intensity with increasing depth in the SC. The marked peak appears near the center of the spectrum because the probe embedded in the first sample stripped has greater freedom of motion. The other two lines of the nitroxide probe overlaid the . Figure 70.10 Experimental EPR spectra of 5-DSA in the first stripped SC from human mid-volar forearm (a), the first stripped SC from human forehead before washing (b), and the first stripped SC from human forehead after washing (c). The short dashed line corresponds to the characteristic signal. The long dashed line corresponds to the probe incorporated into the SC lipids
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central region of the spectrum. The results imply that signals can originate from sebaceous secretion. Further investigations of the characteristic peak were performed. > Figure 70.10a shows the EPR spectrum of the first strip from the SC. The strong and broad peak observed for the SC sheet from the human forehead is shown in > Fig. 70.10b. The peak intensity decreases after washing the SC with soap (> Fig. 70.10c). Thus, the signal can be attributed to sebaceous secretion [7]. The strength of the signal is considered to reflect the abundant sebaceous secretion at the forehead compared with that of the forearm.
Quantitative Order Parameter (S0) Related to SC Lipid Structure One can calculate the angle (g in > Fig. 70.8) between the rotational diffusion symmetry axis (the lipid in the SC) and the z-axis of the nitroxide axis system. > Figure 70.11 represents the schematic illustration of the bilayer distance in relation to the angle. The simulated S0 value of 0.61 is an angle of 30. The value of 0.96 is an angle of 9.4. The angle suggests that the SC lipids align nearly perpendicularly to the bilayer surface. The larger S0 value results from longer distance between the lipid bilayers. The analysis implies that the longer distance of the lipid bilayer can be related to a well-oriented SC structure. > Figure 70.12 presents the typical EPR spectra of 5-DSA incorporated in the SC lipids from human mid. Figure 70.11 Schematic illustration of relative lipid bilayer distances and the values of simulated order parameters related to the angles between the bilayer surface and the single-chain probe
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. Figure 70.12 Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA in the first stripped human SC from midvolar forearm. The EPR spectrum was obtained with the single scan
volar forearm. The characteristic peak indicated by the arrow is prominent only for the first strip. A reasonable agreement of the simulated and experimental spectra was obtained. The S0 value was 0.25, while the S value was 0.54. The simulated S0 value of 0.25 is an angle of 45. > Figure 70.13 shows that human SC stripped from the lower-leg presents a typical EPR spectra of 5-DSA incorporated in the SC lipids. The EPR spectrum of stripping number 1 is slightly different from number 3. The characteristic peak indicated by the arrow in the spectrum is prominent only for the first strip. This peak diminishes in intensity with increasing depth in the SC. The reasonable agreement of the simulated and experimental spectra suggests that simulation analysis can provide comprehensive information regarding the SC lipids. The S0 value changes from 0.28 to 0.60, while the S value is in the range of 0.63–0.64. The S0 value of 0.60 is an angle of 31. The higher S0 value implies that the lower SC lipids have a better-ordered structure than those of the upper SC lipids. Satisfactory agreement between the experimental and calculated spectra can provide a quantitative S0, which reveals the microscopic ordering in association with the structure of the SC lipids.
Other Applications of the EPR Method Effects of Surfactants on SC Lipids Different types as well as different mixtures of surfactants change the fluid structure of lipid bilayer differently. Kawasaki et al. examined the influence of anionic
. Figure 70.13 Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA in human SC stripped from the lower leg. Stripping numbers show consecutively stripped SC from the surface downward. The arrow of stripping number 1 indicates the characteristic peak. The EPR spectra were obtained with the single scan
surfactants, sodium lauryl sulfate (SLS) and sodium lauroyl glutamate (SLG), on human SC by the EPR spin label method [1]. The order parameter obtained by 1.0% wt SLS-treated cadaver SC (C-SC) was 0.52. On the other hand, a high S value of 0.73 for 1.0% wt SLG was obtained. The results suggest clear surfactant effects on the structure of the lipid bilayer. In addition, a reasonable correlation between order parameters and human clinical data (visual scores and transepidermal water loss values) was shown.
Effects of Skin Penetration Enhancers on SC Lipids Interaction of skin penetration enhancer correlates with the fluidity of the intercellular lipid bilayers. Quan and Maibach investigated the effects on a cadaver (C-SC) at three concentrations of laurocapram (1-dodecylazacyclo-
Structure of Stratum Corneum Lipid Studied by Electron Paramagnetic Resonance
heptan-2-one) utilizing the EPR spin probe method [23]. The EPR spectra of laurocapram-treated human SC were totally different from those of untreated C-SC. The results suggest that laurocapram causes an increase in the flexibility and polarity of local bilayers surrounding 5-DSA.
Conclusion EPR along with a modern computational analysis provides quantitative insight into the SC structure as a function of its depth. The EPR spectral pattern contains important information regarding the probe mobility as well as the SC lipid structure. Satisfactory agreement between the experimental and calculated spectrum can provide a quantitative S0, which gives the microscopic lipid ordering of the SC lipid. The SC lipid structures can be related to the SC barrier functions. In addition, the EPR method recognizes sebaceous exudates [6]. Therefore, the EPR technique could in turn provide more comprehensive information, which would further the understanding of various SC structures.
Cross-references > The
Stratum Corneum and Aging
References 1. Kawasaki Y, Quan D, Sakamoto K, Cooke R, Maibach HI. Influence of surfactant mixtures on intercellular lipid fluidity and skin barrier function. Skin Res Technol. 1999;5:96–101. 2. Mizushima J, Kawasaki Y, Tabohashi T, Maibach HI. Effect of surfactants on human stratum corneum: electron paramagnetic resonance. Int J Pharm. 2000;197:193–202. 3. Alonso A, Meirelles NC, Yushmanov VE, et al. Water increases the fluidity of intercellar membranes of stratum corneum: correlation with water permeability, elastic and electrical resistance properties. J Invest Dermatol. 1996;106:1058–1063. 4. Kitagawa S, Ikarashi A. Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. Chem Pharm Bull. 2001;49:165–168. 5. Nakagawa K, Mizushima J, Takino Y, Kawashima T, Maibach HI. Chain ordering of stratum corneum lipids investigated by EPR slowtumbling simulation. Spectrochimi Acta A Mol Biomol Spectrosc. 2006;63:816–820.
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6. Yagi E, Sakamoto K, Nakagawa K. Depth dependence of stratum corneum lipid ordering: A slow-tumbling simulation for electron paramagnetic resonance. J Invest Dermatol. 2007;127:895–899. 7. Bonte˙ F, Saunois A, Pinguet P, Meybeck A. Existence of a lipid gradient in the upper stratum corneum and its possible biological significance. Arch Dermatol Res. 1997;289:78–82. 8. Weerheim A, Ponec M. Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography. Arch Dermatol Res. 2007;293:191–199. 9. Rogers J, Harding C, Mayo A, Banks J, Rawlings A. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 10. Bommannan D, Potts RO, Guy RH. Examination of stratum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol. 1990;95:403–408. 11. Zhang G, Moore DJ, Mendelsohn R, Flach CR. Vibrational microspectroscopy and imaging of molecular composition and structure during human corneocytes maturation. J Invest Dermatol. 2006;126:1088–1094. 12. Bouwstra JA, Gooris GS, van der Spek JA, Bras W. Structural investigations of human stratum corneum as determined by small angle X-ray scattering. J Invest Dermatol. 1991;97:1005–1012. 13. Pilgram GSK, Engelsma-Van Pelt AM, Bouwstra JA, Koerten HK. Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature. J Invest Dermatol. 1999;113:403–409. 14. Marks R, Dawber RP. Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Dermatol. 1971;84: 117–123. 15. Hubbell WL, McConnell HM. Molecular motion in spin-labeled phospholipids and membrane. J Am Chem Soc. 1971;93:314–326. 16. Ge M, Rananavare SB, Freed JH. ESR studies of stearic acid binding to bovine serum albumin. Biochim Biophys Acta. 1990;1036: 228–326. 17. Schneider DJ, Freed JH. Calculating slow motional magnetic resonance spectra. In: Berliner LJ, Reuben J (eds) Biological Magnetic Resonance, Vol. 8. New York: Plenum Press, 1989, pp. 1–76. 18. Budil DE, Lee S, Saxena S, Freed JH. Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm. J Magn Reson Ser A. 1996;120:155–189. 19. Meirovitch E, Igner D, Igner E, Moro G, Freed JH. Electron-spin relaxation and ordering in smectic and supercooled nematic liquid crystals. J Chem Phys. 1982;77:3915–3938. 20. Ge M, Freed JH. Polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different. An ESR study. Biophys J. 1998;74:910–917. 21. Crepeau RH, Saxena S, Lee S, Patyal BR, Freed JH. Studies on lipid membranes by two-dimensional Fourier transform ESR: enhancement of resolution to ordering and dynamics. Biophys J. 1994;66: 1489–1504. 22. Elias PM. Epidermal lipids, barrier function and desquamation. J Invest Dermatol. 1983;80(suppl):44–49. 23. Quan D, Maibach HI. An electron paramagnetic resonance study. I. Effect of Azone on 5-doxyl stearic acid-labeled human stratum corneum. Int J Pharm. 1994;104:61–72.
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72 The Measurement and Perception of Uneven Coloration in Aging Skin Paul J. Matts
Introduction Human beings view the world using sensitive light meters that operate on the basis of ‘‘Contrast Sensitivity’’. ‘‘Contrast’’ can be defined simply as a ratio of adjacent luminance values and ‘‘Contrast Sensitivity’’ is a measure of how faded or washed-out an image can become before it is indistinguishable from a uniform field. It has been determined experimentally that the minimum discernible difference in grey-scale level that the human eye can detect is about 2% of full brightness [1, 2]. This outstanding Contrast Sensitivity allows the world around to be perceived in great detail; indeed, without contrast (and a means for achieving this via a variable-focus mechanism), human beings would effectively be rendered blind. The human eye, therefore, is drawn automatically to areas with high ratios of adjacent luminance – in simple terms, the world is viewed through edges created by contrast. If the human retina responds to only a narrow bandwidth of the electromagnetic spectrum (so-called ‘‘visible light’’, a nominal 400–700 nm), the interaction of these wavelengths with skin, therefore, is of utmost importance in understanding the way individuals perceive others and are themselves perceived. In young skin, reflection from the skin surface is largely diffuse, due to the very large number of reflecting polygonal plateaus that make up ‘‘microrelief ’’, and this has been found to be predictive for perception of soft, firm skin [3]. As microrelief is lost with increasing age and cumulative photodamage, so too is this natural ‘‘soft-focus’’ effect, driving low-contrast optics. In aging human skin, contrast is certainly increased by high ratios of adjacent luminance values due to shadowing formed by high amplitude/low frequency surface topography – and especially so in the case of linear features such as ‘‘lines’’, ‘‘furrows’’ and ‘‘wrinkles’’. Color, however, also plays an important role in the perception of age, health and beauty. It has been firmly established that the processes of intrinsic and, particularly, extrinsic aging drive a steady accumulation of enlarging, localised concentrations of the two colored skin
chromophores, melanin and hemoglobin [4–8]. Contrast can easily be created by color if a homogeneous field is disrupted by colored features of either/both sufficient diameter and ratio of adjacent luminance. In other words, therefore, independent of contrast formed by shape and/or topography, localised concentration of chromophores in aging skin causes a significant increase in contrast, particularly in sun-exposed areas such as the face, neck and de´colletage. While much is known about surface optics and topography, color contrast in skin remains a remarkably unstudied subject. The chapter will review briefly, recent research in this area that sheds new light on the measurement of the molecular basis of color contrast in skin and its effect on perception of age, health and attractiveness.
Measurement of the Molecular Basis of Color Contrast in Skin Human skin coloration is dependent almost exclusively on the concentration and spatial distribution of the chromophores melanin and hemoglobin, where melanin plays the dominant role in driving constitutive coloration [9, 10]. Objective approaches to determining skin color in vivo have centered around spectrophotometric or colorimetric approaches and the use of derived color coordinates such as L*a*b*, and various digital imaging/image analysis techniques, reviewed in full by Pierard [11]. While these measures certainly bring objectivity to the measurement of skin color, they still are not able to fully separate the individual contributions of the chromophores responsible for either the measured, integrated remittance spectrum or the final photographic image (no matter how high a quality it may be). A new measurement capability, SIAscopy™ (Spectrophotometric Intracutaneous Analysis; 12–15), developed by Cotton and Claridge [12] and then Astron Clinica (Cambridge, UK), operates on the principle of ‘‘chromophore mapping’’, that is, the in vivo measurement of the concentration and distribution of eumelanin,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_72, # Springer-Verlag Berlin Heidelberg 2010
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oxyhemoglobin and dermal collagen, to produce mutually exclusive greyscale concentration maps of these chromophores. The ‘‘SIAscope™’’ is now a commercially available instrument and, while it has been shown to have excellent sensitivity and specificity in the early identification of malignant melanoma, the principle of chromophore mapping that it employs can be readily applied to normal, healthy skin [12–15]. The technique is based upon a unique combination of dermatoscopy, contact remittance spectrophotometry and hyper-spectral imaging. In short, the SIAscope™ is able to obtain a high-resolution composite white-light image of the skin over a defined area and provides four additional, mutually exclusive chromophore maps that display the concentration of epidermal melanin and hemoglobin, collagen and melanin in the papillary dermis, pixel by pixel (> Fig. 72.1). The dermal melanin end point is the key diagnostic criterion used in melanoma diagnosis (not of concern with regard to normal skin). The contact SIAscope™ comprises a handheld scanner with a flat glass-fronted probe, placed in contact with the skin using light, but firm, pressure (to avoid blanching).
Further research by Astron Clinica has yielded ‘‘NonContact’’ SIAscopy™ (NCS) that overcomes the limitations of a skin contact probe. By necessity, this approach needs to be insensitive to local geometry and illumination intensity, in other words, the unavoidable artifacts of measuring 3-D objects, rather than flat surfaces. NCS is implemented [16] using an essentially conventional (although finely calibrated) digital camera and lighting system and may be used to acquire large-field eumelanin and oxyhemoglobin chromophore maps. In deploying NCS, the camera is treated not so much as an imaging device, but more as a three-waveband spectrometer, making use of the RGB Bayer filter over the CCD. The spectral power distribution of the light source and the raw response of the CCD are determined accurately over the visible range (400–700 nm) and are supplied as calibration data to the NCS algorithms, based on the SIA™ mathematical Model of Light Transport within skin. In short, for every pixel of the original raw image, NCS calculations are performed to yield exclusive concentrations of eumelanin and oxyhemoglobin. When recombined as an array, a
. Figure 72.1 Example of SIAscope™ II chromophore maps (12mm diameter). (a) composite white light image (b) oxyhemoglobin concentration map (c) eumelanin concentration map (d) collagen concentration map
The Measurement and Perception of Uneven Coloration in Aging Skin
parametric greyscale concentration map is produced, directly analogous to those calculated using the contact technique. It should be noted that a fully cross-polarized lighting system is needed, to eliminate specular reflection (which, by nature, contains no sub-surface information). An example of the NCS technique applied to a whole face can be seen in > Fig. 72.2. The NCS technique now allows routine acquisition of full-face melanin and hemoglobin chromophore maps, and the method has proven an ideal clinical partner. In a recent double-blinded study, NCS was used to provide a quantitative means of measuring the effect of a vehicle containing 2% N-acetyl glucosamine (NAG) and 4% niacinamide (N) vs. a vehicle control, applied topically, full-face, twice-daily for 8 weeks, to two groups of 100 females aged 40–60 respectively, on melanized hyper-pigmented spots [17]. Analysis of the NCS melanin maps demonstrated clear treatment effects for the NAG + N combination vs vehicle control, resulting in a significant (p < 0.05) reduction in melanin spot area fraction and a significant (p < 0.05) increase in melanin evenness. In a separate study [18] an excellent correlation was demonstrated between NCS derived melanin concentrations and eumelanin concentrations in human skin biopsies, spanning Fitzpatrick skin types I–VI. It must be concluded, therefore, that largefield chromophore mapping by NCS brings a new level of sensitivity and specificity to measurement of human skin color and constitutes a true step forward in the measurement of aging human skin and its treatment.
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Color Contrast Plays a Major Role in the Perception of Age, Health and Attractiveness Evolutionary psychologists have proposed that preferences for facial characteristics such as symmetry, averageness, and sexual dimorphism may reflect adaptations for ‘‘mate choice’’ because they probably provide visual cues of health and reproductive ability. Two recent studies found a positive association between homogeneity of skin features and perceived attractiveness. Importantly, however, both studies did not differentiate between visual contrast caused by skin surface topography and skin color distribution. Fink, Grammer and Thornhill [19] demonstrated that women’s facial skin texture affects male judgment of facial attractiveness, and found that homogeneous skin (that is, an even distribution of features relating to both skin color and skin surface topography) is most attractive. Analogous to the manner in which coloration plays a role in mate-choice in birds, therefore, visible color and color distribution in human facial skin may provide an indication of the age, health and attractiveness of the respective individual. More recently, Jones et al. [20] demonstrated that ratings of attractiveness of small areas of skin imaged from the left and right cheeks of male facial images significantly correlated with ratings of facial attractiveness. It was also found that apparent health of skin influences male facial attractiveness, independent of shape information.
. Figure 72.2 Example of full-face Non-Contact SIAscope™ chromophore maps (female subject aged 35). (a) original cross-polarised white-light digital photograph (b) eumelanin concentration map (c) oxyhemoglobin concentration map
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The Measurement and Perception of Uneven Coloration in Aging Skin
Importantly, these studies did not differentiate between skin surface topography and skin color distribution. A unique approach was, therefore, used to investigate the single-variable contribution of skin color contrast to perception of biological age, attractiveness and health [21]. One hundred and sixty-nine Caucasian women aged between 10 and 70 were imaged from front, left and right views using a custom high-resolution digital imaging system. The use of cross-polarised lighting eliminated fine surface texture in this imaging stage. The resulting images were processed using a new, unique series of digital manipulations to create ‘‘stimulus’’ heads where skin color distribution was the only remaining variable. Left and right sides were ‘‘grafted’’ onto the frontal image and then a cloning technique was used to remove any contrast attributable to low-frequency topographical features (lines/furrows, etc.). 2D color maps were than created by fitting the resulting image to a standard 2D template. In this stage, facial features (e.g. pupils, mouth gap, etc.) were standardized geometrically by fitting these to fixed addresses within the 2D template. To generate 3D facial stimuli from 2D color maps, faces were deformed to match a template grid in order to fit on a shape-standardised wire-frame mesh. In the final rendering process, these corrected 2D maps were fitted to the wireframe mesh, akin to a virtual skull. In this process, standardized facial features were added (eyes, nose, mouth, ears, hair, etc.) such that the resulting dataset comprised 169 3D head/face stimuli, standard in every respect apart from the subject’s original skin color distribution. Examples of the end-result process are shown in > Fig. 72.3. These stimuli were shown blind to 430 members of the public (aged 13–76), in Germany and Austria, using
calibrated monitors. Participants were requested to estimate the biological age of each face using a single-step scale ranging from 10 to 60 years. In addition, participants were asked to rate each face for a total of 15 attributes using a ten-point rating scale combining aspects of perceived attractiveness and health and apparent skin condition. The estimated biological age (aggregated estimates from all judges for each face) of facial images ranged from 17.8 to 36.7 years, a span of some 20 years and there was a highly significant positive correlation between the actual biological age of the subjects who provided facial images and the corresponding estimated age of their 3D shape-standardised faces varying only in visible skin color distribution (rho = .721, p < 0.01, 2-tailed). Significant negative correlations emerged between estimated facial age and the global face attributes (attractive: rho = .527, p < .01; healthy: rho = .520, p < .01; youthful: rho = .860, p < .01). In summary, therefore, it can be concluded that skin color distribution alone, independent of facial form, feature and skin surface topography can influence perceived age within a range of 20 years. Furthermore, it also appears to influence significantly perceived ‘‘attractiveness’’, ‘‘youth’’ and ‘‘health’’. It is hypothesized that this is so because color contrast may signal aspects of the underlying physiological condition/health of an individual, relevant for mate-choice.
Conclusion It has been shown that color contrast in human skin, formed by the local distribution and concentration of the
. Figure 72.3 Examples of three ‘‘stimuli’’ with standardized facial form, feature and topography with skin colour distribution of the original Caucasian female faces as the single-variable difference
The Measurement and Perception of Uneven Coloration in Aging Skin
chromophores melanin and hemoglobin, plays a major role in perception of age, health and attractiveness. Strategies to improve the appearance of aging skin, therefore, need to focus not only contrast created by form and topography, but also that created by color distribution and the chromophore targets responsible for this.
Cross-references > Hyperpigmentation
in Aging Skin in Ethnic Groups > The New Face of Pigmentation and Aging > Pigmentation
References 1. Blackwell HR. Contrast thresholds of the human eye. J Opt Soc Am. 1946;36:42–643. 2. Campbell FW, Robson JG. Application of Fourier analysis to the visibility of gratings. J Physiol. 1968;197(3):551–566. 3. Matts PJ, Solechnick ND. Predicting visual perception of human skin surface texture using Multiple-Angle Reflectance Spectrophotometry. American Academy of Dermatology 58th Annual Conference, 2000. 4 American Academy of Dermatology Consensus Conference. Photoaging/Photodamage as a Public Health Concern, Evanston, IL, March 3–4, 1988. 5. National Institutes of Health Consensus Development Conference Statement. Sunlight, Ultraviolet Radiation, and the Skin NIH Consens Statement. May 8–10, 1989;7(8):1–29. 6. Griffiths CEM. The clinical identification and quantification of photodamage. Br J Dermatol. 1992;127(Suppl 41):37–42. 7. Ryan T. The ageing of the blood supply and the lymphatic drainage of the skin. Micron. 2004;35(3):161–171. 8. Montagna W, Carlisle K. Structural changes in ageing skin. Br J Dermatol. 1990;122(Suppl 35):61–70. 9. Anderson RR, Parrish JA. The optics of human skin. J Invest Dermatol. 1981;77(1):13–19.
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10. Bashkatov AA, Genina EA, Kochubey VI, Tuchin VV. Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2000 nm. J Phys D: Appl Phys. 2005;38:2543–2555. 11. Pierard GE. EEMCO guidance for the assessment of skin colour. J Eur Acad Dermatol Venereol. 1998;10(1):1–11. 12. Cotton SD, Claridge, E. Developing a predictive model of human skin colouring. Proc SPIE. 1996;2708:814–825. 13. Cotton SD, Claridge E, Hall PN. Non-invasive skin imaging. In: Duncan J, Gindi G (eds) Proceedings of Information Processing in Medical Imaging, LNCS 1230. New York: Springer, 1997, pp. 501–506. 14. Moncrieff M, Cotton SD, Claridge E, Hall PN. Spectrophotometric intracutaneous analysis – a new technique for imaging pigmented skin lesions. Br J Dermatol. 2002;146(3):448–457. 15. Cotton SD. A non-invasive imaging system for assisting in the diagnosis of malignant melanoma. Ph.D thesis, Birmingham University, Birmingham 1998. 16. Preece S, Cotton SD, Claridge E. Imaging the pigments of skin with a technique which is invariant to changes in surface geometry and intensity of illuminating light. In: Barber D (ed) Proceedings of Medical Image Understanding and Analysis. Sheffield: MIUA 2003, pp. 145–148. 17. Matts PJ, Miyamoto K, Bissett DL, Cotton SD. The Use of Chromophore Mapping to Measure the Effects of a Topical N-Acetyl Glucosamine/Niacinamide Complex on Pigmentation in Human Skin. American Academy of Dermatology 64th Annual Conference, 2006. 18. Matts PJ, Dykes PJ, Marks R. The distribution of melanin in skin determined in vivo. Br J Dermatol. 2007;156(4):620–628. 19. Fink B, Grammer K, Thornhill R. Human (Homo sapiens) facial attractiveness in relation to skin texture and colour. J Comp Psychol. 2001;115(1):92–99. 20. Jones BC, Perrett DI, Little AC, Boothroyd L, Cornwell RE, Feinberg DR, Tiddeman BP, Whiten S, Pitman RM, Hillier SG, Burt DM, Stirrat MR, Law-Smith MJ, Moore FR. Menstrual cycle, pregnancy and oral contraceptive use alter attraction to apparent health in faces. Proc Roy Soc Lond B: Biol Sci. 2005;272(1561):347–354. 21. Fink B, Grammer K, Matts PJ. Visible skin colour distribution plays a major role in the perception of age, attractiveness and health in female faces. Evol Hum Behav. 2006;27(6):433–442.
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Probiotics
77 The Potential of Probiotics and Prebiotics for Skin Health Arthur C. Ouwehand . Kirsti Tiihonen . Sampo Lahtinen
Introduction Microbes are ubiquitous in the environment, and despite last century’s improvement in hygiene, human beings are continuously exposed to them. In fact, humans are not only exposed to microbes, but also hosting them. An adult human being consists of an estimated 1013 eukaryotic cells. At the same time, human body hosts an estimated 1014 microbes, most of them are in the large intestine but also appreciable amounts on the various sites of the skin. The composition of this microbiota (formerly known as ‘‘microflora’’) is complex and influenced by various environmental factors. It is, therefore, not surprising that different parts of the human body, which are exposed to the outside environment, will have a different microbiota. The composition and activity of the skin microbiota will be discussed in more detail below. Aberrancies in the skin microbiota, and also in the intestinal microbiota, may contribute to disease. It may, therefore, be desirable to modulate the composition and/ or activity of this microbiota. The most widely used method to modulate a microbiota is by the use of antibiotics. Antibiotics, although very powerful and sometimes reasonably selective, have the risk of inducing antibiotic resistance and unintentionally modifying other parts of the microbiota, as is evidenced by the induction of, for example, antibiotic-associated diarrhoea. Although antibiotics are likely to remain essential therapeutics, alternative, or complementary treatments to modify a microbiota exist: probiotics and prebiotics. According to a generally accepted definition of an FAO/WHO work group, probiotics are: ‘‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’’ [1]. The most common probiotics are members of the genera Lactobacillus and Bifidobacterium, but probiotics of other genera exist as well (> Table 77.1). It is important to note that only specific strains are probiotic, not the species. Furthermore, different strains may have different probiotic properties and properties from one strain cannot be extrapolated to another strain, not even of the same species.
Prebiotics are commonly defined as: ‘‘non-digestible food ingredients that, when consumed in sufficient amounts, selectively stimulate the growth and/or activity of one or a limited number of microbes in the colon resulting in documented health benefits’’ [2]. Although prebiotics are non-digestible and in many cases would fall under the definition of dietary fiber, they are not synonymous; not all fibers are prebiotic as they may not always be selectively utilized by a limited (and beneficial) part of the microbiota. The most widely investigated prebiotics are the fructo-oligosaccharides (FOS), inulin, and galacto-oligosaccharides (GOS). But, there is a wide range of other (potential) prebiotics, (> Table 77.2). Also, different prebiotics have their specific health benefits that cannot be extrapolated to others. What is, however, currently not clear is whether prebiotics of the same ‘‘class’’, for example, GOS, but produced by different processes (different process conditions, different enzymes, etc.) and therefore, with different degree of polymerization and type of glycosidic linkages will have the same health benefits. Or, should these different types of the same prebiotic ‘‘class’’ be regarded as separate prebiotics, in analogy to probiotic strains? Probiotics and prebiotics are typically included in a variety of functional foods; the definition of the latter does not even consider nonfood applications. Normally administered orally, they may, nevertheless, have beneficial effects on the skin. The intestine is the body’s main immune organ, and the mucosal immune system of the gut is linked to the immune system of the skin (and other mucosae) through migration of immune cells. Probiotics and, to a lesser extent, prebiotics have been documented to modulate the immune system. They may, therefore, also indirectly affect the functionality of the skin, as will be discussed further on. Pre- and probiotics may also influence the bioavailability of nutrients and that way affect the condition of, for example, the skin. Functional foods targeting this ‘‘beauty from within’’ are already on the market and some do have limited scientific evidence suggesting their efficacy. Unfortunately, many such products do not have documentation and their efficacy remains speculative. Topical
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_77, # Springer-Verlag Berlin Heidelberg 2010
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The Potential of Probiotics and Prebiotics for Skin Health
. Table 77.1 Examples of microorganisms commonly used as probiotics. Note that not the species but specific strains within these species maybe probiotic Lactobacilli
Bifidobacteria
Other lactic acid bacteria
Non-lactic acid bacteria
L. acidophilus
B. adolescentis
Enteroccus faecalis
Bacillus subtilis
L. casei
B. animalis ssp. lactis
E. faecium
B. cereus
L. crispatus
B. bifidum
Lactococcus lactis
B. coagulans
L. delbrueckii ssp. bulgaricus
B. breve
Streptococcus thermophilus
Clostridium butyricum
L. fermentum
B. infantis
Escherichia coli
L. johnsonii
B. longum
Propionibacterium freudenreichii
L. paracasei
Saccharomyces cerevisiae boulardii
L. plantarum L. reuteri L. rhamnosus L. salivarius
. Table 77.2 Examples of substances commonly used for their prebiotic properties Prebiotic (candidate)
Abbreviation
Main monomer(s)
Degree of polymerization
Linkage
Fructo-oligosaccharide (Oligofructose)
FOS
Fructose
1–7
b-(1,2)*
Galacto-oligosaccharide (Trans galacto-oligosaccharide)
GOS (TOS)
Galactose
1–6
b-(1,4)*
10–60
b-(1,2)
2
a-(1,4)
Inulin
–
Fructose
Lactitol
–
Galactose, Glucitol,
Lactulose
–
Galactose, Fructose
2
b-(1,4)
Partially hydrolysed guar gum
PHGG
Mannose, Galactose
10–300
b-(1,4) a-(1,6)
Polydextrose
PDX
Glucose
Xylo-oligosaccharide
XOS
Xylose
Resistant starch
RS
Glucose
12–30
(1,6)
2–7
b-(1,4)
10–100
a-(1,4) a-(1,6)
*Depending on the enzyme used in manufacture.
application of probiotics, and especially prebiotics, has received limited attention to date. Topical products on the market usually do not contain live bacteria but fermentates or extracts. The topical application of live microbes may seem unusual; however, the skin has its own microbiota, which thus may be influenced similar as the intestinal microbiota. Concerning prebiotics; also sweat contains small amounts of glycogen which is likely to function as an endogenous prebiotic.
Skin Structure and Function The human skin is the largest organ in a human body and has an average surface area of about 2 m2. Skin structure and function varies between different anatomical sites. The thickness of the skin epidermis varies a substantially from eyelids to soles of the feet. Moreover, the skin secretions mixed with the dead cells varies from one anatomical site to another and provide different environments for
The Potential of Probiotics and Prebiotics for Skin Health
skin microbes. Secretions of sweat and sebaceous glands as well as detached cells from the epidermis are highest on the forehead and upper back. Palms, lips, and soles of the feet do not have sebum-producing glands. Hair follicles and associated sebaceous glands are also part of skin. The human skin has two main layers: the inner dermis and the outer epidermis. The epidermis and its secretions provide the primary protective barrier against external UV radiation, toxins, pathogens, dehydration, and mechanical disturbances. Although the epidermis is continuously replacing itself within 30–40 days, it does not contain blood vessels. Its nutrient and oxygen supply is dependent on diffusion from the dermal blood circulation. The regulation of a balanced epidermal turnover is crucial for skin health. As an example, psoriasis is considered a skin disease with a markedly increased rate of epidermal turnover. Dandruff is also a condition of increased cell turnover partially caused by the metabolites of the yeast Malassezia. The epidermis can be divided into various sublayers with different functional characteristics: basal cell, spinous cell, granular cell, and cornified cell layers. The principle cells in the epidermis are keratinocytes. Other cell types in the epidermis are pigment-producing melanocytes, immunogenic Langerhans cells, and Merckel cells which act as mechanoreceptors. Langerhans cells, part of the dendritic cell family, play an important role in the skin immune system which protects the body from environmental stressors and pathogens. Hyaluronan is the principal extracellular matrix protein in the epidermis, facilitating tissue remodeling, and other cellular and metabolic processes. The dermis consists mainly of collagen and elastin fibers with fibroblast, blood and lymph vessels, muscles, nerves, and glands. Below the dermis is hypodermis, containing adipocytes. During normal differentiation, keratinocytes originating in the basal layer of the epidermis, move toward the skin surface and change their function and composition. At the surface, the keratinocytes undergo apoptosis and become filled with keratin protein. This outermost layer of skin, called stratum corneum, functions as the main barrier against water loss and external disturbances. The special lipid/water lamellar structure in the stratum corneum is important to maintain the permeability barrier of the skin. Natural moisturising factor (NMF) which is mainly composed of water-soluble amino acids prevents water loss from corneocytes. The water content in deeper keratinocyte layers is important for the maturation of the skin cells. Hyaluronan is the most important extracellular matrix regulating tissue remodeling and water balance in the living keratinocyte
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layers. In addition to extracellular matrix, organic osmolytes such as inositol, betaine, and taurine are also known to participate in the osmoregulation of keratinocytes during hyperosmotic stress and UV radiation [3]. Recent findings also indicate that tight junctions regulate skin barrier and thus water balance in living skin cells. Cells of different layers in the epidermis express only selected types of cellular junctions. Granular cells are interconnected with tight junctions whose structural proteins are ZO-1, occludin, claudin-1, and -4. Abnormal distribution of occludin and ZO-1 occurs in psoriasis plaques. Below the epidermis is the dermis which is mainly a connective tissue containing, collagen fibers, elastin, fibroblasts, blood vessels, and cells with immune activity. Fibroblasts are responsible for the collagen synthesis in the dermis and they also play an important role in wound healing.
Clinical Alterations in Skin Aging Typical age-related changes of the skin are wrinkles, dryness, changes in color and structure. The skin structure is mainly dependent on the collagen and elastin fibers which undergo changes during aging. The dryness of the aging skin is caused by the decreased blood circulation as well as decreases in sweat and oil secretion by glands. Moreover, thinness of the skin can be caused by loss of underlying fat and increased loss of water. Thin skin also makes small dilated blood vessels near the skin surface (telangiectasias) more visible. The fragile blood vessels increase blood leakage to skin. Free radicals may accelerate the agerelated alterations in skin. Abnormalities of the expression of tight junction-associated proteins (occludin, ZO-1, claudin-1, and claudin-4) have been identified in malignant disorders of keratinisation. Seborrheic keratoses are non-cancerous growths of the outer layer of skin which are more common in elderly people. Typical changes in aging skin are decreases in keratinocyte proliferation and differentiation, which cause thickening and drying of the stratum corneum. In the aging process, collagen and elastin fibers are not produced correctly by fibroblast or the fibers are degraded enzymatically. Also, numbers of melanocytes and Langerhans cells decrease. Thus, changes in extracellular matrix such as decreases in elastin, collagen, and hyaluronan biosynthesis cause a decrease in skin elasticity. Moreover, blood circulation in the dermic layer slows down, decreasing the delivery of nutrients and oxygen to skin cells. In addition, endocrinological changes and fat redistribution in subcutaneous layer cause loss of skin structure.
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The Potential of Probiotics and Prebiotics for Skin Health
In addition to chronological aging, environmental stressors and diet can cause premature skin aging. Free radicals produced by smoking, dehydration, pollution, and UV radiation are the primary source of premature aging of cell membranes, mitochondria, elastin, and collagen fibers, as well as DNA damage. Decrease of collagen fibers and formation of abnormal elastin fibers speed up the structural aging and premature apoptosis and inflammation the functional disturbances in the skin.
Skin Microbiota Human skin is covered with a continuous layer of microbes, which reside within epidermis, dermis, and the skin-associated glands and follicles, forming a diverse multicellular community known as the normal skin microbiota. The skin microbiota constitutes mainly of different bacteria but also of fungal species. The total number of microbes on the skin surface is typically within the range of 104–106 cells/cm2. The healthy skin microbiota contributes to skin homeostasis and plays a role in both health and disease. The composition of the normal microbiota of the human skin is diverse and differences between the skin microbiota of different individuals are high [4], although some studies suggest a relatively low interpersonal variation [5]. Notably, the composition of skin microbiota also varies between different anatomical sites, which provide different environmental conditions (e.g., moisture, temperature, pH, presence of hairs, follicles and other microbes, sweat, nutrients, exposure to light and oxygen) for microbes to proliferate [5]. Normal skin bacterial microbiota is dynamic over time [6], while the fungal skin microbiota is thought to be more stable [7]. The composition of normal skin microbiota is not fully characterized to date. Most of the conventional knowledge on the microbes associated with skin originates from traditional cultivation studies, in which samples taken from skin (e.g., swabs) are cultured in laboratory conditions and the colony-forming microbes are identified based on growth requirements and phenotypic characteristics. Based on cultivation studies, the healthy human microbiota has been proposed to constitute mainly of Propionibacterium (e.g., P. acnes), Staphylococcus (e.g., S. epidermis and S. hominis), Corynebacterium, Streptococcus, Pseudomonas, Micrococcus, Acinetobacter, Brevibacterium, and Dermabacter hominis, and the yeast Malassezia. The obvious limitation of traditional cultivation studies is that the characterization of skin microbes is biased towards microbes which are readily cultivable using
standard laboratory methods, while ‘‘yet-to-becultivated’’ microbes for which the suitable laboratory growth conditions have not been established remain undetected. The recent expansion in the use of modern molecular techniques in the characterization of skin microbiota has facilitated the research in this field and has provided knowledge on the complexity of the skin microbiota. Most common new methods allow the determination of skin microbiota based on specific DNA sequences. The number of studies relying on culture-independent identification of microbes is small, but nevertheless the existing reports suggest that the skin microbiota is considerably different from what has been earlier suggested based on traditional methods. Dekio et al. [8] demonstrated that in addition to microbes which can be detected by culturebased methods such as P. acnes and Staphylococcus species, skin microbiota also consists of a diverse community of previously unknown and yet-to-be-cultivated microbes. Gao et al. [6] studied swabs of volar forearm skin and suggested that typical human skin bacterial microbiota appears to consist partly of a conserved microbiota, represented by bacterial groups common to most individuals, and partly of a highly diverse microbiota, which accounts for a high level of variation observed between the normal skin microbiota of different individuals. Vast majority (>90%) of the bacteria belong into three phyla: Actinobacteria, Firmicutes, and Proteobacteria. These three phyla as well as the phylum Bacteroidetes are found on skin as well as other mucosal surfaces of humans, but skin is the only mucosal surface on which Actinobacteria dominate. Grice et al. [5] studied the skin of antecubital fossa (inside of the elbow) and suggested that a majority of the microbes belong to Proteobacteria, while Actinobacteria and Firmicutes remain minor components of the skin microbiota. The differences between the results obtained from different studies may be attributed to different skin regions sampled or interpersonal and geographical differences of microbiota of different subjects. At genus level, the studies by Gao et al. [6] suggest that certain genera are conserved and widely represented in normal superficial skin microbiota. These genera, namely Propionibacteria, Corynebacteria, Staphylococcus, and Streptococcus, are abundant and may account up to half of the microbiota of healthy skin. Other than these four genera, interpersonal differences of the microbiota at the genus level are remarkably high. Conversely, studies by Grice et al. [5] suggest that the two genera belonging to Proteobacteria dominate skin microbiota: Pseudomonas and Janthinobacterium. The natural fungal skin microbiota remains poorly defined. Yeasts related to the genus
The Potential of Probiotics and Prebiotics for Skin Health
Malassezia appear to constitute a major proportion of this microbiota, and the species distribution of the skin fungal microbiota is largely host-specific [7]. Skin microbiota plays a role in both maintenance of skin health and development of skin diseases. Examples associated with alterations in the composition of skin microbiota include acne and psoriasis. Healthy skin follicles have been suggested to be almost exclusively colonized by P. acnes while follicles of acne patients also include other microbes such as S. epidermis [9]. P. acnes has been proposed as the causative agent of acne, but current evidence of the causative role of P. acnes in the disease is conflicting [10]. Psoriasis and other inflammatory skin disorders are associated with alterations in skin microbiota. In psoriatic lesions, bacteria belonging to the phylum Firmicutes and genus Streptococcus are overrepresented while bacteria typical of healthy skin, such as phyla Actinobacteria and Proteobacteria and the genus Propionibacterium are underrepresented compared to skin microbiota of healthy persons or uninvolved skin of patients with psoriasis [4]. Skin fungal microbiota may also be altered in psoriatic lesions, although these findings are not consistent [7]. Aberrant skin microbiota has also been linked with atopic dermatitis [10]. Pathogenic microbes associated with various skin diseases are numerous but an extensive review of these is not within the scope of the current chapter. The role of skin microbes in skin health and disease provides rationale for therapies aiming at maintenance or restoration of healthy skin microbiota. Probiotics and prebiotics have been suggested as potential applications of such therapies. It has been suggested that probiotics could also be used to promote skin cell development. Many probiotic strains are also known for their potential to modify host immune responses, providing an additional possible mechanism for probiotics aimed at skin health. Modulation of host immune system function could be achieved locally (topical applications) or through systemic immune functions (e.g., oral applications). Most probiotics currently used are microbes typical of healthy gastrointestinal microbiota, such as strains of Lactobacillus and Bifidobacterium. These are usually administered orally, and are most commonly aimed at promotion of gut health and improvement of immune system function. The established probiotic strains may also have a potential for being used as probiotics for skin health, and indeed some probiotic strains are already being used to prevent or treat allergic skin diseases particularly eczema as will be described below. An alternative approach to skin probiotics is to design probiotics which are strains typical of healthy skin microbiota, specifically targeted for maintenance of
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skin health. Such strains could be isolated from the normal skin microbiota of healthy individuals and used as topical probiotic applications.
Probiotic Applications for Skin Health Eczema The mucosae and skin form a common immunological entity, connected by systemic, and lymphatic immune functions. Hence immune effects elicited in the gastrointestinal tract also influence immune responses in the oral cavity, mammary glands, urogenital tract, and skin. B-cells that are activated in the Peyer’s patches in the intestine may travel to other sites of the body. Also the production of cytokines by immune cells in the gut may have influences in peripheral sites like the skin. It is, therefore, not so surprising that oral consumption of immunoactive components, such as probiotics or allergens may have an influence on immune responses of the skin. Indeed, mouse studies have suggested that orally administered probiotic extracts may influence skin immune abnormalities [11]. Probiotics, and to a lesser extend prebiotics, have been investigated for their ability to treat or prevent atopic eczema. These investigations have been mainly performed in infants and young children. Allergy-related immune effects in children above the age of 2 years and adults appear to be difficult to obtain as the immune system has matured and is difficult to influence. The elderly, however, may have reduced immune function and have been found to be responsive to immune-modulating probiotics. Consumption of probiotic bacteria Bifidobacterium lactis HN019 and Lactobacillus rhamnosus HN001 has been observed to improve natural killer cell and phagocytic activity [12]. It would, therefore, not be unreasonable to hypothezise that specific probiotic strains might also positively influence immunological disorders of the skin in the elderly. To date this has not been investigated though. As an example, the influence of probiotics and prebiotics on atopic dermatitis in infants will be discussed. Under natural circumstances, humans are exposed to an abundance of microbes. Affluent societies have gradually eliminated this exposure through an improvement in hygiene. This has lead to a dramatic reduction in the incidence of infectious diseases but has been accompanied by an increase in allergic and autoimmune diseases. For proper development of the immune system, microbial exposure is essential. Probiotics might form a safe alternative for microbial exposure. Studies have, therefore,
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The Potential of Probiotics and Prebiotics for Skin Health
. Table 77.3 Summary of studies with probiotics aimed at reducing the risk for atopic eczema (AE) in at-risk infants (Compiled and modified after Lee et al. [23]) Probiotic strain
Dose (CFU)
Age at start
Duration (Months)
Short reference
‘‘Outcome’’
Lactobacillus rhamnosus GG
1 ¥ 1010
28 days
6
Reduced incidence of AE
Kallioma¨ki et al. 2001 [24]
L. rhamnosus GG
6 ¥ 109
28 days
6
No effect on AE, increased incidence of wheezing
Kopp et al. 2008 [25]
14–28 days
6
Reduced incidence of AE
Kukkonen et al. 2007 [26]
L. rhamnosus GG+ Lc705+B. breve Bb99 +P. freudenreichii JS (+GOS)
1.2 ¥ 1010
L. reuteri ATCC 55730
1 ¥ 108
28 days
12
No effect on AE, reduced IgE-associated eczema
Abrahamsson et al. 2007 [27]
L. acidophilus LAFTI L10
3 ¥ 109
0–2 days
6
No effect on AE, increased incidence of allergen sensitisation
Taylor et al. 2007 [28]
L. rhamnosus HN001
6 ¥ 109
28–35 days
24
Reduced incidence of AE
Wickens et al. 2008 [29]
Bifidobacterium lactis HN019
9 ¥ 109
28–35 days
24
No effect on AE
Wickens et al. 2008 [29]
Escherichia coli
5 ¥ 108
¼–4
Reduced incidence of AE
Lodinova´Za´dnikova´ et al. 2003 [30]
6
Reduced incidence of AE
Moro et al. 2006 [31]
Galactooligosaccharides + fructo-oligosaccharides
Table 77.4). Similarly, inclusion of certain probiotic strains into exclusion diets has been observed to improve eczema.
To what extend these observations in, sometimes very young, children can be used to prevent immune-related skin disorders in elderly remains to be determined. In addition to eczema-related skin inflammation, mouse studies have suggested that immune modulation by orally administered probiotics [14] or prebiotics [15] may reduce contact-induced skin inflammation.
Treatment Options for Premature Aging of Skin The changes in skin by aging are dependent not only on genetics, but also on nutrition, UV light, environmental pollutants, and stress. The primary treatment for photoaging is photoprotection and secondary antioxidants, in addition to other novel compounds such as osmolytes, polyphenols, probiotics, and prebiotics. Traditionally, products targeted to skin health are applied topically and are, therefore, likely to affect the
The Potential of Probiotics and Prebiotics for Skin Health
77
. Table 77.4 Summary of studies with probiotics aimed at treating atopic eczema (AE) (Compiled and modified after Lee et al. [23]) Probiotic strain
Age at start (months)
Dose (CFU)
Bifidobacterium lactis Bb-12 or L. rhamnosus GG
3–8 ¥ 1010
Lactobacillus rhamnosus or L. rhamnosus GG
Short reference
Improvement
Isolauri et al. 2000 [32]
3 ¥ 108 CFU/g formula 3.8
No effect
Brouwer et al. 2006 [33]
L. rhamnosus GG
1010
19
No effect
Fo¨lster-Holst et al. 2006 [34]
L. rhamnosus GG or L. rhamnosus GG + Lc705 + B. breve Bb99 + P. freudenreichii JS
5 ¥ 109 or 1010
1.4–11.9
Ambiguous
Viljanen et al. 2005 [35]
L. rhamnosus 19070-2 + L. reuteri DSM 122460
2 ¥ 1010
12–120
Ambiguous
Rosenfeldt et al. 2003 [36]
L. rhamnosus HN001 + B. lactis HN019
2 ¥ 1010
13–131
In food allergic children
Sistek et al. 2006 [37]
L. fermentum VRI-003 PCC
109
Mean 10.9
Improvement
Weston et al. 2005 [38]
uppermost skin layers. The skin barrier prevents most compounds and practically all microbes penetrating deeper layers. However, external compounds, microbes, and/or microbial metabolites may have an effect on the function of the skin barrier. Oral applications have different treatment options, as they can affect skin functions via the immune system and vasculature from inside the body. Current applications for anti-aging are targeted to preventing UV damages, increase the skin elasticity, cell renewal, and hydration. Current knowledge on the effects of probiotics and prebiotics in skin care are quite limited. However, some studies on the effects of microbes or their metabolites on skin hydrating and inflammation, UV protection as well as atopic dermatitis in children exist.
Photoaging The clinical alterations caused by UV light include freckles, lentigo solaris, and squamous cell carcinoma. Although freckles have a genetic background, the formation of freckles is triggered by UV-B radiation which activates melanocytes to increase the melanin production. Lentigines are freckles that may not fade in the winter. Typically they form after years of exposure to the sun, becoming more common in older people. Solar keratosis
4.6
Outcome
is a premalignant condition of skin that may be accompanied by the UV damage. Exposure of skin to solar UV radiation can cause skin cancer, photoaging, and other cellular and immunological changes. As a defence against UV radiation, melanocytes produce melanin blocking damage caused by UV. UVB radiation causes direct DNA damage by the free radicals while UVA causes indirect damage penetrating deeper layers. Immunosuppression caused by UV exposure is mediated by interleukin (IL)-10 released by keratinocytes and immune cells.
Probiotics and Protection from Sunburn Protection and recovery from sunburn has been one of the early research targets of probiotics for the skin. These early studies were carried out with fractions of bifidobacteria applied to the skin. The results of these early studies were contradictory, which may relate to the use of different fractions and Bifidobacterium strains, a common complication in probiotic research. Animal studies have shown that oral administration of Lactobacillus johnsonii La1 reduced UV-induced immune suppression. Feeding of the strain was also found to counteract UV-induced Langerhans cell depletion and IL-10 induction. Nonirradiated mice did not show any change in
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these immune markers; indicating that the strain contributes to maintaining skin immunity homeostasis. Subsequent human studies showed that consumption of this strain 56 days prior to experimental UV exposure did not protect the skin from UV-induced damage, but facilitated the recovery, most likely by stimulating the UV-depleted immune function of the skin. This effect was seen only in UV sensitive subject; not in UV tolerant subjects, about half of the volunteers [16]. Such observations are in agreement with what has been described above for the skin immunity modulating potential of certain probiotic strains. In extension to this, one could consider the use of microbes that produce carotenoids; carotenoids are known to protect the skin from UV damage. Many microbes are known to produce carotenoids, such as, for example, the yeast genus Rhodotorula which is present on certain surface ripened cheeses. They could possibly function as a skin-protective probiotic of known safe use. In fact, the skin of frequent sunbathers has been observed to be more colonised with carotenoid containing bacteria then infrequent sunbathers. In addition to providing protection from UV irradiation, the carotenoids would have a potential cosmetic benefit as they might contribute to improved tanning. Interestingly, carotenoids producing Bacillus indicus has been isolated from human faeces and could be considered an endogenous source of these compounds. Microbial carotenoid supplementation does, therefore, seem to be feasible.
Probiotics for Cosmetic Applications The use of probiotics in cosmetic applications has been proposed. Although controlled scientific trials are scarce, several patent applications have been filed in this field. Some in vitro and animal studies relating to potential cosmetic applications of probiotics have been carried out. Probiotics or their metabolites may have a role as moisturizing agents, although other compounds such as betaine are likely to be much more effective in this respect. Baba et al. [17] showed that in an in vitro skin cell model, Lactobacillus helveticus-fermented milk whey enhanced the expression of profilaggrin mRNA, a precursor of filaggrin, a protein which binds to keratin fibers in epithelial cells and contribute to skin moisture retention. In the same study, L. helveticus-fermented milk was observed to induce keratinocyte differentiation in vitro. Hyaluronic acid, a major component of body extracellular matrix, is widely distributed throughout the epithelial tissue and is commonly used in cosmetic applications. Soy milk fermented with Bifidobacterium has been shown to induce
hyaluronic acid production by skin cells in vitro and in an animal model [18]. The potential for probiotics for aging skin is discussed in more detail elsewhere in this book.
Potential Forms of Probiotics for Skin Probiotics are usually consumed as live microbes. It is commonly thought that viable probiotics are more likely to be biologically active than inactivated probiotics, since they maintain metabolic activity, may colonize the host at least transiently, may be more likely to attach to host mucosa and cells than inactivated cells, and are likely to possess better antimicrobial activity against harmful microorganisms. In the case of skin probiotics, it may be speculated that viable probiotics could remain viable on the skin and thereby colonize the skin. Although the possibility cannot be excluded, currently there is no evidence suggesting that topically administered probiotics could proliferate and colonize the skin, thus, becoming members of the host’s normal skin microbiota. Permanent colonization of probiotics aimed at skin health may be considered unlikely, as in the case of orally administered probiotics, it is known that these microbes do not colonize the host permanently and that the effects of probiotics typically last only through the period of administration. While in general viable probiotics are considered to be more active than nonviable probiotics, the latter are not without an effect [19]. In addition, in certain cases the health benefit of a probiotic is attributed to a metabolite produced by the bacteria, not to the cell itself. Inactivated skin probiotics, cell components of inactivated bacteria, or probiotic metabolites may be the preferred choice in cases in which safety and adverse effects are of concern. For example, in the case of wounds, the application of viable probiotics can lead to translocation of bacteria into the bloodstream with the risk of bacteraemia. Moreover, inactivated probiotics are likely to be more stable at room temperature than viable microbes, as the latter in liquid solutions usually require cold storage, which may not be feasible for skin applications. Indeed, inactivated forms of probiotics, components of probiotics, and cellfree extracts of probiotics have already been assessed for different skin applications [11, 14]. Nevertheless, the demonstration of the health benefit is of utmost importance when selecting probiotics for skin application. If a health benefit is demonstrated for a probiotic strain in viable form, the same health benefit cannot be directly assumed for inactivated form of the same strain (and vice versa).
The Potential of Probiotics and Prebiotics for Skin Health
New Skin Targets for Probiotics and Prebiotics The most potential target for the oral probiotics could be regulating abnormal immune responses in skin. As described above, probiotics have already indicated to have beneficial effects on allergy, eczema, and psoriasis. They have also shown to protect from UV-induced immune suppression in vitro. In addition to immune cells, oral probiotics may influence the dermal fibroblasts via blood circulation. Fibroblasts are responsible of the collagen synthesis and they have important role in wound healing.
Anti-aging Probiotics, and especially their metabolites, may have an important role in epidermal dynamics. The balance of skin renewal and repair requires optimal water, oxygen, and nutrient balance as well as growth factors which typically decrease with aging. New targets for probiotic use in anti-aging could be to inhibit formation of fragmented elastin fibers, scavengefree radicals, and activate dermal microcirculation. The role of probiotics in the function of epidermal tight junctions and lipid lamellae between corneocytes may provide new skin applications to probiotics. Topically applied probiotics and their metabolites have shown to increase hyaluronic acid production. Topical probiotics or prebiotics may also resist pathogen invasion to mechanically or chemically irritated skin areas. Furthermore, topical probiotics and their metabolites may also be protective against environmental toxins by binding or degrading them. The permeation barrier of the skin may limit the use of the topically applied prebiotics, probiotics, or their metabolites in skin care. However, novel techniques such as liposomes may facilitate the permeation of these products across the skin barrier. Moreover the effect of living probiotics on technical properties of topical skin care products may need more developmental work.
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barrier repair is needed to evaluate their potential for skin healing. The efficacy of Lactobacillus plantarum and its metabolites against Pseudomonas aeruginosa, a common pathogen of burns and wounds, has been demonstrated [20], suggesting that probiotic metabolites may be useful in topical treatment of burns and wounds. Moreover, topical application of kefir (a fermented dairy product) gel has been shown to shown to enhance wound healing in animal model [21]. The efficacy of probiotics in the care of severe wounds (gun shot wounds) has been assessed in animal model using both topical application (irrigation of the wound with probiotic suspension) and oral application [22]. The oral application is based on the hypothesis that in the case of serious trauma, the intestinal barrier function is dysfunctioning and significant translocation of intestinal bacteria into the body occurs, and this translocation is used as a route for probiotics to enter the deep wounds, where they inhibit the proliferation of pathogenic organisms causing wound infection. It should be noted that application of live bacteria to wounds involves a risk of bacteria entering the blood stream and causing bacteraemia, and increasing the risk of infections. Inactivated microbial cells, isolated cell components, or microbial metabolites may offer a safer alternative, free of the risk of bacteremia. Nevertheless, extensive safety testing of probiotics and related compounds aimed at wound care is required.
Conclusion Pre- and probiotics have been shown to influence parameters of the skin immune system and contribute to relief of atopic eczema and UV-induced immune suppression. Other immune-related skin conditions could be considered. For topical application of pre- or probiotics, a better understanding of the different skin microbiota is necessary. Technologies that have been widely used in the study of the gastrointestinal microbiota should be adapted. Such studies may indicate further targets on the skin for pre- and probiotics.
Wound and Burn Care
Cross-references
The use of probiotics in wound and burn care has been proposed. Inflammation and injuries activate keratinocytes to produce growth factors which are important for wound healing but also in diseases such as skin cancer and psoriasis. In cultured keratinocytes, probiotics have shown to increase filaggrin production which in turn may promote their differentiation. A more thorough understanding of the role probiotics can play in regulating cell renewal and skin
> Atopic
Dermatitis in the Aged in Aging Skin
> Probiotics
References 1. Guidelines for the evaluation of probiotics in food. http://www.who. int/foodsafety/publications/fs_management/probiotics2/en/: 2002.
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2. Ouwehand AC, Ma¨kela¨inen H, Tiihonen K, Rautonen N. Digestive health. In: Mitchell H (ed) Sweeteners and Sugar Alternatives in Food Technology. Oxford: Blackwell, 2006, pp. 44–53. 3. Warskulat U, Flogel U, Jacoby C, Hartwig HG, Thewissen M, Merx MW, et al. Taurine transporter knockout depletes muscle taurine levels and results in severe skeletal muscle impairment but leaves cardiac function uncompromised. FASEB J. 2004;18(3):577–579. 4. Gao Z, Tseng CH, Pei Z, Blaser MJ. Molecular analysis of human forearm superficial skin bacterial biota. Proc Natl Acad Sci USA. 2007;104(8):2927–2932. 5. Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG, Blakesley RW, et al. A diversity profile of the human skin microbiota. Genome Res. 2008;18(7):1043–1050. 6. Gao Z, Tseng CH, Strober BE, Pei Z, Blaser MJ. Substantial alterations of the cutaneous bacterial biota in psoriatic lesions. PLoS ONE. 2008;3(7):e2719. 7. Paulino LC, Tseng CH, Strober BE, Blaser MJ. Molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions. J Clin Microbiol. 2006;44(8):2933–2941. 8. Dekio I, Hayashi H, Sakamoto M, Kitahara M, Nishikawa T, Suematsu M, et al. Detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling. J Med Microbiol. 2005;54(Pt 12): 1231–1238. 9. Bek-Thomsen M, Lomholt HB, Kilian M. Acne is not associated with yet-uncultured bacteria. J Clin Microbiol. 2008;46(10):3355–3360. 10. Dekio I, Sakamoto M, Hayashi H, Amagai M, Suematsu M, Benno Y. Characterization of skin microbiota in patients with atopic dermatitis and in normal subjects using 16S rRNA gene-based comprehensive analysis. J Med Microbiol. 2007;56(Pt 12):1675–1683. 11. Cinque B, Di Marzio L, Della Riccia DN, Bizzini F, Giuliani M, Fanini D, et al. Effect of Bifidobacterium infantis on Interferongamma- induced keratinocyte apoptosis: a potential therapeutic approach to skin immune abnormalities. Int J Immunopathol Pharmacol. 2006;19(4):775–786. 12. Ouwehand A, Lahtinen S, Nurminen P. Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019. In: Lee YK, Salminen S (eds) Handbook of Probiotics and Prebiotics. Hoboken: Wiley, 2009, pp. 473–477. 13. Lee J, Seto D, Bielory L. Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. J Allergy Clin Immunol. 2008;121(1):116–121. 14. Chapat L, Chemin K, Dubois B, Bourdet-Sicard R, Kaiserlian D. Lactobacillus casei reduces CD8+ T cell-mediated skin inflammation. Eur J Immunol. 2004;34(9):2520–2528. 15. Watanabe J, Sasajima N, Aramaki A, Sonoyama K. Consumption of fructo-oligosaccharide reduces 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice. Br J Nutr. 2008;100(2):339–346. 16. Peguet-Navarro J, Dezutter-Dambuyant C, Buetler T, Leclaire J, Smola H, Blum S. et al. Supplementation with oral probiotic bacteria protects human cutaneous immune homeostasis after UV exposuredouble blind, randomized, placebo controlled clinical trial. Eur J Dermatol. 2008;18(5):504–511. 17. Baba H, Masuyama A, Takano T. Effects of Lactobacillus helveticusfermented milk on the differentiation of cultured normal human epidermal keratinocytes. J Dairy Sci. 2006;89(6):2072–2075. 18. Miyazaki K, Hanamizu T, Iizuka R, Chiba K. Bifidobacteriumfermented soy milk extract stimulates hyaluronic acid production in human skin cells and hairless mouse skin. Skin Pharmacol Appl Skin Physiol. 2003;16(2):108–116.
19. Ouwehand AC, Salminen S. The health effects of cultured milk products with viable and non-viable bacteria. Int Dairy J. 1998;8:749–758. 20. Valdez JC, Peral MC, Rachid M, Santana M, Perdigon G. Interference of Lactobacillus plantarum with Pseudomonas aeruginosa in vitro and in infected burns: the potential use of probiotics in wound treatment. Clin Microbiol Infect. 2005;11(6):472–479. 21. Rodrigues KL, Caputo LR, Carvalho JC, Evangelista J, Schneedorf JM. Antimicrobial and healing activity of kefir and kefiran extract. Int J Antimicrob Agents. 2005;25(5):404–408. 22. Nikitenko VI. Infection prophylaxis of gunshot wounds using probiotics. J Wound Care. 2004;13(9):363–366. 23. Lee J, Seto D, Bielory L. Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. J Allergy Clin Immunol. 2008;121(1):116–121. 24. Kallioma¨ki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial. Lancet. 2001;357:1076–1079. 25. Kopp MV, Hennemuth I, Heinzmann A, Urabanek R. Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics. 2008;121(4):850–856. 26. Kukkonen K, Savilahti E, Haahtela T, Juntunen-Backman K, Korpela R, Poussa T, Tuure T, Kuitunen M. Probiotics and prebiotic galactooligosaccharides in th eprevebtion of allergic diseases: A randomized, double-blind, placebo-controlled trail. J Allergy Clin Immunol. 2007;119(1):192–198. 27. Abrahamsson TR, Jakobsson T, Bottcher MF, Fredrikson M, Jenmalm MC, Bjorksten B, Oldaeus G. Probiotics in prevention of IgEassociated eczema: a double-blind, randomized, placebo-controlled trail. J Allergy Clin Immunol. 2007;119(5):1174–1180. 28. Taylor AL, Dunstan JA, Prescott SL. Probiotic supplementation for the first 6 months of life fails to reduce the risk of atopic dermatitis and increases the risk of allergen sensitization in high-risk children: a randomized controlled trail. J Allergy Clin Immunol. 2007;119(1): 184–191. 29. Wickens K, Black PN, Stanley TV, Mitchell, Fitzharris P, Tannock GW, Purdie G, Crane J. A differential effect of 2 probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trail. J Allergy Clin Immunol. 2008;122(4):788–794. 30. Lodinova´-Za´dnikova´ R, Cukrowska B, Tlaskalova´-Hogenova´ H. Oral administration of probiotic Escherichia coli after birth reduces frequency of allergies and repeated infections later in life (after 10 and 20 years). Int Arch Allergy Immunol. 2003;131:209–211. 31. Moro G, Arslanoglu S, Stahl B, Jelinek J, Wahn U, Boehm G. A mixture of prebiotic oligasaccharides reduces the incidence of atopic dermatitis during the first six months of age. Arch Dis Child. 2006; 91(10):814–819 32. Isolauri E, Arvola T, Su¨tas Y, Moilanen E, Salminen S. Probiotics in the management of atopic eczema. Clin Exp Allergy. 2000;30: 1604–1610. 33. Brouwer ML, Wolt-plompen SA, Dubois AE, van der HS, Jansen DF, Hoijer MA, et al. No effects of probiotics on atopic dermatitis in infancy: a randomized placebo-controlled trail. Clin Exp Allergy. 2006;36(7):899–906. 34. Fo¨lster-Holst R, Muller F, Schnopp N, abeck D, Kreiselmaier I, Lenz T, von Ru¨den U, Schrezenmeir J, Christophers E, Weichenthal M. Prospective, randomized controlled trial on Lactobacillus rhamnosus in infants with moderate to severe atopic dermatitis. Br J Dermatol. 2006;155(6):1256–1261.
The Potential of Probiotics and Prebiotics for Skin Health 35. Viljanen M, Savilahti E, Hahtela T, Juntunen-Backman K, Korpela R, Poussa T, Tuure T, Kuitunen M. Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a double blind placebocontrolled trial. Allergy. 2005;60(4):494–500. 36. Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppensen DL, Valerius NH, Paerregaard A. Effect of probiotic Lactobacillus strains in children with atopic dermatitis. J Allergy Clin Immunol. 2003;111 (2):389–395.
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37. Sistek D, Kelly R, Wickens K, Stanley T, Fitzharris P, Crane J. Is the effect of probiotics on atopic dermatitis confined to food sensitized children? Clin Exp Allergy. 2006;36(5):629–633. 38. Weston S, Halbert A, Richmond P, Prescott SL. Effects of probiotics on atopic dermatitis: a randomized controlled trial. Arch Dis Child. 2005;90(9):892–897.
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69 The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods Georgios Stamatas
Introduction The term ‘‘skin aging’’ is usually associated with the changes in skin appearance that occur during one’s adult life, and their causes, whether biochemical or biophysical. Skin, however, is a dynamic organ that undergoes changes throughout life, including the first years. Moreover, the changes and challenges that the skin faces early in life may affect later developments. A case in point is the relationship between the number of childhood sunburns and the likelihood of developing skin cancer later in life [1]. It is reasonable then to extrapolate the hypothesis that early events in skin’s life influence later developments, including photoaging, the capacity of skin to respond to external aggressors, and immune development (e.g., allergic reactions). Skin ontogenesis begins in utero [2]. During the first trimester, skin barrier development begins with the stratification of the epidermis. While epidermal cell maturation occurs continually during the whole of the pregnancy period, important developments in the third trimester like the formation of vernix caseosa are considered to create the right environment for the final steps of barrier maturation. The stratum corneum (SC) and the dermo– epidermal undulation (early papillae structures) become visible at 34 weeks of gestational age. At this point, the first signs of water barrier function can be measured. However, the older notion that skin development occurs only during pregnancy and that this organ is fully mature and capable of performing all its functions at birth or in a few short weeks thereafter [3] has been recently challenged and revised [4, 5]. New data point to the fact that skin continues to evolve and fine-tune its functions through the first years of life. Much research has focused on the development of skin functions, in particular, the water barrier, for infants born prematurely; this is because of the danger of
dehydration [6, 7]. Nevertheless, even when considering a full-term birth, the cutaneous tissue faces a big transformation in external conditions, from the aqueous environment of the womb to the gaseous environment of the earth’s atmosphere. The challenge that it faces is to continue to act as the organ that spatially defines the organism and acts as a barrier against dehydration (keeping water in), and against contamination and infection (keeping pathogens out). Of equal importance is its function as a thermal and immune barrier that also faces a big challenge at birth. The removal of the protective layer of vernix, a common practice at birth, is thought to exacerbate the challenges of the transition [8]. The case of skin adaptation during the first few days to the first month of life has received attention from the scientific community [9–12]. Yet, the continuous evolution of skin function and its underlying causes (structure and composition) during the first years of life have only recently become a subject of research [4, 5]. Finally, there is the obvious interest of the cosmetic value of baby skin in particular, when considering countering the signs and effects of adult skin aging. The aim of this chapter is to discuss the similarities and differences between infant (1–24 months) and adult skin in terms of function and structure. This undertaking will help shed some light both on the underlying factors that contribute to the better cosmetic quality of baby skin and on the particular skin care needs of infants. The requirement for using noninvasive methods to study infant skin in vivo is obvious. Moreover, the measurement procedure should be comfortable for an infant and the measurement time as rapid as possible, without compromising the accuracy and robustness of the method. The methods that fulfill these requirements and have been used in infant skin studies will be presented and discussed.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_69, # Springer-Verlag Berlin Heidelberg 2010
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The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
Skin Appearance and Structure Introduction Skin, like any other biological tissue, is physically and chemically heterogeneous. Its appearance, as well as its mechanical properties (including the way it feels to the touch), depends on its structure at the microscopic level. Documentation of the skin appearance macroscopically can be achieved with the use of high-resolution digital imaging. To better understand the intricacies of this method, the physics of light interaction with the tissue will be discussed. For many years, the study of skin microstructure was limited to the collection of biopsies followed by histological and microscopic analysis using transmission electron microscopy (TEM) or scanning electron microscopy (SEM). Progress in optics and imaging has allowed for the development of noninvasive alternatives that permit direct observation in vivo. These methods include video microscopy and in vivo confocal laser scanning microscopy (CLSM). The details of these methods and the ways that they have been used in the study of infant skin structure are discussed.
Imaging and Microscopy Light–Skin Interactions In any physics handbook one can read about the double nature of light, which means that it can be considered as either a stream of elementary particles (photons) or as waves of electromagnetic radiation. With the word ‘‘light,’’ normally the visible part of the electromagnetic spectrum (400–700 nm) is referred to, though this definition is often extended to the adjacent shorter wavelength region of the ultraviolet (UV) as well as the longer wavelength region of the infrared (IR). These regions are subdivided depending on the energy of the radiation (UVC, UVB, UVA, near-IR, mid-IR, far-IR). Note that the energy is inversely proportional to the wavelength, thus the UV has higher energy than the visible and the visible higher than the IR. Although the applications discussed below are based on visible light, the following discussion about light– tissue interactions can be extended to the UV and IR regions. Assume that a beam of light is directed toward the skin tissue. No sooner that the light particles hit the skin surface, a small part of them (about 4%) will bounce back as specular reflection that can be perceived as glare or
‘‘shine.’’ The majority of light will penetrate the surface and will be able to travel through the tissue. Several phenomena can take place during this travel. The energy carried by part of these photons may be absorbed by the electron clouds of specific molecules (such as melanin and hemoglobin) and dissipated in the form of heat. This phenomenon is called light absorption and gives rise to the perception of skin color. For example, hemoglobin molecules are very efficient in absorbing the blue and green regions of the visible spectrum, whereas they absorb red very weakly and therefore blood appears to be red in color. Light scattering is another phenomenon that relates to the change of direction of the light travel through the tissue. It occurs when light meets the interface of two media with different indices of refraction. In the skin such interfaces include cell membranes, organelles (e.g., melanosomes are strong light scatterers), nuclear membranes, as well as surfaces of large molecular agglomerates such as collagen and elastin fibers in the dermis and compacted keratin structures at the top layers of the tissue. Skin, like most biological tissues, is considered to be a turbid medium, which means that due to the high likelihood of light scattering events, tissue penetration is limited and a large part of the incident light reemerges back out of the skin surface. Capturing this light is typically performed by means of digital imaging.
Macroscopic Imaging and the Use of Polarizers A standard macroscopic imaging setup consists of the illumination source or sources (typically flash units) and the detector (typically a high-pixel-count digital camera). Care should be taken for the positioning of the subject to be imaged: in this case a person’s skin. Different body sites have different requirements for positioning (e.g., the face vs the arm). The geometry of the imaging setup, including angles of illumination with respect to the subject and the camera, the distance of the camera from the subject, and the selection of the numerical aperture of the lens, are critical for the accurate and reproducible documentation of skin appearance. As mentioned above, part of the incident light to the skin gets specularly reflected and contributes to the glare in an image. The amount of collected specularly reflected light by the detector can be enhanced or avoided by the proper use of polarizers. Light waves typically travel in all possible orientations. However, an orientation of preference can be selected by
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
placing a polarizing filter in the path of light travel. Such a filter placed in front of the light source in an imaging setup allows for light traveling only in a preferred plane (polarized light) to be used for imaging. The specularly reflected light will always have the same plane of travel as the incident light, whereas the diffusely reflected light (light that has traveled through the tissue and reemerges at the tissue surface) gets scrambled due to the multiple scattering events it undergoes in the tissue. Therefore, if a second polarizing filter is placed in front of the camera lens at an orientation parallel to the plane of the filter at the light source, it can enhance the contribution of the specularly reflected part, thus enhancing the surface details of the skin. On the other hand, by placing the second filter with its plane orthogonal to the plane of the polarizer at the light source, all specularly reflected light can be blocked, thus enhancing skin color and the inherent information about absorbing structures under the skin surface such as blood vessels and melanin-related structures [13, 14]. Using macroimaging one can study the overall appearance of skin that closely resembles clinical observations. In the case of infant skin, macroimaging can be used to document the condition of dry skin, diffuse skin erythema, inhomogeneities in skin pigmentation, bruising, ectatic vessels, etc.
In Vivo Video Microscopy The same principles of physics that govern macroimaging can be applied to light microscopy with the only difference being that of the geometry of the setup (continuous light source, specialized magnifying lens, short lens-to-subject distance due to short depth of field of the lens, etc.). Advancements in electronics and optics have allowed for the commercial availability of instruments equipped with a handheld probe that comes in contact with the skin site of interest and contains in one unit the lens and the detector. Light can be delivered where it is needed by use of fiber optic cables. Fast rate camera detectors permit not only the capture of a high-quality image, but also of a time-lapse video. In vivo video microscopy can be used to document microrelief line density and patterns, hair follicle density, pore size, local diffuse erythema, melanin distribution in lesions, etc.
In Vivo Confocal Microscopy For the last decade or so commercial instruments are available that made possible the in vivo use of confocal
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laser scanning microscopy (CLSM) in reflectance or fluorescence mode. Since the latter involves the use of externally applied dyes its use in infant skin research is limited. This discussion will focus on the reflectance CLSM [15–17]. Reflectance CLSM captures light that has gone through a single scattering event in the tissue. The light source in this case is a beam of laser that is scanned (with the aid of a rotating mirror) in a horizontal plane that optically sections the tissue. Photons that went through a single scattering event are bouncing straight back to the lens that focuses the captured light to the camera detector. By means of moving vertically the objective lens, one is able to optically section the tissue at different depths. The limiting factor for getting a signal from deep in the tissue is that the likelihood of single scattering events decreases almost exponentially with depth. This means that this limits the observation of the whole epidermis, the dermal papillae, and the dermal structures that are found just below them. The contrast in the image then arises from the areas where a change in index of refraction occurs. As mentioned above, these areas in the skin correspond to the compacted keratin of the SC, keratinocyte cell membranes, nuclei, and melanosomes in the epidermis, as well as collagen and elastin fibers in the dermis. Erythrocytes in the dermal capillaries also give a strong signal in reflectance CLSM, allowing for observations of blood flow in these vessels. In vivo CLSM can be used in the study of skin organization at the microscopic level. Some of the parameters that can be calculated from confocal images are: the cell projected area at different epidermal layers, the thickness of these layers, the depth of microrelief lines, the shape and distribution of dermal papillae, etc.
Other Methods Relating to Skin Structure High-frequency ultrasound imaging (or ultrasonography) has also been proposed as a noninvasive method for the study of skin structure at the microscopic level. This method provides images that correspond to vertical cross sections through the skin. The resolution is rather low for detecting any structures in the epidermis and therefore it is best suited for the study of deeper structures. Moreover, the uncertainty of the origin of the signal often renders image interpretation difficult. Optical coherence tomography (OCT) is another method that has been used in the study of skin structure. Like in ultrasonography, OCT images represent vertical
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The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
optical cross sections of the skin. Moreover, similar to in vivo reflectance CLSM, OCT is based on optical properties of the tissue and primarily scattering. Although it was met with big success in the field of ophthalmology, OCT use in dermatology has been limited to research only. One of the reasons is the relatively low resolution and image quality compared to the in vivo CLSM. Finally, information regarding skin structure can come from sources other than imaging. For example, in vivo Raman microspectroscopy (to be discussed later in this chapter) can be used to extract information about the water concentration profile through the top layers of the skin. The shape of this profile contains inherently information regarding the thickness of the SC [18].
Comparison Between Infant and Adult Skin Structure Overview of Skin Structure The structure of inter-follicular skin is typically described as being composed of two substantially different layers: the epidermis on top and the dermis below. The epidermis is much thinner (50–100 mm, excluding palms and soles where it is about 1.5 mm) than the dermis (0.3– 3 mm). In contrast to the largely noncellular dermis, the epidermis is composed of densely packed specialized epithelial cells, the keratinocytes, with very few and small extracellular domains in between. Other cells that reside in the epidermis in fewer numbers than the keratinocytes are melanocytes (responsible for melanin production), Langerhans cells (cells of the immune system), and Merkel cells (neuronal mechanoreceptor cells). The keratinocytes begin their life at the lower-most layer of the epidermis (the basal layer), where they are attached to the basal lamina, the membrane that separates the epidermis from the dermis. Basal keratinocytes are smaller compared to their counterparts in the upper epidermal layers and they have the ability to proliferate. At a certain point in its life, the keratinocyte loses its contact with the basal lamina – and with it, the ability to divide – and moves upward toward the skin surface. As it does so, it undergoes several morphologic and metabolic changes that define the epidermal layers (above the basal layer are first, the spinous and later, the granular layer). The last transition for the keratinocyte is to undergo programmed cell death, lose its nucleus, and get compacted by almost doubling its projected area to become a corneocyte, the dead cell that eventually flakes off of the skin surface. Although considered dead, the corneocytes are critical in performing the
major task of building the skin barrier. Corneocyte desmosomes, as well as the extracellular lipid structures and the corneocytes themselves, make up the physical components of the barrier (brick and mortar model) [19]. The skin surface is not however perfectly flat. The superficial SC structures are permeated by lines known as skin microrelief. These lines are thought to give flexibility to the skin surface and are very important as reservoirs of externally applied substances. They are also the preferential location for normal skin microflora. The hair follicles with the attached sebaceous glands and the sweat ducts are appendages that are lined with cells of epidermal origin. Their secretions (sebum and sweat) are considered to play a role in the immune and water barrier through antimicrobial lipids, control of skin surface pH, and production of components of the natural moisturizing factor (NMF). Underneath the epidermis is the dermis that consists primarily of extracellular matrix material, including collagen and elastin fibers and extrafibrillar matrix (ground substance). The dermis is sparsely populated by fibroblasts. The blood vessels that provide nutrients for the whole skin tissue are found here, along with lymphatic vessels that drain the dermis of excess water. It is common to recognize two distinct layers in the dermis: the papillary layer on top closer to the epidermis and the reticular layer underneath. Each of these layers is characterized by the size of the collagen and elastin fibers (with the reticular dermis having thicker fibers arranged parallel to the skin surface), which give rise to different mechanical properties. In general, everything that has been described so far regarding skin structure can be observed equally well in adult and infant skin. The following paragraphs focus on the observed differences.
Skin Surface If healthy infant skin surface is compared to that of adult using video microscopy, it can be readily observed that the micro relief lines and the SC island structures in between look different (> Fig. 69.1). In infant skin the lines are thinner and more densely arranged than in adult skin. The SC islands are plump and round in infant skin, whereas in adult skin they look larger and flat, with the early signs if flaking at the corners. In parallel, it is generally accepted that the number of follicles remains constant throughout life. Given their smaller body size, the total skin area in infants is greatly smaller than adults (maybe up to ten times) and therefore
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
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. Figure 69.1 The infant skin surface shows many differences compared to adult skin. Typical video microscopy images of (a) infant and (b) adult skin
the density of hair follicles must be equally larger than adults. This fact needs to be taken into account when considering, for example, the permeability of infant skin to foreign substances.
Epidermal Layer Thickness and Cell Size As discussed above the epidermal thickness can be measured using in vivo reflectance CLSM. The SC thickness in infants has been reported to be about 30% less than in adults and the overall thickness of the suprapapillary epidermis is 20–30% less in infants compared to adults [20]. Digital analysis of CLSM images has revealed that infant corneocytes and granular cells are smaller than their adult counterparts. The two observations taken together, along with the fact that infant keratinocytes proliferate at a higher rate than adult (see later in this chapter), may indicate that the life cycle of a keratinocyte is shorter in infant skin compared to adult. Either due to this shorter life cycle or possibly due to mechanisms that have not been developed yet in infant skin, adult SC appears to be more cornified and even drier than infant skin (> Fig. 69.1).
Dermal Papillae The dermal papillae, the undulating structures that characterize the interface between the dermis and the epidermis appear to be different in infant skin compared to
adult when using CLSM (> Fig. 69.2). In infant skin the cross sections of these structures appear to be denser and have more uniform size distribution than in adult. Considering that the capillaries in the papillae provide the nutrients for the epidermis, the implication of this observation is that the epidermal nourishment may be different in infants compared to adults. One can understand the elevated requirement for nourishment for an epidermis that is proliferating at a higher rate.
Dermis Beyond the epidermis and its structures CLSM can be used to extract information about the dermis. When the average image intensity of a CLSM image stack through the skin is plotted against the corresponding depth, the exponential decay of the signal is briefly interrupted by a small maximum at a depth corresponding to a level within the dermis [21]. It has been shown that this maximum occurs due to the increased collagen fiber size in the reticular dermis compared to the smaller fiber size of the papillary dermis. Interestingly, this maximum can only be observed in adult skin and is not evident in infants [20]. The absence of reticular dermis in babies, observed noninvasively by in vivo CLSM, has been previously observed in histological sections [22]. It can be conjectured that the formation of thicker collagen fibers occurs later in life and possibly requires accumulation of products of cross-linking reactions.
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The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
. Figure 69.2 Infant dermal papillae look different from those of an adult. Typical CLSM images of (a) infant and (b) adult (biological mother) skin at about 40 mm from the skin surface. The arrows point to the lumen of dermal papillae structures. The papillae appear to be denser and their shape is more uniform in infant skin
Skin Functions Introduction The further skin research progresses, the more the plurality of functions that the skin has to perform is being appreciated. A more obvious function is to provide the interface between the organism and the external world and thus define the shape and volume of the individual in space. A separate, though not unrelated, function of the skin is to provide a protective barrier from external insults, as well as a barrier that maintains internally the required water content level (about 70% per weight) that is vital for the survival of internal organs. The water barrier is localized within the upper most layer of the epidermis, the SC. The notion of comprising a sort of barrier can be applied to skin for a variety of physical and biochemical factors, for example, barrier to external toxic molecules, barrier to UV radiation, barrier to pathogenic microorganisms, immunologic barrier, and thermal barrier. Maintaining a healthy barrier is a dynamic process and it requires the epidermal layer to continuously replenish itself as the cells of its top layer, the SC, get continuously sloughed off. Therefore, one of the critical functions of the epidermis is its own self-renewal, which does not cease throughout an individual’s life. Skin has been considered as an extension of the immune system as the first line of defense, but even more
astonishingly as an extension of the central nervous system (much recent work has been focused on the skin– brain connection; for a review see [23]). Furthermore, the psychological aspect of skin appearance should not be underestimated. Finally, in the study of infant skin it is mandatory to mention the importance of touch stimulation for healthy physiological and psychological development of the infant [24, 25] or even its effect as analgesic [26]. In the interest of this chapter, however, the discussion to the skin functions that relate to physiology would be limited.
Electric and Spectroscopic Methods Electric Methods Relating to Water Content The water-handling properties of the SC are implicated in a variety of the skin qualities ranging from its optical and mechanical properties to the plurality of the barrierrelated functions. The need for accurate documentation of these properties is evident. The first step is to be able to measure the water content of the SC, based on the idea that although there is a constant flux of water molecules through the SC, there is a value that can be assumed to be relatively constant when the individual is at rest. Skin water content has traditionally been measured by indirect methods that relate the skin electric properties (impedance, capacitance, and conductance) to the
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
amount of water in the SC [27]. The better hydrated the SC is, the easier an electric current can flow through the top layers of the epidermis due to the higher ionic mobility in an aqueous environment. There are, however, limitations to electrical methods. Natural skin lipids or externally applied oils may hinder the electric flow and thus may actually decrease the skin conductance values and increase the measured skin capacitance. Moreover, the concentration of total ionic species found in the SC can influence the electric conductivity or equivalently decrease the skin capacitance value. In spite of these possible artifacts, the portability of electric probes and their ease of use contributed to their general acceptance making them the most commonly used methods in scientific literature regarding skin hydration measurements.
In Vivo Confocal Raman Spectroscopy Although the electrical methods give an estimation of the SC hydration at the point of measurement, this is an integrated value. To study the actual distribution of water concentration in the SC noninvasively, spectroscopic methods and in particular in vivo confocal Raman microspectroscopy need to be relied. Raman spectroscopy is based on measuring the energy shift (in wavenumbers) resulting from inelastic scattering of light by electronic vibrations of chemical bonds. Adaptation of this type of spectroscopy on a confocal arrangement allows for the calculation of concentration profiles of certain substances through the SC in vivo [28]. The requirement for a molecule to have a characteristic signal in Raman spectroscopy is that the electronic vibrations of its bonds change their polarizability when encountering an electromagnetic field (typically visible or infrared light). Water is such a molecule and one can use this method to look at the distribution of water through the first 20–30 mm from the skin surface [29]. The water-holding capacity of SC is thought to depend on certain highly hydroscopic filaggrin breakdown products, such as small amino acids, urea, pyrrolidone carboxylic acid, ornithine, citruline, urocanic acid, and others. These molecules are considered to be the SC’s own humectants and are known in the literature as ‘‘natural moisturizing factor’’ (NMF). Many of these molecules have characteristic signals in Raman spectroscopy and therefore their concentration profiles through the SC can be monitored using a confocal Raman instrument [28].
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Methods Relating to Skin Water Barrier Arguably, the most studied type of skin barrier is that of keeping water inside and preventing tissue dehydration. The integrity of other types of skin barrier, such as that against the penetration of foreign chemical substances, has been inferred to from measurements of the water barrier [30]. Typically, the quality of the SC water barrier function is assessed by measurements of the transepidermal water loss (TEWL) through the SC, which in turn is inferred to by the measurement of water vapor flux density through a cylindrical chamber that is placed in contact with the skin surface [31]. Low values of TEWL are indicative of good barrier function, whereas high values are associated with compromised or poor barrier, such as in diseases with skin barrier abnormalities (eczema, psoriasis, etc.) [32, 33] or following barrier perturbation (tape stripping, washing by harsh cleansers, occlusion of detergents, etc.) [34]. There have been at least four types of instruments measuring TEWL presented in the literature that differ in the design of the probe: open chamber, closed chamber, closed ventilated chamber, and closed chamber with condenser. Measurements of TEWL may be confounded by factors not relating to water barrier itself, but of other internal (skin temperature, sweat gland activity, and subject stress level) or external (environmental temperature and humidity) factors [35, 36]. Another way that has been proposed in the literature to study the water barrier is that of measuring skin moisture content during an sorption/desorption test [37]. Typically, the protocol involves initial measurement of skin hydration followed by application of a drop of water at the site of interest. The drop is left for 10 s on the skin and then blotted off. Sequential measurements of skin hydration follow at defined time points (typically every 10–15 s) until the value stabilizes. The described method provides at least two pieces of information. The initial rise in measured skin hydration between baseline and immediately following blotting of the externally applied water (sorption) indicates how hydrophilic the SC is. In other words, the lower the sorption part of the skin hydration evolution curve, the better is the water barrier function. The second piece of information relates to the time that it takes for the skin hydration value to return to baseline (desorption). This parameter relates to the water-holding capacity of the SC. Moreover, information relating to the mechanisms involved in water desorption can be inferred from the kinetics of the desorption curve [4].
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The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
Fluorescence Spectroscopy It has been mentioned that self-renewal is a critical function of the epidermis. Epidermal stem cells are believed to give rise to transient amplifying cells that reside at the basal layer of the epidermis. These cells are responsible for replenishing the number of cells that are lost due to natural sloughing of the corneocytes at the top of the SC. Moreover, the replicative rate can increase in the case of injury to rapidly repair the damage. The epidermal cell proliferation rate has been shown to correlate with a signal that can be followed in vivo using fluorescence spectroscopy coupled to a bifurcated fiber optic probe. This signal (295 nm excitation) corresponds to the fluorescence of tryptophan moieties and it has been shown to be exceedingly high in skin diseases involving abnormally high keratinocyte proliferation rate, like in psoriasis [38]. When the epidermis is induced to increase its replenishing rate (either by mechanical or by chemical means), the tryptophan signal increased accordingly [39]. Finally, this signal has been shown to be inducible following biochemical stimulation [40] or exposure to UV radiation [39]. Interestingly for the study of skin aging, the tryptophan signal decreases with the individual’s age (in adults) reflecting the decreased capacity of epidermal cells to proliferate [41]. Moreover, skin sites that are relatively more exposed to environmental factors, such as solar radiation, wind, and cold temperature, are induced to respond by increasing their epidermal proliferation rate, compared to relatively less exposed skin sites. This inducible capacity to respond to external aggressors also decreases with aging [41].
Comparison Between Infant and Adult Skin Functions Firstly, the activity of skin appendages in infants and adults would be examined and then the functions of interfollicular skin and in particular the SC water barrier and the keratinocyte proliferation rate would be compared.
Sebaceous and Sweat Gland Activity The organogenesis of skin appendages like the hair follicles and the sebaceous and sweat glands is complete at birth, which means that the number of these organs remains the same throughout life (or even decreases as in the case of eccrine sweat glands) [42]. However, the
smaller total skin surface area relative to body size in infants means that the density of the appendages is higher compared to adults. The sebaceous glands are well developed and functional in full-term newborns and it is believed that their production of sebum at levels comparable to those of adult skin is due to the residual influence of maternal hormones [43]. A few days after birth though, they shut down for a long quiescent period that lasts until puberty. Analysis of skin lipids therefore shows differences between infants and adults that can be explained at least in part due to the minimal sebaceous activity for the infants older than 1 month [44]. Although the size and structural maturity of sweat glands is similar in infants and adults, their responses differ. While the sweating that results in response to arousal and pain (palmar-plantar or emotional sweating) is detectable from the early days after birth, the sweating response to overheating (as means of cooling) is minimal [45]. Moreover, during the first year of life the number of active glands is small and their function is irregular.
Water Barrier The water-handling properties of preterm infant and neonatal skin have been studied in extend elsewhere [46]. Here the properties of infant skin beyond the first month of life would be reviewed. It has been recently reported that infant and adult skin differ in their water-handling properties, which is evidence to the fact that skin continues to develop and fine-tune its functions during the first years of life [4]. Whether measured by TEWL or by the sorption/desorption test, the water barrier function of infant SC was found to be lacking behind the integrity of the adult barrier. Interestingly, the water barrier function is evolving with the age of the infant, tending toward adult values as the infant gets older. A higher variability was also observed in the infant TEWL data compared to the adult data indicating that the homeostatic mechanisms are not yet fully developed. Moreover, the sorption/desorption test showed that infant skin absorbs more water than adult skin, but also loses this water following faster kinetics. Interestingly, adult skin appears to involve a single mechanism of desorption, while baby skin apparently involves a second one that is more rapid than the first. Similar to the TEWL data, using skin conductance measurements, higher values for baseline skin hydration in infants compared to adults [4] were reported. This is important because it distinguishes healthy infant skin
The Structural and Functional Development of Skin During the First Year of Life: Investigations Using Non-invasive Methods
water barrier from that of an abnormal condition (e.g., atopic dermatitis or psoriasis). In the case of a barrier impairing skin disease the TEWL is high and the skin hydration is low. Infant skin on the other hand shows both TEWL and skin hydration to be higher compared to adult skin. This can be an indication that infant skin barrier is not completely compromised, but that the involved mechanisms may be different than those in adult skin and that these mechanisms continue to develop during the first years of life. A further observation that leads to the same conclusion is the fact that although skin hydration is higher in infants compared to adults, the concentration of NMF in infant SC (measured by Raman microspectroscopy) is lower. Therefore, the water-holding capacity of infant SC must rely on a reservoir other than the NMF. Furthermore, the NMF concentration profile through the SC depends on the infant age, with older infants resembling closer the profiles of their mothers. Raman microspectroscopy measurements moreover confirmed the higher water content of infant SC measured by skin conductance, as well as the higher hydrophilicity following topical application of a drop of water.
Epidermal Cell Proliferation Infant granular cells and corneocytes are smaller than adult ones. This observation is an evidence to the fact that the turnover rate of epidermal cells in the infant is faster than in the adult skin. Using in vivo fluorescence spectroscopy it has been confirmed that this is indeed the case [20]. The fluorescence band linked to keratinocyte proliferation is up to ten times higher in young infants ( Table 67.1). Nevertheless, the majority of published studies [38–46] note a significant decrease of TEWL with age, especially after 60 years. A study performed by Leveque [39] on 145 healthy volunteers reports a significant decrease of TEWL on the forearm during the first 20 years of life and a second decrease after 70 years of age as compared with adulthood levels. The decreased baseline TEWL in the elderly was also confirmed by Wilhelm et al. [40]. TEWL was demonstrated to be significantly lower in an aged group than
. Table 67.1 Age dependence of TEWL Group size (n)
Anatomic site
Result
Reference
87
Lower water loss in individuals older than 70 years
–
Baker (1971) [41]
39
TEWL tended to decrease with increasing age, however, correlation was not statistically significant (age range of the group was not indicated)
Abdomen
Grice and Bettley (1967) [32]
21
Significant negative correlation between TEWL and age (22–71 years)
Onychial
Jemec et al. (1989) [44]
21
Lower TEWL in 66–81 year old individuals than in 19–26 year olds
Leg and forearm
Kligman (1979) [46]
Decreased TEWL after 60 years of age
Forearm
Leveque et al. (1989) [39]
33
No correlation between baseline TEWL and age (range: 19–85 years); on the other hand, four out of six radiolabeled drugs showed significant decreased percutaneous penetration in aged individuals
Forearm
Roskos et al. (1989) [47, 48]
23
No difference in TEWL between age groups (20–30 vs 65–80 years); the Upper arm percutaneous penetration of benzoic acid, however, was significantly reduced in aged individuals
Rougier et al. (1988) [37]
22
TEWL decreased in the aged group (19–22 vs 61–85 years)
Pretibial
Tagami (1988) [45]
43
TEWL showed no correlation with age (age range: 20–48 years)
Forearm
Tupker et al. (1989) [27]
29
Significant decrease with age on most anatomical regions (mean age 26.7 vs 70.5 years)
Eleven different regions
Wilhelm et al. (1991) [40]
20
No significant difference between groups (mean age 29.8 vs 73.6 years)
Seven different regions
Marrakchi et al. (2007) [51]
15
In all sites, the older group had higher TEWL values, but none were significant Eight different Shriner et al. regions (1996) [50]
145
158
TEWL decreases slightly over the entire age scale (8-89 years)
Ventral arm
Leveque et al. (1984) [63]
Transepidermal Water Loss and Aging
in young individuals in 9 of 11 anatomic sites. TEWL of the palm and postauricular region did not differ between age groups. In the same study, SC hydration, measured as capacitance, did not differ between groups in any of 11 sites. Of note, the postauricular region was the most hydrated site, while the dorsal forearm was the least. Given the above data, one may be tempted to believe that the barrier function to water improves or matures with age. However, Wilhelm et al. suggested that age-related epidermal atrophy and a resulting smaller water reservoir may explain decreased TEWL in elderly subjects. Some reports have failed to demonstrate any significant correlation between age and TEWL [27, 37, 43, 47]. The critical reader might wonder how results can be so conflicting despite similar methodologies and group sizes. While no information about the age of the study group was provided by Grice and Bettley [43], Tupker et al. [27] studied individuals of age 20–48 years, representing mid-adulthood levels. A correlation between age and TEWL could not be expected because TEWL apparently does not decrease before 60–70 years [39]. Studying individuals of age 60 years and older, Roskos and Guy [47] and Rougier et al. [37] failed to demonstrate significant age-related changes in TEWL, possibly due to high ambient temperatures (23 2 C) in the first study or high relative humidity (70%) in the second study. Thus, eccrine sweat contamination may have resulted in falsenegative TEWL data. Both groups, however, demonstrated increased skin barrier integrity in aged individuals by reduced percutaneous penetration of topical compounds (see below) [37, 48]. Roskos and Guy [47] also examined the water barrier in young and old individuals under occlusion (‘‘stress’’), which prevents normal passive water diffusion. It was proposed that removal of occlusion would result in elevated TEWL, which would return to baseline over time. After 15 and 30 s of 24-h polypropylene chamber occlusion, TEWL rates were significantly higher in the old than in the young. Additionally, relaxation (recovery) of ‘‘stressed’’ TEWL to baseline TEWL was significantly slower in the aged group. The authors explained that lower water content in the aged SC may account for their findings. Older tissue would require more water to achieve the new, occlusion-induced hydration equilibrium. After occlusion, aged SC may have more water to eliminate, prolonging relaxation to baseline. Their results confirmed earlier work by Tagami [45], who used conductance measurements in a ‘‘water sorption–desorption test’’ to distinguish between young and old skin. Whether the procedure of hydrating SC, either by occlusion or water application, and then
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measuring TEWL truly reflects barrier properties remains open for debate. Sunwoo et al. determined that under relative humidity (RH) levels of 10% and 30%, hydration of the face decreases similarly in young and elderly men [49]. TEWL of the face increased under 10% RH compared to 30% and 50% in both groups; it stabilized at 60 min, and did not increase any further in either group. There were no significant differences in the change of hydration state or TEWL between groups under any given RH.
TEWL of Aging Skin: Anatomic Studies Examining ten younger (23–47 years) and five older patients (72–90 years), Shriner found the perioral region to have the greatest baseline TEWL values in both groups [50]. The nose and nasalabial regions also had notable TEWL, while the forearm had the lowest values. Unfortunately, precise values were not given; rather, values were plotted on a histogram. TEWL values for each of eight anatomical sites were greater (though not statistically significant) in the younger group than in the older group. In both groups, the neck and perioral regions had the highest level of hydration, while the forehead and forearm were the least well hydrated [50]. No differences were noted between age groups. Additionally, baseline TEWL and hydration correlated positively and significantly with irritation (cutaneous blood flow after closed application of 2.5% benzoic acid for 20 min) in the younger group. In the older group, only hydration correlated positively and significantly with irritation. Marrakchi examined TEWL at seven locations (primarily on the face) between ten young (24–34 years) and ten old (66–83 years) volunteers [51]. The perioral and nasolabial areas showed the highest TEWL values in both groups. No significant TEWL differences were seen between age groups, although the young group demonstrated higher mean TEWL values in the perioral area, neck, and forearm, and the old group demonstrated higher mean TEWL values in the nasolabial area, upper eyelid, forehead, chin, and nose. The neck had a significantly higher capacitance (SC hydration) than all other areas in both age groups [51]. Capacitance of the upper eyelid was significantly higher in the young group. Forearm capacitance was significantly higher in the old group. The authors subdivided the face into three areas based on their findings: (1) areas with a lack of water influx in the epidermis: low capacitance and TEWL (nose and forehead); (2) areas with excess of water
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Transepidermal Water Loss and Aging
evaporation: high TEWL and low capacitance (nasolabial and perioral areas); and (3) areas with high water-holding capacity: high capacitance and low TEWL (the neck).
Percutaneous Penetration and Skin Aging A significant correlation between TEWL and percutaneous absorption of diverse drugs has been demonstrated in several studies [28, 37, 38]. Decreased TEWL in aged individuals apparently reflects a less permeable membrane to topically applied compounds [37, 38, 48, 52, 53]. Rougier et al. [37] report a significantly decreased percutaneous absorption of [14C]-benzoic acid, a highly watersoluble compound, in older subjects (65–80 years). Roskos et al. [48] confirm significantly decreased penetration of four of six radioisotope-labeled substances in aged individuals (>65 years) compared to young ones (22–40 years). Penetration of hydrocortisone, benzoic acid, acetylsalicylic acid, and caffeine was significantly lower in the aged group. Roskos et al. concluded that aging can affect percutaneous absorption, and that relatively hydrophilic compounds are particularly sensitive. The diminished surface lipid content of aging skin results in a diminished dissolution medium for compounds administered topically. This physiologic change may especially impact permeants with lower lipid solubility. This reduced penetration is only compounded by the reduced hydration of aged SC. Conversely, highly lipid-soluble chemicals may still dissolve readily into the SC even when the available lipid medium is reduced. In fact, the percutaneous penetration of testosterone and estradiol (the most lipophilic compounds studied) did not differ significantly between groups in the study by Roskos et al. Thus, like the results of Rougier et al. with benzoic acid, hydrophilic compounds were less absorbed in the elderly. This supports earlier studies by Christophers and Kligman [52] and Tagami [53] who concluded that the barrier function of human skin in vivo increases with increasing chronological age. Despite evidence of decreased drug absorption with age, a recent study demonstrated the rate and extent of salicylic acid (2% cream) absorption to be similar in aged and normal skin [54].
Proclivity to Skin Irritation of the Elderly Epidemiological data suggest a lower incidence of irritant contact dermatitis with increasing age [55]. This may be explained by decreased exposure to or avoidance of
cutaneous irritants. In addition, experimental studies confirmed a decreased reactivity to cutaneous irritants with increasing age [42, 56–59]. Irritant response severity has been investigated after 24-h 0.25% sodium lauryl sulfate (SLS) application on ten anatomic sites of 15 female subjects divided into two age groups (young [mean age of 25.9 1.4 years] and old [mean age of 74.6 1.9 years]) [56]. The severity of SLS-induced skin irritation was quantified by visual erythema scores and TEWL measurements. Elderly individuals had significantly lower irritant responses on 5 of 11 sites; furthermore, these individuals still had lower (though not significant) responses in most of the remaining sites compared to the young group. At some anatomic sites (thigh and dorsal forearm), aged individuals completely failed to demonstrate erythematous reactions. TEWL measurements at these sites, however, demonstrated that, despite a lack of visual signs, barrier damage still occurred. Thus, a disparity exists between visual inflammation and nonvisual function (TEWL), particularly in the elderly group. A similar study demonstrated age-related decrease in inflammation after Rhus allergen contact in previously sensitized persons [60]. As many as 8/9 young (18–25 years) subjects reacted by 24 h after exposure, compared with 1/6 elderly (65–84 years) subjects [60]. An epidemiological study found age-related differences in immediate (type I, urticarial) hypersensitivity responses using prick tests to common allergens [61]. Peak prevalence of reactivity occurred in the third decade (52%), decreasing slowly until 50 years, and then rapidly afterwards (16% in those older than 75) [61]. This may be explained by a true decline in immunologic reactivity or in the skin’s ability to respond to immunologic challenge [61]. Grove et al. demonstrated lower visual inflammatory scores to dimethyl sulfoxide, histamine, ethyl nicotinate, chloroform-methanol, and lactic acid in old versus young (ages not given) subjects [57]. Ghadially et al. elegantly examined the effects of barrier disruption on young and aged human and murine skin [11]. Barrier insults (acetone treatment and adhesive tape stripping) demonstrated significant differences in barrier integrity and recovery between groups. Barrier perturbation (TEWL 20 g/m2/h) occurred after 18 2 strippings in aged human skin versus 31 5 strippings in young human skin. Additionally, after 24 h, 50% barrier recovery (based on baseline TEWL) occurred in young subjects compared with 15% in old subjects. ‘‘The aged epidermal permeability barrier is both easier to perturb and slower to repair’’ [11].
Transepidermal Water Loss and Aging
Ghadially et al. also demonstrated a 30% decrease in total lipid content of aged murine SC; lipid profile and distribution were similar in aged versus young murine SC [11]. The aged epidermis secreted fewer lamellar bodyderived contents; furthermore, these contents failed to form continuous multilamellar bilayers in the SC interstices. This may contribute to a porous SC matrix, accounting for altered barrier integrity and repair. Barrier disruption may be attributable to a depletion or alteration in intercellular lipids. Exogenous lipid formulations have demonstrated promising results. Coderch et al. compared liposomal lipid formulations to Ceraphyl 45 lipid formulations; the lipid formulations examined were: synthetic stratum corneum lipid mixture (25% palmitic acid, 25% cholesterol, 40% ceramide III, and 10% cholesterylsulfate) and internal wool lipid extract (22.3% free fatty acids, 24.5% cholesterol, 21.9% ceramides, and 9.8% cholesterylsulfate) [62]. Liposomal formulations demonstrated superior water-holding capacity to Ceraphyl 45 formulations. Following SLS exposure, aged skin treated with liposomal lipid formulations had lower TEWL values than control and placebo skin, indicating acquired protection of the skin barrier against detergentinduced dermatitis.
Other Skin Changes Associated with Aging Leveque et al. determined that skin thickness begins to decrease after 35 years for women and 45 years for men, skin elasticity (torsion extensibility) decreases significantly after 35 in both sexes, SC (corneocyte) shedding increases after 60, and average depth of skin imprint lines (furrows) increases greatly after 60 though their density decreases [63]. Cua et al. determined that the dynamic friction coefficient (m) did not vary with age in 29 volunteers in two age groups (young [seven females with a mean age of 24.9 1.1 years and seven males with a mean age of 28.7 0.5 years] and old [seven females with a mean age of 75.3 2.4 years and eight males with a mean age of 73.8 1.9 years]) [64]. The forehead and postauricular region had the highest m of 11 anatomic areas, while the abdomen had the lowest. A significantly lower capacitance was also found only on the palms of the ‘‘old’’ group. TEWL was generally lower in the elderly everywhere except the postauricular region and palm. A significant correlation was demonstrated between m and capacitance for most anatomic regions, but between m and TEWL only on the palm and thigh.
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Murine Experiments To better understand the exacerbation of chronic xerosis in the elderly during wintertime, Choi et al. studied the skin barrier of aged hairless mice in a dry environment [65]. Epidermal thickness and SC layer number in aged mice increased in a dry environment, though TEWL did not differ between dry and normal environments. TEWL was lower in aged mice compared with young mice in normal humidity (40–60%). Contrary to aged mice, young mice had increased TEWL in a dry environment ( 80 mg/cm2/h1) to study barrier function [65]. In young mice, recovery rate was delayed at 24 h after acetone exposure in a dry environment. The recovery rate of the aged skin barrier after acetone treatment was delayed in both environments, compared to young skin barrier. However, no difference in aged skin barrier recovery occurred between environments. After barrier perturbation in the aged mice, changes in SC layer number and epidermal thickness were similar in both environments. Under normal humidity, change in epidermal thickness after barrier perturbation was more marked in aged mice than in young mice. Compared to young mice, aged mice had slightly thinner intercellular lipid layers on electron microscopy [65]. Secretion and number of lamellar bodies did not differ between both environments in aged mice. Ghadially et al. further examined the effect of lipids on SC barrier function [66]. As described previously, SC of aged mice displays decreased lipid content and extracellular bilayers. This may result in impaired barrier recovery after insult (18.7% vs 60.8% recovery by 24 h in aged vs young mice). Upon further examination, Ghadially et al. determined that cholesterol synthesis is decreased significantly under basal conditions. Furthermore, sterologenesis fails to reach absolute levels obtained in young epidermis following tape stripping perturbation. A 40% decrease in the activity of HMG-CoA reductase, the rate-limiting enzyme in sterologenesis, was observed under basal conditions in aged mice. Despite a greater than 100% increase in HMG-CoA reductase activity after barrier perturbation in aged mice, absolute levels did not attain those reached in treated, young epidermis. Ghadially et al. also supplemented aged murine SC with an equimolar mixture of SC physiological lipids (cholesterol/ceramide/linoleic acid/palmitic acid) or cholesterol alone [66]. Either mixture, applied once, accelerated barrier recovery (assessed by TEWL measurement) at
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Transepidermal Water Loss and Aging
both 6 and 24 h after disruption with cellophane tape stripping (6–8 strips). Additionally, after four applications of either mixture, electron microscopy demonstrated repletion of extracellular spaces with normal lamellar bilayer structures.
Discussion Despite a predisposition to dryness, aged skin does not demonstrate perturbed water permeability at baseline (as indicated by TEWL). In contrast, most studies displayed lower TEWL in individuals of age 60 and older [39–42, 44–46]. An explanation for reduced TEWL in the aged is not obvious. Skin anatomy, physiology, and biochemistry change markedly with increasing age [67]. Barrier function, undoubtedly, changes as well (> Table 67.2). Reduced sweating associated with aging [68] probably does not contribute to decreased TEWL, as sweating fails to significantly alter baseline TEWL at rest in a cool environment [69]. Berardesca proposes that increased corneocyte size and SC thickness, secondary to impaired desquamation and corneocyte accumulation, may account for decreased TEWL after age 60 [70]. It is explained that a concomitant decrease in TEWL and SC hydration distinguishes dry aged skin from dry pathological skin, which has high TEWL and decreased hydration [70].
Nevertheless, studies on isolated corneocytes have not demonstrated an inverse relationship between corneocyte size and TEWL [71]. In the case of skin senescence, there seems to be no clear correlation between these parameters [63]. Although the thickness of the SC is not altered by age, its renewal time is greatly prolonged. In young adults, SC transit time as estimated by the dansyl chloride staining method is about 20 days, while it is more than 30 days in older adults [72]. Whether increased renewal time in aged SC is relevant to its barrier properties is still unknown. Decreased density and efficiency of the skin microvasculature [73] resulting in decreased skin temperature may explain diminished TEWL in elderly individuals [63]. However, Wilhelm et al. [39] corrected TEWL measurements to a standard skin reference temperature of 30 C, and still noted lower TEWL values in the elderly. Altered SC lipid composition may also contribute to the decreased TEWL values in elderly individuals [74].
Physicochemical Interpretation The simplest way to model both percutaneous absorption and TEWL is to apply Fick’s first law of diffusion, even though the SC is not an inert membrane. The equation often appears as: dQ=dt ¼ DKp c=h
. Table 67.2 Summary of age-dependent changes relevant to the permeability barrier Structure/ parameter
SC thickness Unchanged SC intercellular lipids
Influence on permeability barrier
Change with skin aging –
Reference Holbrook KA, Odland GF (1974) [8], Plewig G, et al. (1983) [9]
Changed composition Possible influence on (decrease of sterol esters) and partitioning and triglycerides) diffusivity
Miyake I. (1988) [73]
Decreased density
Same as above; experiments done on mice
Ghadially R, et al. (1995) [11]
SC water content
Subtle decrease
Decreased partitioning Tagami H. (1988) [45], Surber C, et al. (1990) [76] of lipophilic compounds into SC
SC turnover/ renewal
Prolonged
Unknown
Ghadially R, et al. (1995) [11], Roskos KV, Guy RH. (1989) [47], Leveque JL, et al. (1984) [63], Marks R et al. (1981) [71]
Epidermis
Atrophy
Decreased water reservoir
Grove GL, Kligman AM (1982) [72]
Transepidermal Water Loss and Aging
where dQ/dt = rate of skin penetration (TEWL); D = effective diffusion coefficient of drug (water) in SC; Kp = partition coefficient of drug (water) between membrane and solution; c = concentration gradient of drug (water in this case); and h = effective thickness of the skin barrier. For water diffusing through the SC in the opposite direction, dQ/dt is measurable at the skin surface as TEWL. Thus, all factors relevant to dQ/dt apply to TEWL. SC thickness (h) is not altered by age [8, 9] and may not account for the increased in vivo barrier. It may also be assumed that c is constant, although age-related epidermal atrophy and reduced tissue hydration may decrease the concentration gradient for water [73]. SC lipid composition and concentration may change with increasing age [11, 74], but the relevance of this finding to barrier properties is not completely clear. Roskos confirmed an altered SC lipid composition in aged individuals. Additionally, she found an overall diminution of SC epidermal lipid content with increasing age in humans in vivo by means of attenuated total reflectance infrared spectroscopy (ATR-IR) [75]. Decreased epidermal lipid content in the elderly would reduce the partitioning (Kp) of water and hydrophilic compounds in SC [76], thus explaining the reduced TEWL and the decreased percutaneous absorption of hydrophilic compounds. SC water content also influences Kp. No significant differences in SC water content between young and old individuals were demonstrated by capacitance or conductance measurements [45, 50, 56]. Using more sensitive ATR-IR spectroscopy instrumentation, however, it was demonstrated that the SC is drier in the elderly [77]. Tagami also confirmed reduced water-binding capacity in aged SC using a ‘‘water sorption–desorption’’ test [45]. Reduced SC hydration in the elderly would imply that aged skin is less attractive to hydrophilic molecules and to water. This would result in a decreased Kp, and, thus, a decreased dQ/dt.
Conclusion A dynamic membrane, the SC is constantly changing and adapting as people age. It is now believed that barrier function, represented by TEWL, correlates directly with age. Age-associated improvement in permeability barrier function is incompletely understood; numerous theories abound. This has important implications for transdermal drug delivery in the elderly. Despite superior baseline integrity, aged SC may be disrupted more easily and take longer to recover under stressed conditions. Further
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research clearly describing age-related changes in barrier function will be invaluable.
Cross-references > Bioengineering > Hydration
Methods and Skin Aging of the Skin Surface
References 1. Rutter N. The immature skin. Br Med Bull. 1988;44(4):957–970. 2. Hammarlund K, Sedin G, Stromberg B. Transepidermal water loss in newborn infants. VIII. Relation to gestational age and post-natal age in appropriate and small for gestational age infants. Acta Paediatr Scand. 1983;72(5):721–728. 3. Wilson DR, Maibach HI. An in vivo comparison of skin barrier function. In: Maibach HI, Boisits EK (eds) Neonatal Skin, Structure, and Function. New York: Marcel Dekker, 1982, pp. 101–110. 4. Nikolovski J, et al. Barrier function and water-holding and transport properties of infant stratum corneum are different from adult and continue to develop through the first year of life. J Invest Dermatol. 2008;128(7):1728–1736. 5. Blank IH, Scheuplein RJ. Transport into and within the skin. Br J Dermatol. 1969;81:4–10. 6. Scheuplein RJ. Percutaneous absorption after twenty-five years: or ‘‘old wine in new wineskins.’’ J Invest Dermatol. 1976;67(1):31–38. 7. Elias PM. Epidermal lipids, membranes, and keratinization. Int J Dermatol. 1981;20(1):1–19. 8. Holbrook KA, Odland GF. Regional differences in the thickness (cell layers) of the human stratum corneum: an ultrastructural analysis. J Invest Dermatol. 1974;62(4):415–422. 9. Plewig G, et al. Thickness of corneocytes. In: Marks R, Plewig G (eds) Stratum Corneum. Berlin: Springer-Verlag, 1983, pp. 171–174. 10. Michaels AS, Chandrasekaran SK, Shaw JE. Drug permeation through human skin, theory and in vivo experimental measurement. J Am Inst Chem Eng. 1975;21:985–996. 11. Ghadially R, et al. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95(5): 2281–2290. 12. Grubauer G, Elias PM, Feingold KR. Transepidermal water loss: the signal for recovery of barrier structure and function. J Lipid Res. 1989;30(3):323–333. 13. Proksch E, Elias PM, Feingold KR. Regulation of 3-hydroxy-3methylglutaryl-coenzyme A reductase activity in murine epidermis. Modulation of enzyme content and activation state by barrier requirements. J Clin Invest. 1990;85(3):874–882. 14. Jackson SM, et al. Effect of cutaneous permeability barrier disruption on HMG-CoA reductase, LDL receptor, and apolipoprotein E mRNA levels in the epidermis of hairless mice. J Lipid Res. 1992;33 (9):1307–1314. 15. Bommannan D, Potts RO, Guy RH. Examination of stratum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol. 1990;95(4):403–408. 16. Eriksson G, Lamke LO. Regeneration of human epidermal surface and water barrier function after stripping. A combined study with electron microscopy and measurement of evaporative loss. Acta Derm Venereol. 1971;51(3):169–178.
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17. Bowser PA, White RJ. Isolation, barrier properties and lipid analysis of stratum compactum, a discrete region of the stratum corneum. Br J Dermatol. 1985;112(1):1–14. 18. Blank IH. Further observations on factors which influence the water content of the stratum corneum. J Invest Dermatol. 1953;21 (4):259–271. 19. Blank IH, Gould E. Location and reformation of the epithelial barrier to water vapor. Arch Dermatol. 1962;78:702–714. 20. Blank IH, Gould E. Study of mechanisms which impede the penetration of synthetic anionic surfactants into skin. J Invest Dermatol. 1962;37:311–315. 21. Thune P. Evaluation of the hydration and the water-holding capacity in atopic skin and so-called dry skin. Acta Derm Venereol Suppl (Stockh). 1989;144:133–135. 22. Frost P, et al. Ichthyosiform dermatoses. 3. Studies of transepidermal water loss. Arch Dermatol. 1968;98(3):230–233. 23. Silverman RA, Lender J, Elmets CA. Effects of occlusive and semiocclusive dressings on the return of barrier function to transepidermal water loss in standardized human wounds. J Am Acad Dermatol. 1989;20(5 Pt 1):755–760. 24. Grice K, Sattar H, Baker H. The cutaneous barrier to salts and water in psoriasis and in normal skin. Br J Dermatol. 1973;88(5):459–463. 25. Serup J, Staberg B. Differentiation of allergic and irritant reactions by transepidermal water loss. Contact Dermatitis. 1987;16(3):129–132. 26. van der Valk PG, Nater JP, Bleumink E. Skin irritancy of surfactants as assessed by water vapor loss measurements. J Invest Dermatol. 1984;82(3):291–293. 27. Tupker RA, et al. Baseline transepidermal water loss (TEWL) as a prediction of susceptibility to sodium lauryl sulphate. Contact Dermatitis. 1989;20(4):265–269. 28. Lotte C, et al. In vivo relationship between transepidermal water loss and percutaneous penetration of some organic compounds in man: effect of anatomic site. Arch Dermatol Res. 1987;279(5):351–356. 29. Onken HD, Moyer CA. The water barrier in human epidermis. Physical and chemical nature. Arch Dermatol. 1963;87:584–590. 30. Murahata RI, Crowe DM, Roheim JR. The use of transepidermal water loss to measure and predict the irritation response to surfactants. Int J Cosmet Sci. 1986;8:225–231. 31. Wilson DR, Maibach HI. Transepidermal water loss: a review. In: Leveque JL (ed) Cutaneous Investigation in Health and Disease. Noninvasive Methods and Instrumentation. New York: Marcel Dekker, 1989, pp. 113–134. 32. Grice KA. Transepidermal water loss. In: Jarret A (ed) The Physiology and Pathophysiology of the Skin. London: Academic, 1980, pp. 2115–2127. 33. Nilsson GE. Measurement of water exchange through skin. Med Biol Eng Comput. 1977;15(3):209–218. 34. Pinnagoda J, et al. Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis. 1990;22(3):164–178. 35. Tagami H, Kobayashi H, kikuchi K. A portable device using a closed chamber system for measuring transepidermal water loss: comparison with the conventional method. Skin Res Technol. 2002;8:7–12. 36. Nuutinen J. Measurement of transepidermal water loss by closedchamber systems. In: Serup J, Jemec GBE, Grove GL (eds) Handbook of Non-invasive Methods and the Skin. Boca Raton: Taylor & Francis, 2006. 37. Rougier A, et al. Relationship between skin permeability and corneocyte size according to anatomic site, and sex in man. J Soc Cosmet Chem. 1988;39:15–26.
38. Dupuis D, et al. In vivo relationship between percutaneous absorption and transepidermal water loss according to anatomic site in man. J Soc Cosmet Chem. 1986;37:351–357. 39. Leveque JL. Measurement of transepidermal water loss. In: Leveque JL (ed) Cutaneous Investigation in Health and Disease. New York: Marcel Dekker, 1989, pp. 135–152. 40. Wilhelm KP, Cua AB, Maibach HI. Skin aging. Effect on transepidermal water loss, stratum corneum hydration, skin surface pH, and casual sebum content. Arch Dermatol. 1991;127(12):1806–1809. 41. Baker H. Deperdition d’eau par voie trans-epidermique. Ann Dermatol Syphiligr. 1971;98:289–296. 42. Elsner P, Wilhelm D, Maibach HI. Sodium lauryl sulfate-induced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal woman. J Am Acad Dermatol. 1990;23:648–652. 43. Grice K, Bettley FR. Skin water loss and accidental hypothermia in psoriasis, ichthyosis and erythrodermia. Br Med J. 1967;4:195–201. 44. Jemec G, Agner T, Serup J. Transonychial water loss. Relation to sex, age and nailplate thickness. Br J Dermatol. 1989;121:443–446. 45. Tagami H. Aging and the hydration state of the skin. In: Kligman AM, Takase Y (eds) Cutaneous Aging. Tokyo: University of Tokyo Press, 1988, pp. 99–109. 46. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39–46. 47. Roskos KV, Guy RH. Assessment of skin barrier function using transepidermal water loss – effect of age. Pharm Res. 1989;6:949–953. 48. Roskos KV, Maibach HI, Guy RH. The effect of aging on percutaneous absorption in man. J Pharmacokinet Biopharm. 1989;17: 617–630. 49. Sunwoo Y, et al. Physiological and subjective responses to low relative humidity in young and elderly men. J Physiol Anthropol. 2006;25(3):229–238. 50. Shriner DL, Maibach HI. Regional variation of nonimmunologic contact urticaria. Functional map of the human face. Skin Pharmacol. 1996;9(5):312–321. 51. Marrakchi S, Maibach HI. Biophysical parameters of skin: map of human face, regional, and age-related differences. Contact Dermatitis. 2007;57(1):28–34. 52. Christophers E, Kligman AM. Percutaneous absorption in aged skin. In: Montagna W (ed) Advances in Biology of Skin. Oxford: Pergamon, 1965, pp. 163–175. 53. Tagami, H. Functional characteristics of aged skin. 1. Percutaneous absorption. Acta Dermatol (Kyoto). 1971/1972;66/67:19–21. 54. Davis DA, et al. Percutaneous absorption of salicylic acid after repeated (14-day) in vivo administration to normal, acnegenic or aged human skin. J Pharm Sci. 1997;86(8):896–899. 55. Malten KE, et al. Occupational dermatitis in five European dermatological departments. Berufsdermatosen. 1971;19(1):1–14. 56. Cua AB, Wilhelm KP, Maibach HI. Cutaneous sodium lauryl sulphate irritation potential: age and regional variability. Br J Dermatol. 1990;123(5):607–613. 57. Grove GL, et al. Use of nonintrusive tests to monitor age-associated changes in human skin. J Soc Cosmet Chem. 1981;32:15–26. 58. Soschin D, Kligman AM. Adverse subjective responses. In: Kligman AM, Leyden JJ (eds) Safety and Efficacy of Topical Drugs and Cosmetics. New York: Grune and Stratton, 1982, pp. 377–387. 59. Coenraads PJ, Bleumink E, Nater JP. Susceptibility to primary irritants: age dependence and relation to contact allergic reactions. Contact Dermatitis. 1975;1(6):377–381. 60. Lejman E, et al. Age differences in poison ivy dermatitis. Contact Dermatitis. 1984;11(3):163–167.
Transepidermal Water Loss and Aging 61. Barbee RA, et al. Immediate skin-test reactivity in a general population sample. Ann Intern Med. 1976;84(2):129–133. 62. Coderch L, et al. Efficacy of stratum corneum lipid supplementation on human skin. Contact Dermatitis. 2002;47(3):139–146. 63. Leveque JL, et al. In vivo studies of the evolution of physical properties of the human skin with age. Int J Dermatol. 1984;23(5):322–329. 64. Cua AB, Wilhelm KP, Maibach HI. Frictional properties of human skin: relation to age, sex and anatomical region, stratum corneum hydration and transepidermal water loss. Br J Dermatol. 1990;123 (4):473–479. 65. Choi EH, et al. The skin barrier state of aged hairless mice in a dry environment. Br J Dermatol. 2002;147(2):244–249. 66. Ghadially R, et al. Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice. J Invest Dermatol. 1996; 106(5):1064–1069. 67. Gilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol. 1989;21(3 Pt 2):610–613. 68. Sato K, Timm DE. Effect of aging on pharmacological sweating in man. In: Kligman AM, Takase Y (eds) Cutaneous Aging. Tokyo: University of Tokyo Press, 1988, pp. 111–126. 69. Pinnagoda J, et al. Transepidermal water loss with and without sweat gland inactivation. Contact Dermatitis. 1989;21(1):16–22.
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70. Berardesca E, Maibach HI. Transepidermal water loss and skin surface hydration in the non invasive assessment of stratum corneum function. Derm Beruf Umwelt. 1990;38(2):50–53. 71. Marks R, Nicholls S, King CS. Studies on isolated corneocytes. Int J Cosmet Sci. 1981;3:251–258. 72. Grove GL, Kligman AM. Age-associated changes in human epidermal cell renewal. J Gerontol. 1982;38:137–142. 73. Miyake I. Histological aging of facial skin. In: Kligman AM, Takase Y (eds) Cutaneous Aging. Tokyo: University of Tokyo Press, 1988, pp. 571–588. 74. Saint Leger D, et al. Age-associated changes in stratum corneum lipids and their relation to dryness. Dermatologica. 1988;177 (3):159–164. 75. Roskos KV. The effect of skin aging on the percutaneous penetration of chemicals through human skin. Dissertation. University of California, San Francisco, 1989. 76. Surber C, et al. Optimization of topical therapy: partitioning of drugs into stratum corneum. Pharm Res. 1990;7(12):1320–1324. 77. Potts RO, Buras EM. In vivo changes in the dynamic viscosity of human stratum corneum as a function of age and ambient moisture. J Soc Cosmet Chem. 1985;36:169–1985.
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Microbiology
83 Aging Skin Microbiology Duane. L. Charbonneau . Yen L. Song . Cheng Xu Liu
Introduction The skin, the largest organ of the body, helps maintain homeostasis by regulating heat and moisture transfer and is the body’s first line of defense against environmental insults such as temperature extremes and pathogen invasion. Structurally, it is composed of multiple layers of epithelial cells (> Fig. 83.1). The outer layer of the skin, the epidermis, arises from a generative basal layer of cells; the daughter cells terminally differentiate and keratinize as they rise to the skin surface. Thus, about 90% of the epidermis consists of dead keratinocytes embedded in a lipid-rich, hydrophobic ‘‘mortar’’ that creates a relatively impervious barrier with the environment. Other epidermal cell types include melanocytes (pigmented, UVabsorbing cells), sensory Merkel cells, and Langerhans cells of the immune system. Below this, the dermis contains structural proteins (such as collagen and elastin), hair follicles and sebaceous glands, other eccrine and apocrine glands, adipose tissue, and blood vessels. In the average adult, the overall skin surface area is between 1.5–2.0 m2. Average skin thickness ranges from 2–3 mm. Microorganisms colonize the skin surface and other structural elements such as the sebaceous glands and hair follicles. Historically, microbial populations associated with the human body were characterized by techniques that identified only those organisms amenable to selective culture. However, recent advances in genomic analysis yield a more comprehensive and complex picture of the body’s microbial inhabitants [1]. Several anatomical habitats (e.g. the skin, the oral and nasal cavities, the gut, and the vagina) support highly diverse populations of symbiotic and commensal organisms – collectively known as the microbiota – thought to play a role in both health and disease [1, 2]. The chapter will describe the present understanding of the microbiota associated with human skin and review what is currently known about changes to such communities as the skin ages.
Skin Microbiota Although the unborn baby’s skin is considered to be sterile in utero, the skin becomes microbially colonized
following birth. The skin harbors large numbers of microbes: cell densities of bacteria on forehead skin, for example, range from 104 to 106 cfu cm 2, as assessed by culture [3]; similarly, quantitation of microbial DNA from forehead skin swabs yields estimates of 104 cm 2 bacteria, based on the molecular weight of the average bacterial genome [4]. (For perspective, traditional techniques quantify live organisms amenable to culture conditions; genomic techniques identify a more comprehensive spectrum of microbes and their relative isolation frequencies, but do not discriminate between viable and nonviable organisms.) Currently the U.S. National Institute of Health is conducting a major project to assess the composition of the skin micobiota using metagenomic analysis [7].
Transient and Resident Biota Microbes that inhabit the skin are considered to be either transient or resident biota [5]. Transient biota become attached to the skin as a result of contact with contaminated items or environmental surfaces; for example, by handling raw foods, or by resting the forearms on a table. Such microbes are inconsistently isolated from the skin. The transfer of transient biota to and from fomites and from person to person plays a critical role in infectious disease transmission. Studies have documented transfer of such organisms from an individual’s hands to other parts of the body as well as transfer between individuals. The classic example is the work of Gwaltney and colleagues [6] which demonstrated the importance of hand-to-hand transmission of the common cold virus. In contrast, resident skin biota are the complex communities of microbes that consistently inhabit the skin and are not routinely removed by washing with non-medicated soaps. Resident bacterial species include Staphylococcus, Micrococcus, Corynebacterium, Rhodococcus, Propionibacterium, Brevibacterium, Dermatobacter and Actinobacter [5]. The community composition of resident skin biota is believed to be as essential to the health of the skin as gut microorganisms are to overall health of the individual [2]. Resident biota promote health through pathogen inhibition, immune modulation, and by sustaining the integrity
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_83, # Springer-Verlag Berlin Heidelberg 2010
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Aging Skin Microbiology
. Figure 83.1 Diagram of the skin architecture
of the skin barrier; however, under the right conditions, certain constituents of the skin biota, such as Staphylococcus aureus, Streptococcus pyogenes, Candida spp., and Actinobacter spp., may become opportunistic pathogens [5]. This will be discussed later in the chapter.
Composition of the Skin Microbiota Characterization of the skin microbiota by cultivationindependent, genomic techniques is under way [4, 7–10], with the goals of fully characterizing community composition and defining the roles of resident and transient microbes in health and disease [1, 2]. Historically, the skin was considered to harbor rather simple microbial communities, but modern molecular tools have unlocked the true complexity of the skin microbiota [4, 6–12]. The bacterial biota of the skin include a vast array of anaerobic and aerobic communities; among the fungal biota, yeasts of the genus Malassezia appear to predominate [8] (> Tables 83.1 and > 83.2). Metagenomic analysis, typically via amplification of small subunit rRNA gene sequences (16s rDNA), allows identification of bacteria to the phylum, genus, or species level [13]. Bacterial taxa represented in the skin microbiota of healthy subjects include Actinobacteria, Proteobacteria, Firmiculites, Bacteroidetes, Cyanobacteria, Deinococcus-Thermus, Thermomicrobia, Spirochaetes, and some as yet unclassified [4, 7, 10].
Studies have found compositional differences by site (forehead, volar forearm, inner elbow, outer ear, vulva), by individual, and by time [4, 7, 10–12]. Although the data reveal a broad spectrum of organisms and a substantial degree of intra- and interindividual diversity, a significant majority (about 80%) of clones identified by genomic analysis correspond to known, culture-defined species [4, 10]. A subset of these clones are previously identified skin biota, such as Propionibacterium acnes, Staphylococcus epidermidis, and Actinobacter johnsonii; however, these species, while often dominant in cultivationdependent analyses, do not necessarily represent the majority of genetic clones isolated. Moreover, although all subjects bear diverse communities of skin biota, only a few species were consistently found on the skin of every subject. Consequently, it has been suggested that the skin biota form a scaffold that rests on a small number of resident genera and species, but supports a high frequency of diverse and variable transients [10]. Site-specific differences in community composition (e.g., volar forearm, inner elbow, outer ear, and vulva) may be influenced by differences in sebum production, moisture, occlusion, and exposure to light or environmental contaminants. For example, pseudomonads, organisms not traditionally thought of as skin microbes, are constituents of the skin biota of the inner elbow [7]. Typically found in soil, water, and decomposing organic materials, pseudomonads require moist environments for growth, which
Aging Skin Microbiology
. Table 83.1 The resident skin microflora identified by culture-based methods I. Gram-positive bacteria
. Table 83.1 (Continued) I. Gram-positive bacteria Propoinibacterium
P. avidum Coagulase-positive Staphylococcus
Brevibacterium
S. aureus
Dermabacter
Coagulase-negative Staphylococcus S. epidermidis
II. Gram-negative bacteria
S. hominis
Acinetobacter
A. calcoaceticus
S. haemolyticus
A. johnsonii
S. capitis
S. junii
S. midis
Pseudomonas
S. warneri
III. Mycoflora
S. pyrogenes S. saprophyticus S. simulans S. xylosus
Pityrosporum
M. slooffiae M. restricta
M. luteus
M. obusta
M. varians
M. sedentarius M. roseus
M. furfur M. globosa
S. sciuri
M. kristinae
P. ovale
M. sympodialis
S. saccharolyticus
M. lylae
P. aeruginosa
P. orbiculare Malassezia
S. cohnii
Micrococcus
P. acnes P. granulosum
Micrococcaceae Staphylococcus
83
M. pachydermatis Candida
C. albicans C. tropicalis C. parapsilosis
M. nishinomiyansis M. agieis Coryneforms Corynebacterium
C. diphtheriae C. diphtheriae gravis C. diphtheriae mitis C. diphtheriae belfanti C. diphtheriae intermedius Nondiphtheriae corynebacteria (diphtheroids) C. minutissimum C. tenuis C. xerosis C. jeikeium (CDC group JK) C. striatum C. afermentans
may explain their association with this particular anatomical site. However, pseudomonads were not significant constituents of the bigta of vulvar skin [12], although this site is also more moist and occluded than exposed skin. Vulvar skin biota tended to mirror the vaginal biota, and also included constituents of enteric origin. Investigators have also examined differences in skin biota from various skin layers. Sampling of the skin biota of the inner elbow by three methods (superficial swab, skin scrapping of the epidermis, and punch biopsy of the full thickness of epidermis and dermis) all provided similar profiles of community membership [7]. Lastly, skin structures (e.g. the hair follicles and the sebaceous, eccrine, and apocrine glands) may represent sub-habitats with somewhat distinct microbiota. For example, Propionibacterium acnes bacteria, etiologic agents in acne vulgaris, reside within the pilosebaceous
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. Table 83.2 Skin microflora bacteria species only detected by 16S gene clone library (Cited data from Dekio et al. [4], Gao et al. [10]) Phyla/genus
. Table 83.2 (Continued) Phyla/genus Rothia
R. mucilaginosa R. dentocariosa
C. coyleae
Nakamurella
N. multipartita
C. mucifaciens
Microlunatus
M. phosphovorus
C. lipophiloflavum
Atopobium
A. vaginae
C. appendicis
Firmicutes
C. imitans
Anaerococcus
A. prevotii
C. sundsvallense
Peptoniphilus
P. harei
C. glaucum
Peptostreptococcus
P. anaerobius
C. glucuronolyticum
Veillonella
V. dispar
C. matruchotii C. durum
V. parvula Lactobacillus
C. pseudotuberculosis
Mycobacterium
R. aeria R. nasimurium
Genus/species
Actinobacteria Corynebacterium
Genus/species
L. crispatus L. jensenii
C. pseudogenitalium
Leuconostoc
L. argentinum
C. tuberculostearicum
Streptococcus
S. infantis
C. accolens
S. gordonii
C. simulans
S. parasanguinis
C. aurimucosum
S. sanguinis
C. nigricans
S. cristatus
C. singulare
S. salivarius
C. amycolatum
S. intermedius
C. kroppenstedtii
S. agalactiae
M. chlorophenolicum
Gemella
M. obuense Tsukamurella
T. tyrosinosolvens
Rhodococcus
R. erythropolis
G. haemolysans G. morbillorum G. sanguinis
Facklamia
R. corynebacterioides
F. hominis F. languida
Dietzia
D. maris
Eremococcus
E. coleocola
Gordonia
G. sputi
Granulicatella
G. elegans
G. bronchialis
Enterococcus
E. faecalis
G. terrae
Bacillus
B. licheniformis
Mobiluncus
M. curtisii subsp. holmesii
Actinomyces
A. neuii
Proteobacteria
A. naeslundii
Methylobacterium
B. subtilis M. extorquens
Gardnerella
G. vaginalis
Brevibacterium
B. paucivorans
Bradyrhizobiaceae
Bradyrhizobiaceae spp.
Janibacter
J. melonis
Rhizobiales
Rhizobiales spp
Tetrasphaera
T. elongata
Pedomicrobium
P. australicum
Kocuria
K. marina
Hyphomicrobium
H. facile
K. palustris
Paracoccus
Paracoccus spp.
K. rhizophila
Sphingopyxis
Sphingopyxis spp.
M. mesophilicum
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. Table 83.2 (Continued) Phyla/genus
Genus/species
Sphingobium
S. amiense
Caulobacteraceae
Caulobacteraceae spp.
Paracraurococcus
Paracraurococcus spp.
Brevundimonas
B. aurantiaca B. vesicularis
Pasteurellaceae
Pasteurellaceae spp.
Haemophilus
Haemophilus spp.
Serratia
S. marcescens subsp. sakuensis
Diaphorobacter
D. nitroreducens
Acidovorax
A. temperans
Aquabacterium
Aquabacterium spp.
Pseudomonas
P. saccharophila
Burkholderiales
Burkholderiales spp.
Neisseria
Neisseria subflava
Neisseriaceae
Neisseriaceae spp.
Betaproteobacteria
Betaproteobacteria spp.
Stenotrophomonas
S. maltophilia
Xanthomonadaceae
Xanthomonadaceae spp.
Acinetobacter
A. ursingii A. haemolyticus
Alkanindiges
Alkanindiges spp.
Enhydrobacter
E. aerosaccus
Gammaproteobacteria
Gammaproteobacteria spp.
Pseudomonas
P. stutzeri P. tremae
Bacteroidetes Hymenobacter
Hymenobacter spp.
Chitinophaga
Chitinophaga spp.
Flavobacteriaceae
Flavobacteriaceae spp.
Porphyromonas
Porphyromonas spp
Prevotella
P. corporis P. disiens P. bivia P. melaninogenica
Cyanobacteria Deinococcus-Thermus Thermomicrobia
unit of the hair follicle and sebaceous gland. Utilizing sebum as a nutrient source, the microbes form biofilms, encasing themselves with a protective, extracellular polysaccharide lining that limits antimicrobial concentrations.
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This creates an ecological niche where the species selectively thrives [14, 15].
Contributions of Skin Microbiota to a Healthy Host The manner in which the skin microbiota contribute to the health of the host is not fully defined, but the existence of microcolonies and biofilms on the skin may imply the existence of consortia of microbial symbionts and commensals that function in concert with the host to assure homeostasis. Some mechanisms by which the biota contribute to the health of the host are discussed below.
Pathogen Inhibition Although the skin harbors opportunistic pathogens ubiquitous to the environment [4], it is effective in preventing pathogen invasion. The keratinized outer layer of the epidermis creates a relatively impervious barrier. Moreover, the skin turns over very 4 weeks; the shedding of dead keratinocytes from the skin surface also limits colonization by pathogens. In addition, low moisture and acidic pH conditions on the skin surface exert selective pressures to establish healthful microbiota [16]. Compromised skin is susceptible to infection by pathogenic bacteria and fungi, and the incidence of skin infection increases with age as skin becomes more fragile [17]. Recently, however, the emergence of more virulent skin pathogens, such as Community Acquired Methicillin Resistant Staphylococcus (CA-MRSA) has created a major societal burden, as these organisms have a unique propensity to infect young, otherwise healthy people [18]. Pathogen inhibition by symbionts and commensals is accomplished by various mechanisms. The first is a passive mechanism – competitive exclusion – whereby resident biota occupy skin sites that could be inhabited by pathogenic microorganisms. For example, Staphylococcus epidermidis occupies receptors on the host cell that also can be recognized by more virulent bacteria such as Staphylococcus aureus. A second mechanism is the scavenging of nutrients essential for the growth and proliferation of pathogenic bacteria. For example, Corynebacterium jeikeium produces siderophores that sequester iron and also has specialized mechanisms for acquiring of manganese. Manganese acquisition is essential for protecting this bacterium from superoxide radicals. Superoxide dismutase, a manganese-dependent enzyme, may protect both the resident bacterium and the host from oxidative damage. The sequestering of iron and manganese by resident
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bacteria robs potential pathogens of these growth-limiting substrates. A third mechanism is the production by resident biota of secondary metabolites with antimicrobial properties. For example, S. epidermidis produces lanthionine-containing antibacterial peptides, also called bacteriocins, that act as antibiotics. Some of these peptides are: epidermin, epilancin K7, epilancin 15X Pep5, and staphylococcin 1580. Other peptides produced by S. epidermidis counteract intra- and interspecies competitors. The peptides are effective against S. aureus, group A streptococcus, and Streptococcus pyogenes. Staphylococcus aureus can be a commensal inhabitant of the skin, especially in the nasal area. It is estimated that 32% of the US population carries S. aureus without infection [2]. Certain strains of S. aureus produce bacteriocins (e.g., staphylococcin 462 peptide) that inhibit the growth of other S. aureus strains. Thus, colonization by S. aureus can result in a positive benefit for the host. Propionibacteria also produce bacteriocins and bacteriocin-like compounds. These compounds include propionicin PlG-1, jenseniin G, propionicins SM1, SM2 T1, and acnecin, which are inhibitory toward lactic acidproducing bacteria, gram-negative bacteria, yeasts, and molds. Pseudomonas aeruginosa is a member of the resident skin biota in certain moist areas of the skin [7]. These bacteria produce potent compounds that inhibit the growth of other microorganisms. P. aeruginosa has been shown to suppress the growth of fungal species such as Candida krusei, C. kefr, C. guilliermondii, C. tropicalis, C. lusitaniae, C. parapsilosis, C. pseudotropicalis, C. albicans, Saccharomyces cerevisiae, Torulopsis glabrata and Aspergillus fumigatus. A classic example is the production of pseudomonic acid A by P. fluorescens. Pseudomonic acid A is the active ingredient in the commercial topical antibiotic, Mupirocin1, commonly used in the treatment and prevention of streptococcal and staphylococcal infections.
Immune Modulation Growing evidence suggests that the resident bacterium, S. epidermidis, supports immune system function in the skin. This organism may exert a protective role by influencing the innate immune response of keratinocytes through Toll-like receptor (TLR) signaling. The TLRs are pattern recognition receptors that specifically identify pathogenic moieties: the presence of S. epidermidis on the skin increases keratinocyte response to pathogens. Similarly, the presence of certain S. aureus strains among the skin
biota may prime the host immune system to recognize other more virulent strains of this bacterial species [2].
Maintaining Skin Integrity Group A streptococci produce a cytolytic toxin known as streptolysin O (SO) that is considered part of the pathogenic arsenal of this organism. Despite its toxigenic effects, SO also plays a role in wound healing by stimulating keratinocyte migration. Low concentrations of SO induce the cell adhesion molecule, CD44, and subsequently increase the production of collagen, hyaluronate, and other extra cellular matrix components that facilitate wound healing. The cytotoxin, streptokinase, also derived from streptococcal species, is now used clinically for fibrinolysis. Consequently, it is becoming apparent that certain virulence factors produced by group A streptococci also may be an asset to the host.
Skin Biota and Disease Although the resident skin biota typically may play a role in protecting the host, under certain circumstances constituents of the resident biota may themselves be pathogenic [5]. Yeast vulvovaginitis is a common example of pathogenesis by constituents of skin and mucosal biota. Candida albicans and other Candida species commonly colonize the vagina and external genitalia of healthy women without frank infection, but disruptions to the community composition of the vulvovaginal microbiota can lead to overgrowth of these organisms and the development of yeast vulvovaginitis [19]. In the facial region, P. acnes colonizes hair follicles of healthy individuals [15], but a combination of host and bacterial factors may precipitate the development of acne [14]. Breaches in skin integrity provide opportunities for infection by cutaneous biota. S. aureus and S. pyogenes are the two major organisms responsible for common skin and soft tissue infections [20]. Similarly, CA-MRSA infections are commonly associated with breaches in the skin integrity [21]. Staphylococcus epidermidis, long considered an important member of the resident skin biota, is a nosocomial pathogen associated with infections of surgical wounds and medical implants [22]. Other resident biota, such as S. warneri, Corynebacterium spp., and Pseudomonas spp., have also been demonstrated to cause infections. Emerging evidence indicates that characteristics of the skin microbiota may play a role in chronic dermatoses such as such as atopic dermatitis (AD), seborrheic
Aging Skin Microbiology
dermatitis (SD), dandruff, and psoriasis [23]. For example, yeasts of the genus Malassezia, constituents of the skin biota [24–28], tend to colonize sebaceous skin regions. These yeasts are a causative factor in pityriasis versicolor [29] and are believed to contribute to and exacerbate the development of atopic dermatitis, seborrheic dermatis, and dandruff. Moreover, the intestinal biota play a critical role in immune system development, and changes to these biota appear to be central to the development of atopy. For example, shifts in the composition of gut microbiota during infancy may alter immune responses to environmental allergens (including allergens associated with skin biota) and thereby may contribute to the development of atopic dermatitis [30, 31]. Patients with atopic dermatitis or atopic eczema are more likely to raise Malasseziaspecific IgE antibodies than healthy controls [32, 33], with adults showing higher rates of sensitization than children [34]. Furthermore, the higher cutaneous pH of patients with atopic dermatitis may enhance the release of allergens from some Malassezia species [35]. Interestingly, although certain Malassezia species (e.g., M. globosa and M. restricta) are found in both AD patients and healthy subjects, specific genotypes are more highly associated both with AD [28] and with seborrheic dermatitis [36]. In the case of psoriasis, no clear dichotomy in fungal microbiota has been found between patients and healthy subjects [8], but significant alterations have been observed in the community composition of bacterial skin biota: bacterial diversity was higher in psoriatic lesions than on healthy skin of the same patient or on the skin of disease-free subjects, with overrepresentation of the Firmiculites phylum and underrepresentation of the Actinobacteria and Proteobacteria phyla [37]. Most strikingly, the genus Propionibacterium, including the major cutaneous species P. acnes, was highly underrepresented on affected skin. Hence, psoriasis lesions are accompanied by a significant ecological shift in associated skin microbiota, but the mechanisms underlying this shift remain to be determined. Finally, skin biota also play a role in disease transmission. Differentiating between transient and resident biota has shed light particularly on the role that skin biota on the hands play in the transmission of infectious disease. Frequent exposure to certain transient microbes may lead to their becoming established constituents of the resident skin biota. For example, nurses performing similar tasks within a hospital exhibit similarities among their resident biota, but the skin biota of those assigned to different tasks have distinct constituents [38]. Similarly, bacterial constituents of the resident skin biota of homemakers often are identical to those of isolates identified within the
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home environment [39]. The association of hands with infection transmission dates back to the mid-1800s. Studies by Ignaz Semmelweis in Vienna, Austria, and Oliver Wendell Holmes in Boston, USA established that hospitalacquired diseases were transmitted via the hands of health care workers HCWs [40]. In a seminal 1847 study, Semmelweiss observed substantially higher maternal mortality from puerperal fever at one clinic versus another (16% versus 7%). Noting that in the former, doctors often went directly to the delivery suite after performing autopsies, Semmelweis instituted aggressive hand hygiene with chlorination; mortality rates fell below 3% in the more affected clinic and remained low thereafter. Since then, multiple studies have established an association of skin biota on the hands with hospital-acquired and community-acquired infections [6, 41–43].
Microbiology of Skin Aging Intrinsic and Extrinsic Aging Two primary processes, known as intrinsic and extrinsic aging, contribute to aging skin. Intrinsic aging is the result of genetically programmed cellular aging processes that occur naturally. As the skin ages, changes in skin physiology alter the associated microbiota, with implications for skin integrity and susceptibility to infection. Extrinsic aging, also known as premature aging, refers to degeneration associated with exogenous causes, some of which include sun exposure, poor nutrition, smoking, alcohol consumption, and pollution. The best-studied extrinsic aging process is caused by solar UV radiation, resulting in wrinkling, altered pigmentation and, potentially, neoplasia. Prematurely damaged or aged skin also presents challenges to the attendant microbiota, with consequences for the health of the host.
Skin Microbiota in Childhood Through Adolescence Much of what is known about the impact of age on skin microbiota derives from traditional culture-based methods of detection. The genesis of the skin microbiota begins at birth. The composition of the microbiota will differ depending on the baby’s mode of delivery. Babies born by Caesarean section are initially sterile, and subsequently become colonized by microbes from the external environment [44]. In contrast, babies born by
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vaginal delivery are colonized prior to birth by microorganisms encountered in the birth canal [44]. The microorganisms found at birth are usually present in small numbers, except for Staphylococcus epidermidis, acquired from the vulvovaginal biota just prior to birth. Newborns carry micrococci, coryneform bacteria, and gram-negative organisms more frequently and in larger numbers than older children and adults. For example, coryneform bacteria comprise a large portion of the resident skin flora of the newborn, but unlike S. epidermidis, they become established several hours after delivery [44]. Gram-negative bacteria are commonly cultured from the skin of children at the age of 6 weeks; this is distinct from the situation in the adult, where limited numbers of Gram-negative bacteria are detected on the skin by culture. Yeast components of the skin microbiota in children are predominantly Candida species (non-lipophilic yeasts) and Malassezia species (lipophilic yeasts) [45]. In preterm infants, staphylococci colonize the skin earlier than Propionibacterium and Malassezia species, as the latter have a slower growth rate and might require greater maturation of epidermal structures [46]. In babies, most skin infections are associated with S. aureus (which colonizes the umbilicus and nasopharynx of many infants) or with either S. epidermidis, coliforms, Pseudomonas, or yeasts [47]. Malassezia species are significant in that they have been implicated in certain systemic neonatal infections [48]. In the 1970s, changes in facial skin biota with age were studied. The levels of resident aerobic and anaerobic bacteria on the face depend on age [44], and changes seem to be related to sebum production. For example, concentrations of anaerobic diphtheroids and surface aerobic micrococci were higher in the infants than in young children. At puberty, cell densities of microorganisms on facial skin increased, especially during late adolescence. The observed changes in skin microbiota are may be the result of hormonal or physiological changes that alter the skin environments the skin matures. An obvious example is the increased incidence of acne and other skin infections in adolescents compared to prepubertal children. Moreover, host factors can also affect the types of metabolites produced by the skin microbiota. For example sex hormones have been shown to have an impact on the compositions of gingival biota [49].
Skin Microbiota in Adults The microbiota of adult human are both complex and variable based on site, age, sex, and ethnicity. Within the inhabitant microbial communities, the relationship with
the host ranges from mutualistic to parasitic. The skin resident microbiota are thought to play a critical role in establishing homeostasis within the host. However, the balance between the host and the microbiota is delicate and can change rapidly following breaches in skin integrity, leading to infection. Bacterial and fungal species and strains on the skin vary considerably depending on the skin environment and immune status. With the advent of culture-independent molecular techniques, the range of microorganisms identified from human skin has expanded significantly [4, 7, 9], and understanding of the composition of the skin microbiota will likely continue to increase as these techniques become routine. Recent studies have shown that there is a core set of microorganisms that comprise most of the resident skin microbiota, but less common bacteria comprising the balance of the population may be transients that differ significantly amongst individuals and between sample times [7].
Important Constituents of the Skin Microbiota Gram-Positive Bacteria
Staphylococcus spp. on the skin are considered commensals that may, in some cases, become opportunistic pathogens [2]. The most common staphylococcus species on the skin are distinguished by their inability to produce coagulase, an enzyme that plays a role in virulence. There are 32 species of coagulase-negative staphylococci, of which 15 are exclusive to humans, and 10 routinely isolated from normal glabrous skin. Staphylococcus epidermidis and Staphylococcus hominis are the species most frequently isolated from the skin by culture dependent techniques. S. epidermidis colonizes the upper part of the body preferentially and constitutes over 50% of the resident staphylococci identified with these methods. Occasionally, these organisms cause nosocomial infections in patients with indwelling foreign bodies such as heart valves and intravenous catheters. Staphylococci also can spread from one person to another in patient care settings, setting off a cascade of infection among susceptible individuals. Such has been the case in several hospital outbreaks, where improper handwashing by care providers facilitates transmission of staphylococcus infection. For as long as microorganisms have been cultured from the skin and from sites of infection, Staphylococcus aureus, a coagulase positive staphylococcus, has been identified as a constituent of the skin biota. The organism is considered transient because it is not routinely isolated from all
Aging Skin Microbiology
skin sites. However, it is often found on the hands, perineum and in the vaginal biota, and, as noted earlier, is considered a normal constituent of the biota of the nasal cavity [50, 51]. An estimated that 86.9 million people in the US are colonized with S. aureus in the nares, where the organisms appears to be acting as a commensal [52]. S. aureus are important human pathogens, particularly those strains that produce superantigenic toxins. The organism causes clinical outcomes ranging from minor and self-limited skin infections to invasive and life-threatening diseases. S. aureus expresses many virulence factors (both secreted and cell-surface associated) that contribute to evasion. At present, S. aureus infections are treated with antibiotics; unfortunately, many antibiotic resistant strains have arisen, including methicillin-resistant S. aureus (MRSA), now found in both hospital and community settings [53]. Vancomycin-intermediate and vancomycin-resistant S. aureus strains (VISA and VRSA) have been documented [54–56], a critical development as vancomycin has been regarded an antibiotic of last resort. Other gram-positive bacteria are worthy of mention. Although less frequently detected by culture-dependent techniques than the staphylococci, at least eight different Micrococcus species have been identified from human skin [5]. Micrococcus luteus is by far the most common of the micrococci detected by culture, followed by Micrococcus varians. Coryneforms are gram-positive pleomorphic bacilli included in the classification of skin commensals. This group includes the Corynebacterium spp., Propionibacterium spp., Dermabacter spp. and Brevibacterium spp. Propionibacterium acnes, abundant on the skin of the scalp, forehead, and back, is by far the most predominant coryneform. Gram-Negative Bacteria
Gram-negative rods are not commonly isolated from normal healthy adult skin, probably because the skin is too dry an environment. Gram-negative bacterial species on the skin often derive from the gastrointestinal system and contaminate other areas of the body. These microbes occasionally become resident flora in moist intertriginous areas such as the axilla and toe webs, and also may become established on mucosal surfaces of the nose. In addition, Acinetobacter and Pseudomonas species are Gram-negative constituents of the skin biota; as noted earlier, their presence seems to be site-dependent [7]. Mycobiota
Fungi and yeast are often detected in the skin microbiota of healthy people, particularly Malassezia, which inhabit
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the hair follicles. Pityrosporum species are most numerous on the back and chest, with highest numbers paralleling areas of highest sebum excretion. Yeasts such as Candida species, normally found on up to 40% of mucous membranes, seldom colonize healthy skin. Increased skin colonization by these yeasts is seen in immunosuppressed patients, diabetics, and patients with psoriasis or atopic dermatitis [8].
Skin Microbiota in the Aged Several structural and functional changes in the skin occur intrinsically as skin ages. The skin becomes drier, barrier function is reduced, skin turnover rates diminish, and structural proteins, such as collagen, become more disorganized. These, as well as other cellular changes, are a natural consequence of passing time. Other physiological effects, such as hormonals changes, changes in immune function, and lower rates of wound healing, affect the skin’s ability to function as the interface between internal and external environments [59]. Aged skin has a higher pH, is less hydrated, less elastic, more permeable, and more susceptible to infection. A dysregulation of immune function and a possible decline in cell-mediated immunity (signalled by reduced numbers of epidermal Langerhans cells that process and present antigen to T-cells) likely affects host resistance to infections. In some populations, malnutrition, obesity, institutional care, and dementia also become risk factors for infection. Consequently, the skin of older adults is structurally and functionally different from that of younger age groups. For example, a study showed that the prevalence of skin colonization by Proteus mirabilis and Pseudomonas aeruginosa in people over 65 is 25% higher than that in younger people [57]. Yeasts are also recovered more often encountered in the flora of the skin of the elderly [58]. The microbiota of aged populations has not been completely characterized. However, older adults are more susceptible to a number of skin infections. For example, cellulitis, a bacterial infection of the lower dermis and subcutaneous tissue, is more common among the aged. Breaches in skin integrity from various causes can be precipitating factors for this infection. S. pyogenes (b-hemolytic streptococci belonging to Lancefield group A) and S. aureus are the most common pathogens associated with cellulitis [59]. Infections causing deep tissue necrosis, which can lead to sepsis, also are more commonly associated with elderly patients. They can be attributed to a dysbiosis, or imbalance, that occurs between the microbiota and the host.
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Polymicrobial necrotizing infections (type I necrotizing fasciitis) are typically associated with combinations of Gram-positive organisms such as streptococci, and either staphylococci, enterococci, enteric gram-negative bacteria, or anaerobes. In contrast, monomicrobial necrotizing infections (type II necrotizing fasciitis), are predominately associated with either a group A streptococci or S. aureus. Fungal infections, which usually are not serious, can occur in older adults when there is trauma, chaffing, or poor hygiene. Candidiasis develops in moist, intertriginous areas such as the groin, perineum and axillae. Ringworm (dermatophytosis) and tinea versicolor are also seen is this population.
Conclusion The skin is the first line of defense against environmental insults and pathogenic microbes. The resident skin microbiota plays a critical role in maintaining the health of the skin by inhibiting pathogen invasion, stimulating host innate immunity, and maintaining skin barrier function. Much of the knowledge of the characteristics of the skin biota derives from culture dependent methods of detection. The intrinsic and extrinsic processes that contribute to skin aging are well understood, but the impact of these changes on the composition and function of skin microbiota has not been studied extensively. Recent advances in cultivation independent tools for the characterizing microbial communities are expected to shed more light on this subject. It is thought that the relationship between the host and the resident skin microbiota is mutualistic, in a manner resembling the relationship between the microbiota of the GI tract and the host. The constituent microbiota of the GI tract support the immune function and overall health of the host: environmental factors, such as antibiotic use, disrupt the ecological balance within the gut and create a state of dysbiosis that adversely affects the health of the host. An analogous symbiosis may exist between the host and the skin microbiota to promote healthful skin physiology and maintain the body’s external defense against pathogens. Currently, probiotics are being evaluated in patients with apparently disrupted GI biota to restore the normal balance and improve the overall patient health. In a similar fashion, as knowledge of the characteristics and functionality of the skin microbiota expands and lifetime changes in the skin biota are better understood, it may become possible to develop probiotic or other targeted forms of therapy to reduce skin infections in the aged and to help maintain skin health throughout a lifetime.
Cross-references > The
Vaginal Microbiota in Menopause
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Aging Skin Microbiology 19. Sobel JD. Vulvovaginal candidosis. Lancet. 2007;369:1961–1971. 20. Elston DM. Epidemiology and prevention of skin and soft tissue infections. Cutis. 2004;73:3–7. 21. Redziniak DE, Diduch DR, Turman K, et al. Methicillin-resistant Staphylococcus aureus (MRSA) in the Athlete. Int J Sports Med. 2009. 22. O’Gara JP, Humphreys H. Staphylococcus epidermidis biofilms: importance and implications. J Med Microbiol. 2001;50:582–587. 23. Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL, Jr. Skin diseases associated with Malassezia species. J Am Acad Dermatol. 2004;51:785–798. 24. Gupta AK, Kohli Y. Prevalence of Malassezia species on various body sites in clinically healthy subjects representing different age groups. Med Mycol. 2004;42:35–42. 25. Gupta AK, Kohli Y, Summerbell RC, Faergemann J. Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses. Med Mycol. 2001;39: 243–251. 26. Hernandez Hernandez F, Mendez Tovar LJ, Bazan Mora E, Arevalo Lopez A, Valera Bermejo A, Lopez Martinez R. Species of Malassezia associated with various dermatoses and healthy skin in the Mexican population. Rev Iberoam Micol. 2003;20:141–144. 27. Lee YW, Kim SM, Oh BH, Lim SH, Choe YB, Ahn KJ. Isolation of 19 strains of Malassezia dermatis from healthy human skin in Korea. J Dermatol. 2008;35:772–777. 28. Sugita T, Tajima M, Amaya M, Tsuboi R, Nishikawa A. Genotype analysis of Malassezia restricta as the major cutaneous flora in patients with atopic dermatitis and healthy subjects. Microbiol Immunol. 2004;48:755–759. 29. Difonzo EM, Faggi E. Skin diseases associated with Malassezia species in humans. Clinical features and diagnostic criteria. Parassitologia. 2008;50:69–71. 30. Penders J, Stobberingh EE, van den Brandt PA, Thijs C. The role of the intestinal microbiota in the development of atopic disorders. Allergy. 2007;62:1223–1236. 31. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiota composition and development of atopic manifestations in infancy: the KOALA Birth Cohort Study. Gut. 2007;56:661–667. 32. Darabi K, Hostetler SG, Bechtel MA, Zirwas M. The role of Malassezia in atopic dermatitis affecting the head and neck of adults. J Am Acad Dermatol. 2009;60:125–136. 33. Casagrande BF, Fluckiger S, Linder MT, et al. Sensitization to the yeast Malassezia sympodialis is specific for extrinsic and intrinsic atopic eczema. J Invest Dermatol. 2006;126:2414–2421. 34. Takahata Y, Sugita T, Kato H, Nishikawa A, Hiruma M, Muto M. Cutaneous Malassezia flora in atopic dermatitis differs between adults and children. Br J Dermatol. 2007;157:1178–1182. 35. Selander C, Zargari A, Mollby R, Rasool O, Scheynius A. Higher pH level, corresponding to that on the skin of patients with atopic eczema, stimulates the release of Malassezia sympodialis allergens. Allergy. 2006;61:1002–1008. 36. Tajima M, Sugita T, Nishikawa A, Tsuboi R. Molecular analysis of Malassezia microflora in seborrheic dermatitis patients: comparison with other diseases and healthy subjects. J Invest Dermatol. 2008; 128:345–351. 37. Gao Z, Tseng CH, Strober BE, Pei Z, Blaser MJ. Substantial alterations of the cutaneous bacterial biota in psoriatic lesions. PLoS ONE. 2008;3:e2719. 38. Aiello AE, Cimiotti J, Della-Latta P, Larson EL. A comparison of the bacteria found on the hands of ‘homemakers’ and neonatal intensive care unit nurses. J Hosp Infect. 2003;54:310–315.
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39. Larson EL, Gomez-Duarte C, Lee LV, Della-Latta P, Kain DJ, Keswick BH. Microbial flora of hands of homemakers. Am J Infect Control. 2003;31:72–79. 40. Rotter ML. Semmelweis’ sesquicentennial: a little-noted anniversary of handwashing. Curr Opin Infect Dis. 1998;11:457–460. 41. Bloomfield SF, Aiello AE, Cookson B, O’Boyle C, Larson EL. The effectiveness of hand hygiene procedures in reducing the risks of infections in home and community settings including handwashing and alcohol-based hand sanitizer. Am J Infect Control. 2007;35 (10 Suppl 1):S27–64. 42. Hadaway LC. Skin flora and infection. J Infus Nurs. 2003;26:44–48. 43. Larson E. Skin hygiene and infection prevention: more of the same or different approaches? Clin Infect Dis. 1999;29:1287–1294. 44. Leyden JJ, McGinley KJ, Mills OH, Kligman AM. Age-related changes in the resident bacterial flora of the human face. J Invest Dermatol. 1975;65:379–381. 45. Nobel WC. Microbiology of Human Skin, 2nd ed. London: Lloyd Luke Medical Books, 1981. 46. Bernier V, Weill FX, Hirigoyen V, et al. Skin colonization by Malassezia species in neonates: a prospective study and relationship with neonatal cephalic pustulosis. Arch Dermatol. 2002;138:215–218. 47. Venkatesh MP, Placencia F, Weisman LE. Coagulase-negative staphylococcal infections in the neonate and child: an update. Semin Pediatr Infect Dis. 2006;17:120–127. 48. Juncosa Morros T, Gonzalez-Cuevas A, Alayeto Ortega J, et al. Cutaneous colonization by Malassezia spp. in neonates. An Esp Pediatr. 2002;57:452–456. 49. Klinger G, Eick S, Klinger G, et al. Influence of hormonal contraceptives on microbial flora of gingival sulcus. Contraception. 1998; 57:381–384. 50. Peacock SJ, de Silva I, Lowy FD. What determines nasal carriage of Staphylococcus aureus? Trends Microbiol. 2001;9:605–610. 51. von Eiff C, Becker K, Machka K, Stammer H, Peters G. Nasal carriage as a source of Staphylococcus aureus bacteremia. Study Group. N Engl J Med. 2001;344:11–16. 52. Mainous AG, 3rd, Hueston WJ, Everett CJ, Diaz VA. Nasal carriage of Staphylococcus aureus and methicillin-resistant S aureus in the United States, 2001–2002. Ann Fam Med. 2006;4:132–137. 53. Goetghebeur M, Landry PA, Han D, Vicente C. Methicillin-resistant Staphylococcus aureus: a public health issue with economic consequences. Can J Infect Dis Med Microbiol. = Journal canadien des maladies infectieuses et de la microbiologie medicale/AMMI Canada. 2007;18:27–34. 54. Aligholi M, Emaneini M, Jabalameli F, Shahsavan S, Dabiri H, Sedaght H. Emergence of high-level vancomycin-resistant Staphylococcus aureus in the Imam Khomeini Hospital in Tehran. Med Princ Pract. 2008;17:432–434. 55. Oliveira GA, Dell’Aquila AM, Masiero RL, et al. Isolation in Brazil of nosocomial Staphylococcus aureus with reduced susceptibility to vancomycin. Infect Control Hosp Epidemiol. 2001;22:443–448. 56. Tiwari HK, Sen MR. Emergence of vancomycin resistant Staphylococcus aureus (VRSA) from a tertiary care hospital from northern part of India. BMC Infect Dis. 2006;6:156. 57. Lertzman BH, Gaspari AA. Drug treatment of skin and soft tissue infections in elderly long-term care residents. Drugs Aging. 1996;9:109–121. 58. Somerville DA. The normal flora of the skin in different age groups. Br J Dermatol. 1969;81:248–258. 59. Klein NC, Cunha BA. Skin and soft tissue infections. In: Yoshikawa TT Norman DC (eds) Infectious Disease in the Aging. Totowa: Human Press, 2001, pp. 139–145.
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Scales and Typing System
87 Assessing Quality of Ordinal Scales Depicting Skin Aging Severity Fabien Valet . Khaled Ezzedine . Denis Malvy . Jean-Yves Mary . Christiane Guinot
Introduction In dermatology and in plastic surgery, severity rating scales have been extensively used to describe and quantify the severity of skin disorders [1–4], to assess treatment outcomes, efficacy of cosmetic surgical procedures, and even patients’ concern and satisfaction [5–7]. With the development of aesthetic procedures, assessment of severity before and after treatment of skin aging features [8] as well as the identification of significant determinants of skin aging [9, 10] (UV exposure, smoking, genetic polymorphisms) are increasingly reported. Among these scales, ordinal severity scales illustrated by photographs have been widely developed to help plastic surgeons and dermatologists in more objective assessments [1, 9]. For the use of ordinal rating scales, in addition to validity and sensitivity to changes, reproducibility is also required [11]. Reproducibility can be defined as the ability to obtain similar results when several measurements of the same objects are performed. In particular for patients, the reproducibility of ratings is a major issue, because their classification into one of the different categories of an ordinal scale may have important consequences on their therapeutic follow-up, and possibly on their quality of life. Therefore, it is of prime importance to analyze the variability of ratings resulting from the use of such scales, and to investigate the reproducibility of these ratings as a major component of the quality of this scale. With this aim, the same signs are usually rated independently by the same observer at two distant times (intra-observer ratings) and also by several observers (inter-observer ratings). The reproducibility evaluation of the intra- and interobserver variations is usually performed through the weighted Kappa statistic [12]. This method provides a global evaluation of the degree of agreement between two observers, but gives no information on the quality of the scale and the possible defects within its structure. In 2007, Valet et al. proposed a method to estimate distinguishabilities between all adjacent categories of the scale [13]. This method is able to highlight difficulties in
distinguishing some of the categories of the scale among observers, that is, any specific reproducibility defect in the scale. Skin aging results from a number of processes including intrinsic factors, such as chronological age, and extrinsic or environmental factors, such as chronic ultraviolet (UV) exposure and smoking habits. Aging is accelerated in the areas exposed to UV radiation, a process known as photoaging. Sun-induced cutaneous changes (photoaging) are superimposed on intrinsic aging and are characterized clinically by wrinkles, roughness, laxity, irregular pigmentation (lentigines), elastosis, and telangiectasia [14]. To assess the effects of chronic sun exposure on the skin, different photographic scales have been proposed [15–18]. Standardized grading systems for photodamage are mandatory, in particular because they can improve the quality of related clinical studies and can be used in large epidemiologic surveys [19]. In this chapter, two methods proposed for the assessment of the quality of an ordinal rating scale are described: the weighted Kappa statistic and Valet’s method. The properties of both methods are illustrated using intradermatologist and inter-dermatologists ratings resulting from the use of Larnier’s photoaging scale [15]. Through this example, the benefits of the use of each statistical method for the analysis of the quality of ordinal scales are highlighted.
Study Population In the context of the SU.VI.MAX study – a randomized placebo-controlled trial, conducted on French middle-aged adult volunteers, that focused on the effects of vitamin and mineral supplementation on health [20, 21] – an ancillary cross-sectional study was undertaken to investigate the expression of facial skin aging features. This study was conducted on a sub-sample of 567 females (age range 44–70 years), who lived in the Paris area and who agreed to participate in this ancillary protocol.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_87, # Springer-Verlag Berlin Heidelberg 2010
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Collection of Digital Images The digital images database was collected in 2002. Standardized images of the whole face were taken from the 567 volunteers. The women had to follow specific skin care instructions: no application of detergent or cosmetic products on the facial skin for at least 12 h before the photograph was captured. The digital images were taken with a Kodak DSC 760 camera, which provides highresolution images (2,036 3,060 pixels), combined with a 105 mm camera lens. The camera was mounted on a monopod to allow standardized positions of the camera. A specifically designed chair was used to allow standardized positions of the women’s faces. Lightening conditions were also standardized by using two symmetrical lamps, placed at a 45 angle to each side of the volunteer’s face, which provided a continuous daylight spectrum.
Study Design and Assessment of Severity of Photoaging Larnier’s scale is a 6-grade ordinal scale of photodamage, each grade being depicted by three reference photographs that illustrate the diversity and range of pigmentation disorders, wrinkling, and looseness [15]. Due to logistic planification, among the 567 photographs a first set of 314 was rated by a trained dermatologist (D) twice (D1 and D2), at a one-year interval (> Table 87.1). Following
. Table 87.1 Ratings of the first set of 314 photographs of women faces, by one dermatologist at two successive times, D1 and D2, using Larnier’s scale
this, four experimented dermatologists (A, B, C, and D) independently rated the remaining 253 photographs (> Table 87.2). All ratings were made independently, using the reference photographs responding to each grade of the scale.
Methods Estimating Degree of Agreement: Kappa Statistic To estimate the degree of agreement between two ordinal ratings – made by two independent observers or by the same observer at two different times – the weighted Kappa statistic is generally used [12]. The Kappa statistic measures the percentage of data values in the main diagonal (percentage of agreement) of the table, and then adjusts these values for the amount of agreement that could be expected due to chance only (percentage of agreement due to chance). For ordinal ratings, the weighted Kappa statistic is an improvement on the classical Kappa statistic, as it can attribute different weights to each possible combination of discordant observations. In this study, equalspacing weights as defined by Cicchetti and Alisson [22] were used. Kappa values range from 1 to 1, 0 indicating null agreement, 1 and 1 accounting for perfect agreement and perfect disagreement, respectively. To interpret the level of agreement, the five-level nomenclature proposed by Landis and Koch [23] was used: kappa values of 0–0.2, 0.21–0.40, 0.41–0.60, 0.61–0.80, and 0.80–1.00 can be interpreted as poor, fair, moderate, substantial, and perfect agreement, respectively.
Estimating Degree of Distinguishability Between Adjacent Categories: Valet’s Method The degree of distinguishability (DD) between two categories of an ordinal scale is the raters’ ability to distinguish between these two categories. In other words, it expresses the distance between these categories on the scale. Defined by Darroch and McCloud [24], the DD between two categories ranges from 0 to 1:1 indicates a perfect distinguishability between these categories, whereas a value of 0 indicates that these categories are not distinguishable. Recently, Valet et al. proposed a new method to estimate DD between adjacent categories [13]. The authors argued that the DD between different pairs of adjacent categories of an ordinal scale (i.e. 1 and 2, 2 and 3, and so on) are not
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. Table 87.2 Ratings of the second set of 253 photographs of women faces, by four dermatologists (A, B, C, and D) using Larnier’s scale
necessarily equal and may vary all along the scale. Therefore, the analysis of the DD variations may reveal heterogeneity within the structure of the scale, and can indicate where agreement failed. For this reason, estimation of DD is of prime interest to analyze the structure of agreement, and hence the quality of the scale. The structure of the
scale can be easily illustrated through the graphical display of its DD estimates. For example, an almost perfect scale would have all its DD estimates close to 1 (> Fig. 87.1a). In a less perfect scale, DD estimates between adjacent categories would be identical but with a lower value, stressing that these adjacent categories are equal but
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. Figure 87.1 Examples of DD variations for five-grade severity scales. Scale a.: Depicts a perfect scale. In this case, the DD values are equal to 1 all along the scale, which is indicated by dashed lines of similar length. The other scales illustrate four examples of unperfected scales. For these scales, distinguishabilities are indicated by black arrows with lengths proportional to DD values. Scale b.: Distinguishability smaller than 1, but homogeneous all along the scale. Scale c.: Perfect distinguishability between the extreme adjacent grades, but low distinguishability between intermediate adjacent ones. Scale d.: Perfect distinguishability between the intermediate adjacent grades, but low distinguishability between extreme adjacent ones. Scale e.: Distinguishability smaller than 1 but non-homogeneous all along the scale, with a very low distinguishability between two adjacent intermediate grades
difficult to distinguish (> Fig. 87.1b). In problematic scales, DD estimates would appear heterogeneous, highlighting the observers’ difficulties in distinguishing between some adjacent categories, compared to others (> Figs. 87.1c–e).
Computation and Expression of the Results The weighted Kappa statistic and the DD estimates have been computed using SASß software release 9.1.3 (SAS Institute Inc., SAS Campus Drive, Cary NC 27513, USA, FREQ procedure option AGREE, and GENMOD procedure, respectively), and R software release 8 (R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, Kappa function package vcd, and glm function, respectively). Both software programs provided identical results (codes are available on request). For each time of assessment (D1 and D2 of the intradermatologist ratings) and for each of the six pairs of
dermatologists (inter-dermatologists ratings), the Kappa and DD estimates were expressed with their 95% confidence intervals.
Results Due to the age range of the sample, category 6 was pooled with category 5 (> Tables 87.1 and > 87.2). > Table 87.3 shows estimates of weighted Kappa (Kw) and degrees of distinguishability (DD), for each pair of ratings in intradermatologist and inter-dermatologists studies. Furthermore, to illustrate the heterogeneity of DD values all along the scale, a graphical display of the DD estimates from > Table 87.3 is proposed in > Fig. 87.2.
Intra-Dermatologist Study The weighted Kappa estimate was equal to 0.62 (i.e. a substantial agreement according to the Landis and Koch
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. Table 87.3 Estimates of weighted Kappa (Kw) and degrees of distinguishability (DD) between successive adjacent categories i & i + 1, for each pair of ratings. Standards errors (s.e.) are given for each estimate DD (s.e.) between adjacent categories Ratings
Kw (s.e.)
1&2
2&3
3&4
4&5
0.62 (0.07)
0.70 (0.08)
0.70 (0.08)
0.70 (0.08)
0.92 (0.07)
A&B
0.43 (0.07)
0.68 (0.20)
0.68 (0.20)
0.90 (0.07)
0.90 (0.07)
A&C
0.54 (0.07)
0.69 (0.12)
0.69 (0.12)
0.86 (0.09)
0.86 (0.09)
A&D
0.58 (0.08)
0.84 (0.08)
0.47 (0.33)
0.84 (0.08)
0.84 (0.08)
B&C
0.35 (0.07)
0.85 (0.06)
0.85 (0.06)
0.85 (0.06)
0.85 (0.06)
B&D
0.47 (0.07)
0.78 (0.08)
0.78 (0.08)
0.78 (0.08)
0.78 (0.08)
C&D
0.51 (0.07)
0.77 (0.08)
0.77 (0.08)
0.77 (0.08)
0.77 (0.08)
D1 &
D2a
a
D1 and D2 are ratings from dermatologist D at two different times
nomenclature). Moreover, the estimate of the degree of distinguishability between categories 4 and 5 (0.92) was much larger than that for adjacent categories from 1 to 4 (0.70). These results, illustrated in > Fig. 87.2, suggested that the dermatologist distinguished adjacent categories 4 and 5 more easily than the other adjacent ones.
Inter-Dermatologists Study The Kappa statistic estimates ranged from 0.35 to 0.58 (fair to moderate agreement). The DD estimates between all adjacent categories were equal all along the scale and greater than 0.60, except for the pairs involving dermatologist A. In these particular cases, DD estimates were found to be lower between the first three adjacent categories (adjacent categories 1 and 2, and 2 and 3) than between the others adjacent ones (adjacent categories 3 and 4, and 4 and 5). These results are illustrated in > Fig. 87.2.
Discussion From the perspective of evidenced-based medicine, it is of prime importance to provide measurement tools that allow the detection of clinically relevant changes that are due to disease evolution or therapeutic responses, rather than measurement errors [25]. The primary objective of an ordinal scale is to produce an efficient, valuable, and practical tool. In this context, the reproducibility of ratings is the main component of its quality. To date, a number of dermatological rating scales (with or without photograph standards) have been developed to accurately evaluate skin aging features [26–30]. However, despite the
wide use of ordinal scales for skin aging assessment, only a very few have been investigated for their reproducibility, and most of them have been stated as ‘‘validated’’ with a Kappa threshold greater than 0.60. Nevertheless, none of them has been investigated for reproducibility using tools that provide some information on the structure of the scale. In this study, the intra-dermatologist degree of agreement reached 0.62; this value is similar to those found in the original paper [15] (median Kappa value = 0.63, calculated from eight intra-dermatologist ratings). Both values correspond to substantial degrees of agreement. Concerning the inter-dermatologists results, the Kappa estimates ranged from 0.35 to 0.58 (fair to moderate degrees of agreement). Moreover, the degrees of agreement between ratings involving dermatologist B were systematically lower than the others (0.35–0.47), highlighting that this investigator had some difficulties in using this scale. In the original paper [15], the ratings of each dermatologist were compared to the ratings of a consensus panel on two different occasions (median Kappa values = 0.58 and 0.61). Therefore, these last results cannot be compared to the results of this study, as the study designs are definitely different. Using Valet’s method, differences within the degrees of distinguishability between adjacent categories were evidenced, suggesting that dermatologist D experienced some difficulties in distinguishing between the three first adjacent grades. In the inter-dermatologists variation study, the DD estimates were all lower than 1, but homogeneous all along the scale, except for comparisons involving dermatologist A. Indeed, dermatologist A experienced some difficulties in distinguishing between the three first adjacent grades. Except for this dermatologist, the DD estimates between adjacent
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. Figure 87.2 DD estimates for each pair of ratings. Lengths of black arrows are proportional to the DD values. The first scale depicts a perfect scale, with DD values equal to 1 all along the scale, which are indicated by dashed lines of similar lengths. The other scales illustrate DD estimates between adjacent categories for each pair of ratings indicated by black arrows with lengths proportional to DD estimates
categories are identical all along the scale, but with lower values than a perfect scale. The experimental conditions of the reproducibility study were fairly suitable. Indeed, the raters were welltrained, experienced dermatologists. In addition, one of the strengths of this study is that the sample size was rather large (314 individuals in the intra-dermatologist study and 253 in the inter-dermatologists study) in comparison to similar published studies [26–30]. Nevertheless, due to the women’s characteristics, in particular the age range (44–70 years), the last category of the scale was almost unused. For that reason, the extreme adjacent categories were not tested. An additional limitation should be underlined, which concern both methods in the case of more than two observers. Indeed, for Valet’s method, the DD estimates between the scale categories must be calculated for each possible pair of observers. No method has yet been developed that can estimate a ‘‘global’’ DD between the scale categories for more than two observers. Similarly, the estimate of the degree of agreement using the Kappa statistic method can also be calculated between each possible pair of observers. A global Kappa statistic has been proposed to deal with more than two observers, corresponding roughly
to an average Kappa [31]. However, as the contingency tables associated to each pair of dermatologists are not independent, the interpretation of this global statistic is problematic. The method developed by Valet et al. provides estimates of the DD between each pair of adjacent categories of an ordinal scale. Therefore, this method also provides an indication of the degree of agreement through the DD estimates between adjacent categories. Indeed, an equal and high DD value between all adjacent categories of the scale would clearly lead to a lower variability within ratings and hence a better agreement between them. In addition, contrary to the Kappa statistic method, this method can highlight DD heterogeneities between adjacent categories, and may suggest some modifications to improve the quality of the scale. For example, a low DD between two adjacent categories would clearly suggest that they should be pooled or redefined in order to improve the reproducibility – and hence the quality – of the scale in forthcoming studies. The Kappa statistic is useful for rapid tests and has widespread use, even though its interpretation using the nomenclature of Landis and Koch is questionable. Several authors have pointed out the limits of this global index of
Assessing Quality of Ordinal Scales Depicting Skin Aging Severity
agreement [32, 33], and for weighted Kappa statistics, the choice of weights may be arbitrary and may strongly influence the Kappa level estimates. When using the squared error weights on this data set, as proposed by Fleiss [34, 35], the weighted Kappa levels would be higher (indicating substantial or almost perfect agreements) than those estimated with equal-spacing weights (linear error). In addition, as highlighted by Valet et al. [36], the Kappa statistic may indicate an almost perfect degree of agreement even if a major scale defect exists. Indeed, this method does not provide any indication that could be used for scale improvement.
Conclusion According to the present findings, the reproducibility of Larnier’s scale appears to be questionable. It appears that some dermatologists had difficulties in distinguishing between some adjacent categories, highlighting some major scale defects. Consequently, the quality of this scale could be improved. Among suggestions for these improvements, the reference photographs should be discussed in order to redefine the three first categories and illustrate the concerned categories using new reference photographs.
Acknowledgment The authors gratefully acknowledge the dedicated efforts of the SU.VI.MAX participants, and the investigators and staff involved in this study, in particular Dr. Raymonde Danila and Dr. Randa Jdid. The authors wish also to gratefully acknowledge Dr. Catherine Larnier for her kind interest in this research.
References 1. Coenraads PJ, et al. Construction and validation of a photographic guide for assessing severity of chronic hand dermatitis. Br J Dermatol. 2005;152(2):296–301. 2. Berth-Jones J, et al. A study examining inter- and intrarater reliability of three scales for measuring severity of psoriasis: Psoriasis area and severity index, physician’s global assessment and lattice system physician’s global assessment. Br J Dermatol. 2006;155(4): 707–713. 3. Hanifin JM, et al. The eczema area and severity index (EASI): Assessment of reliability in atopic dermatitis. EASI Evaluator Group. Exp Dermatol. 2001;10(1):11–18. 4. Witkowski JA, Parish LC. The assessment of acne: an evaluation of grading and lesion counting in the measurement of acne. Clin Dermatol. 2004;22(5):394–397.
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5. Ching S, et al. Measuring outcomes in aesthetic surgery: A comprehensive review of the literature. Plast Reconstr Surg. 2003; 111(1):469–480. 6. Most SP, Alsarraf R, Larrabee WF. Outcomes of facial cosmetic procedures. Facial Plast Surg. 2002;18(2):119–124. 7. Heckmann M, Schon-Hupka G. Quantification of the efficacy of botulinum toxin type A by digital image analysis. J Am Acad Dermatol. 2001;45(4):508–514. 8. Alsarraf R, et al. Measuring cosmetic facial plastic surgery outcomes: a pilot study. Arch Facial Plast Surg. 2001;3(3):198–201. 9. Chung JH, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol. 2001;137(8):1043–1051. 10. Guinot C, et al. Relative contribution of intrinsic vs extrinsic factors to skin aging as determined by a validated skin age score. Arch Dermatol. 2002;138(11):1454–1460. 11. Shoukri MM. Measures of interobserver agreement. Boca Raton: Chapman & Hall/CRC, 2004. 12. Cohen J. Weighted kappa: nominal scale agreement with provision for scaled disagreement or partial credit. Psychol Bull. 1968; 70:213–220. 13. Valet F, et al. Log-linear non-uniform association models for agreement between two ratings on an ordinal scale. Stat Med. 2007;26 (3):647–662. 14. Rabe JH, et al. Photoaging: Mechanisms and repair. J Am Acad Dermatol. 2006;55(1):1–19. 15. Larnier C, et al. Evaluation of cutaneous photodamage using a photographic scale. Br J Dermatol. 1994;130(2):167–173. 16. Griffiths CE, et al. A photonumeric scale for the assessment of cutaneous photodamage. Arch Dermatol. 1992;128(3):347–351. 17. Helfrich YR, et al. Effect of smoking on aging of photoprotected skin: Evidence gathered using a new photonumeric scale. Arch Dermatol. 2007;143(5):397–402. 18. Carruthers A, et al. A validated grading scale for crow’s feet. Dermatol Surg. 2008;34(Suppl. 2):S173–S178. 19. Malvy D, et al. Epidemiologic determinants of skin photoaging: Baseline data of the SU.VI.MAX cohort. J Am Acad Dermatol. 2000;42(1) Pt 1:47–55. 20. Hercberg S, et al. A primary prevention trial using nutritional doses of antioxidant vitamins and minerals in cardiovascular diseases and cancers in a general population: the SU.VI.MAX study-design, methods, and participant characteristics. SUpplementation en VItamines et Mineraux AntioXydants. Control Clin Trials. 1998;19(4): 36–351. 21. Hercberg S, et al. The SU.VI.MAX Study: a randomized, placebocontrolled trial of the health effects of antioxidant vitamins and minerals. Arch Intern Med. 2004;164(21):2335–2342. 22. Cicchetti DV, Allison T. A new procedure for assessing reliability of scoring EEG sleep recordings. Am J EEG Technol. 1971;11:101–109. 23. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33(1):159–174. 24. Darroch JN, McCloud PI. Category distinguishability and observer agreement. Aust J Stat. 1986;28:371–388. 25. Weyers W. The 21st century – time for a reliable method for diagnosis in clinical dermatology. Arch Dermatol. 2000;136(1):103–105. 26. Lemperle G, et al. A classification of facial wrinkles. Plast Reconstr Surg. 2001;108(6):1735–1750. 27. Ryu JS, et al. Improving lip wrinkles: Lipstick-related image analysis. Skin Res Technol. 2005;11(3):157–164. 28. Day DJ, et al. The wrinkle severity rating scale: a validation study. Am J Clin Dermatol. 2004;5(1):49–52.
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29. Kim EJ, Reeck JB, Maas CS. A validated rating scale for hyperkinetic facial lines. Arch Facial Plast Surg. 2004;6(4):253–256. 30. Morizot F, et al. Development of photographic scales documenting features of skin aging based on digital images. Ann Dermatol Venereol. 2002;129(10) Pt 1:1s402. 31. Fleiss JL. Measuring nominal scale agreement among many raters. Psychol Bull. 1971;76:378–382. 32. Feinstein AR, Cicchetti DV. High agreement but low kappa: I. The problems of two paradoxes. J Clin Epidemiol. 1990;43(6):543–549. 33. Cicchetti DV, Feinstein AR. High agreement but low kappa: II. Resolving the paradoxes. J Clin Epidemiol. 1990;43(6):551–558.
34. Fleiss JL, Cohen J. The equivalence of weighted kappa and the intraclass correlation coefficient as measures of reliability. Educ Psychol Meas. 1973;33:613–619. 35. Fleiss JL. Statistical Methods for Rates and Proportions. New York: Wiley, 1981. 36. Valet F, et al. Quality assessment of ordinal scale reproducibility: Log-linear models provided useful information on scale structure. J Clin Epidemiol. 2008;61(10):983–990.
82 Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly Prashant Rai . Daniel S. Marsman . Susan P. Felter
Introduction Disposable diapers for adults are widely used in many parts of the developed world, to safely and effectively manage urinary and fecal incontinence. Their usage is relatively uncommon in the developing world, although adult diapers are manufactured and exported from and used in India and China, to a limited extent. It is estimated that 90–95% of adult incontinent diapers that are used in developed nations are of the disposable kind. These products are in direct contact with the skin of the individual when worn, and given that adult diapers are changed three times a day on average, an incontinent adult may be exposed to approximately a thousand disposable diapers per year. The significant exposure of incontinent adults to disposable diapers necessitated the development of robust and practical methods for assessing the safe and effective use of these important products. A generalized risk assessment (RA) paradigm was established by the National Academy of Sciences in 1983 [14], which consists of a four-step process: hazard identification, dose-response assessment, exposure assessment, and risk characterization. Although the application of this process varies slightly and is described using different terminologies by different organizations and institutions, the basic principles are the same [6, 8, 16]. Kosemund et al. [10] have described the use of this paradigm for the safety evaluation of baby diaper products. This manuscript provides an overview of the application of quantitative risk assessment principles to the safety evaluation of adult care disposable diapers.
Design and Use of Disposable Diapers: Implications for Safety Assessment The modern adult care disposable diaper benefits from a number of innovations in design and materials. These improvements have resulted in products that are highly
absorbent and ensure a snug, comfortable, and discreet fit. These improvements also allow for the manufacture of a product that promotes improved skin health and hygiene by effectively containing urine and excreta, and thus maintaining skin health by reducing direct exposure to these bodily wastes.
Adult Incontinent Diaper Design A number of different material layers are employed in the construction of a typical adult care disposable diaper. In general, these diapers consist of absorbent layers in the diaper core, contained within an outer structure or chassis that ensures proper fit (> Fig. 82.1). These adult diapers are also available in the form of insert pads, which are worn inside the larger outer disposable diaper. These insert pads (which are quite similar to, but larger than, feminine protection pads that are used by women for menstrual fluid management) are commonly used by care institutions that provide adult care facilities and by individuals who do not wish to change the more expensive outer diaper at every instance of soiling. Components of a typical diaper include a polypropylene topsheet that is in direct contact with the skin, cellulose pulp, super absorbent polyacrylate granules contained within the adult diaper core away from direct skin contact, and barrier leg cuffs that ensure fit and prevent leakage, as illustrated in > Fig. 82.1. Different kinds of adult incontinence management products are illustrated in > Fig. 82.2. Additional components include an outer protective barrier layer of polyethylene film, elastic materials that improve fit, and adhesive or loop tapes for fastening the diaper. Adhesives are commonly used throughout the diaper to hold the layers in proper position. Some diaper variants may also feature lotion on the topsheet, which helps to improve the softness/ smoothness properties of diapered skin, and reduces the incidences of adverse skin reactions. Additionally, the
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_82, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 82.1 Cross-section of a typical adult care disposable diaper. NW = Non woven; SAP = superabsorbent polymer; AQL = acquisition layer; BS = backsheet; CBWL = cellulose based wicking layer
. Figure 82.2 Types of adult diaper products *Presented at the 5th International Society of Gerontology Meeting, Nagoya, Japan, 2005 by Mr. Yukio Heki, P&G Japan
topsheet may also be treated with very low levels of mild surfactants to overcome the otherwise hydrophobic nature of the polymer topsheet, allowing urine to pass through more readily. Most of the diaper weight is made up of large molecular weight polymers of natural or synthetic origin.
Raw materials used in diaper construction, including the topsheet and super absorbent materials, have all shown low irritancy and no evidence of sensitization potential in standard skin testing. Because these polymeric materials are solid materials that are biologically inert and have low bioavailability, they enjoy a highly
Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly
favorable human safety profile. Diapers may also contain small quantities of low molecular weight ingredients or residual materials that are part of the manufacturing process. These materials may come into contact with the skin, either through direct exposure or as a result of urine extraction, and therefore are relevant to the assessment of diaper safety. Overall, the safety assessment of an adult disposable diaper involves the identification of ingredients of toxicological relevance in individual raw materials (including impurities and manufacturing aids), and their evaluation via the quantitative risk assessment (QRA) process. Given the potential for skin contact and dermal absorption, the hazard evaluation process involves confirmation of safety for both dermal and systemic toxicological endpoints.
Adult Disposable Diaper Choice and Effects on Skin A 1999 NAFC (National Association for Continence) [15] survey showed that about 60% of incontinent individuals used some type of disposable liner or pad or underwear. It is noteworthy that, at that time, 26.8% of all women with incontinence mentioned that they used sanitary napkins, and 17.4% of all women used tissues, paper towels, or toilet paper in lieu of any specially designed absorbent product. Similarly, 26.5% of all men who responded said they used reusable pads, diapers, or other reusable briefs. Adult diaper manufacturers have designed several types of disposable diapers to suit varying needs. Choice of an adult diaper product type is largely dependent upon the severity and type of incontinent symptoms suffered by an individual. The age of the incontinent individual is often not a major consideration in this case. The mobility of an incontinent individual also is a significant factor when choosing an adult incontinence management product. Younger, normally mobile, lightly-incontinent individuals (30–50 years of age) typically tend to choose more discreet products, like thin insert pads, light incontinence pads, or pull-up pants-type diapers for management of urinary incontinence. Typically, these lighter and thinner products are changed frequently, and as a result prolonged skin contact with the same (wet) product is often reduced. However, if the volume of incontinent urine in these individuals is high, a heavier, thicker pad or diaper may be the product of choice. In contrast, for older, less mobile, moderate to severely incontinent individuals (60–90 years of age), where the volume of incontinent urine may be high, the choice of product may be restricted only to thicker and heavier types of diapers/pads. Use of a thicker, highlyabsorbent product also does not necessitate frequent
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change, a condition which is well suited to less mobile or bedridden individuals. Separately, incontinent individuals may also be cared for by other family members at home or professional caregivers/nurses in senior-citizen institutions, where the choice of incontinence-management product may be made by the caregiver. Here too, depending on the mobility of the individual and the extent of incontinence, the caregiver may choose to use a lighter, thinner product if symptoms of incontinence are mild, and a heavier/thicker product if the extent of incontinence is moderate to severe. With regard to adult incontinence-related skin problems, it is very important to apply a holistic perspective to understand the influences due to the individual’s extent of incontinence and skin condition, as well as the hygiene and skin care measures practiced at home, or provided professionally. Individuals with frail, sensitive skin or with pre-existing skin diseases may preferably have to use highabsorbency, lotion-containing, thicker diaper products (with super absorbent polymers and moisture-permeable back sheets) to minimize the risk of skin complications. Also, diaper topsheets have been engineered creatively by manufacturers, to minimize the risk of skin conditions. For example, immobile incontinent individuals have benefited greatly from products that contain the high performance aperture formed film (T type) type of topsheet, which helps to quickly wick away incontinent fluids (due to larger pore size), thus keeping the skin clean and dry for longer periods of time (> Fig. 82.3). In contrast, products engineered with the common aperture formed film (M type) type of topsheet exhibit slower wicking speeds (due to reduced pore size), and hence are known to provide reduced skin management benefits. In contrast, adult diapers that are manufactured with more conventional nonwoven topsheets are known to provide better long-term skin comfort benefits when compared to aperture formed film types of topsheets, which are excellent for absorption, but do not perform well in regions with high temperatures and high humidity.
Exposure Parameters Being a complex, layered product, with each layer having a different function, the exposure potential for various diaper components ranges from continual direct skin contact for some materials (e.g., the topsheet) to materials with transient or very minimal/negligible skin contact (e.g., the fasteners). As noted above, the majority of chemical components in a diaper are large, inert polymers
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. Figure 82.3 Aperture formed film M and T types of product topsheets
that are not absorbed through the skin. These materials are of little concern when evaluating diaper safety. In contrast, low molecular weight materials that may be present in the topsheet need to be thoroughly assessed. Ingredients/monomers may also be released from diaper core components, solubilize in urine, and become bioavailable at the surface of the adult diaper, and become relevant for exposure analysis.
Materials in Direct Skin Contact Adult diaper raw materials that are in direct contact with the skin include the topsheet, lotion ingredients, the barrier leg cuffs, and the waistband. Skin transfer of chemicals or materials from these components does not necessarily require solubilization in body fluids that may be loaded onto the diaper. Chemicals may be deposited or transferred onto the skin directly, or via solubilization in sweat, urine, or sebum. However, even with materials in direct contact with skin, only a fractional amount of topsheet ingredients are expected to be transferred onto the skin. This is due to the integration of the raw materials/chemicals within the polymeric matrix resin of the topsheet. Typically, obtaining precise analytical data on the leachability of the chemical/raw material from the topsheet is recommended, in order to calculate exposure. Studies have shown that only approximately 10% of a typical topsheet coating material, such as a lotion, is actually transferred to the skin of the adult diaper wearer (P&G Unpublished data). Thus, this lotion transfer factor can be used as a reasonable and conservative estimate for
transfer of all other topsheet ingredients. Residual monomers and contaminants incorporated into the substrate of the topsheet are presumed to be transferred in substantially less quantities than this worst-case estimate.
Materials Not in Direct Skin Contact Diaper components beneath the topsheet are not in direct contact with the skin. These materials include the acquisition layer, the super absorbent polymer, non-woven core wrap material, core and chassis glues. These materials may contain ingredients with the potential to migrate to the skin, typically being carried toward the skin via urine. Exposure to these materials can occur via extraction or solubilization in urine and other body fluids, followed by these fluids resurfacing to the skin. Therefore, urine that resurfaces to the skin may be assumed to be the key carrier for these ingredients. In the absence of experimental data from which to estimate the migration of low molecular weight materials from the diaper interior to the skin surface, a highly conservative default assumption of 100% migration may be used for exposure calculations. A refined exposure evaluation would include actual quantification of the extractable ingredient from the fluid matrix.
The Reflux Approach Reflux (also known as rewet or retention of wetness) is a relevant factor for quantifying exposure to non-skin-contact
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materials. Reflux is defined as the amount of liquid (i.e., urine) that resurfaces to the top of a diaper, and has the potential to come into contact with the skin. Diaper core ingredients that are not in direct skin contact require urine as an aqueous vehicle or carrier to resurface to the skin. This urine that resurfaces to the topsheet is assumed to be the most relevant carrier for ingredients that may be located deep within the diaper. Current diaper risk assessments are based upon the presence and use of highly absorbent core technologies, such as super absorbent polymer (SAP), which have reduced the reflux level of adult care disposable diapers. No default reflux values are recommended for exposure calculations for these types of raw material ingredients. Taped adult diapers show the highest actual reflux value (1.6%), whereas a reflux value of 0.1% is measured for all other types and variants of adult care disposable diapers. The difference between these values reflects the difference in construction of the core of these diapers. All taped diaper variants typically contain an additional blue-colored cellulose-based wicking layer placed just above the absorbent core. Due to the presence of this layer, the core is constructed with slightly reduced amounts of SAP, thus resulting in higher rewet values. The reflux factor has been measured, typically using an absorbing filter paper or a collagen sheet, under pressure, on top of a moist diaper. The diaper is typically loaded with synthetic urine or other aqueous solvents. The filter paper/collagen sheet utilizes the high capillary forces of cellulose fibers to identify very small differences in absorbency between different products. Reflux values measured by using filter paper are more conservative, and in the range of 2–2.5%. Reflux values obtained by using the collagen method (Post Acquisition Collagen Reflux Method; PACORM) are generally considered more realistic and are similar to actual diaper in-use conditions, as collagen closely mimics the re-absorbency characteristics of the upper epidermal layers of the skin. The PACORM uses a stretched diaper on a fastening plate [5]. Following diaper loading (typically, four large gushes of 150 mL each of synthetic urine or saline, depending on diaper size), four circular collagen sheets (diameter 9 cm each, area 64 cm2 each) are stacked onto the topsheet and pressure is applied (approximately 236 g/cm2). Reflux is calculated as the weight difference of the collagen sheets before and after exposure to the topsheet. One limitation of this method is that reflux is measured only under the 64 cm2 collagen sheet area, and not from the entire diaper core surface which is in direct skin contact (1,660 cm2). However, the reflux number obtained is still considered reasonably conservative, as collagen sheets are placed on the central loading
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area with the highest liquid concentration in the diaper core.
Consumer Usage Data and Assumptions for Quantitative Risk Assessment Different diaper usage habits are relevant parameters when determining overall exposure to diaper raw materials and chemicals. Normal consumer habits and practices for diaper usage are varied across different geographies. Procter & Gamble consumer research (unpublished data) indicates that the highest number of adult care disposable diapers used per day is observed in Japan, while fewer diapers are used per day in countries such as Italy, Spain, and Portugal. Adult care disposable diaper usage in the USA shows that numbers are slightly higher than those observed in Europe. For adult care diaper exposure assessments, the following parameters are particularly relevant: ● The adult body weight is assumed to be 60 kg, to account for differences in sex and global diversity. ● The number of adult diapers used per day (diaper change frequency) differs in different geographies. The consumer data from Japan suggests that the highest change frequency is observed with insert pads (3.5–4.0 times per day), whereas outer taped briefs or pull-on diapers are changed less frequently (1.5–1.75 times per day). Italy, Spain, and Portugal demonstrate similar diaper change frequencies, with approximately 2–3 pads being worn per day, irrespective of type. Approximately 3–5 disposable briefs are worn by consumers in the USA in institutional and home care settings. For simplification, a conservative average diaper changing frequency of 3 is assumed in the calculations to account for change frequencies and usage of all adult disposable diaper types. ● The topsheet transfer factor (10%) is the amount of an ingredient that is transferred from the topsheet of the diaper to the skin of the adult. As noted earlier, this is a conservative default value of direct contact ingredient transfer from data generated in support of diaper topsheet lotions, where the intention is to transfer components to the skin during adult diaper wear as a skin health benefit. This value is based upon measurements of actual lotion transfer observed during clinical studies with adult diapers in Europe (P&G, unpublished data). It is assumed that transfer of any substance from the topsheet will not be greater than the transfer of an intentionally added-on ingredient such as lotion.
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● The reflux factor is 1.6% for taped diapers and 0.1% for all other diaper types. The super absorbent core of adult diapers is not in direct contact with the skin of the individual, and is made up of a polymeric gelling material that quickly soaks up urine and contains it within the core. This core technology is specifically engineered to minimize/eliminate any re-exposure of the skin to urine. However, minute amounts of urine might still resurface to the skin, via the phenomenon of reflux (> Fig. 82.4). Thus resurfacing of ingredients from the lower layers of the diaper, albeit remote, is still possible, and resurfacing urine may be assumed to be the carrier of any chemicals that might reside in these core lower-layer materials that are not in direct skin contact.
Materials with Negligible Skin Contact Several diaper materials used on the outside of the diaper chassis (e.g., outer polypropylene liners, graphic printed surfaces, fastening tapes, disposal tapes) have negligible or very minimal direct skin contact. In terms of exposure assessment, these materials are generally judged to have negligible exposure, and can often be considered to contribute insignificantly toward overall exposure to materials during diaper wear.
Principles of Quantitative Risk Assessment (QRA) Examples Adult care disposable diapers are mainly constructed of inert, polymeric materials. In general, these materials are considered safe, and no inherent toxicity issues are
anticipated. However, low levels of non-polymeric components or monomers may be present in the product, or these may be residuals from the diaper raw material manufacturing process, or they could be components of aesthetic materials such as perfumes or lotions that are added to the diaper at low levels. The following section provides examples of exposurebased QRAs for materials in adult care disposable diapers. Citral, a perfume raw material, and acrylic acid, a residual monomer in super absorbent polymers that form the core of the diaper, serve as case studies to showcase the principles of QRA as applied to an adult diaper during consumer use.
Risk Assessment for Citral Citral (CAS # 5392–40–5) (> Fig. 82.5) is a well known flavoring agent, and is the main constituent of lemon and orange oils. It is commonly used in perfumes, and may be a low-level component of the overall complex perfume formula that may be added in a diaper. Perfumes may be added to diapers, typically between the absorbent core and the backsheet, to enhance the overall aesthetics of the diaper. Given the deep-layer application of the perfume in a diaper, it is recommended to use a reflux level of 1.6% for determining potential exposure. Citral is classified as a weak sensitizer, but exposure to low levels does not pose any health risk. The identification of such a safe level is achieved through the sensitization QRA approach for fragrance ingredients. This approach follows the same four basic steps of the risk assessment paradigm defined by the National Academy of Sciences: hazard identification, dose response assessment, exposure assessment, and risk characterization [14].
. Figure 82.4 Illustration of adult care disposable diaper reflux measurement
Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly
. Figure 82.5 Citral (C10H16O)
Recently, a common recommended methodology has been adopted for the QRA of fragrance ingredients by Api et al. [1], and by the European Cosmetics, Toiletry, and Perfumery Association (COLIPA) Toxicology Advisory Group and the Joint COLIPA/International Association for Soaps, Detergents and Maintenance Products (AISE)/ European Flavor and Fragrance Association (EFFA)/International Fragrance Association (IFRA) Perfume Safety group [17]. Clear guidance on different elements of the dermal sensitization QRA process (e.g., uncertainty factors or sensitization assessment factors) is also offered in these references. A Weight-of-Evidence (WoE) approach is used to determine a No-Expected-Sensitization-InductionLevel (NESIL), which introduces a more robust approach to allergen potency evaluation for use in risk assessment. Additionally, Sensitization Assessment Factors (SAFs) within the exposure-based QRA process are based on published data. The SAFs take into account three parameters: inter-individual variability (the same as in general toxicology), vehicle/product matrix effects, and use considerations (specific for dermal sensitization). A Consumer Exposure Level (CEL) is determined for the perfume raw material, and it is essential to express this entity in units of dose/cm2 of skin. This updated process of QRA for perfume ingredients represents an important step forward in skin sensitization risk assessment. Once the NESIL, SAF, and CEL for citral are defined, it is possible to confirm its safety via quantitative risk assessment, and specifically a lack of sensitization hazard, as a result of exposure to citral. There are two key elements involved in this process of risk characterization: ● The establishment of an Acceptable Exposure Level (AEL): The AEL is determined by dividing the WoE NESIL by the SAF, i.e., AEL = WoE NESIL/SAF. Identification of the WoE NESIL is shown in > Table 82.1. ● Comparison of the AEL to the CEL: This is determined by dividing the AEL by the CEL, which is an indication of the acceptability of the CEL relative to the AEL. The percent concentration of the fragrance ingredient, citral, in a diaper is acceptable if the ratio of AEL to CEL is favorable to support safe use of citral.
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Assumptions to calculate CEL for citral as a result of diaper wear: diaper = 0.01/diaper = 0.019 g = 19 mg Amount of citral in perfume = 0.026% Amount of citral/diaper = 0.005 mg Three diapers worn/day (based on Habits and Practices data) Reflux factor = 1.6% (or alternatively 0.1%) Skin contact area = 1,000 cm2 (surface area of smallest adult care disposable diaper) Therefore, CEL for citral = 0.005 mg x 0.016 3/1,000 cm2 = 0.00000024 mg/cm2/day = 0.00024 mg/cm2/day The QRA for citral in adult diapers is summarized in > Table 82.2. It is observed that the AEL is greater than the actual CEL by a factor of 60,000, indicating that use of citral as a perfume ingredient in adult care disposable diapers is safe. A margin of safety of 60,000, while providing overwhelming safety assurance, also indicates that citral as a perfume ingredient may be safely used at much higher levels in adult care disposable diapers, without the risk of skin sensitization.
Exposure to a Diaper Absorbent Core Raw Material – Acrylic Acid Although adult disposable diapers are mostly composed of large molecular weight polymeric materials, there may be unreacted, low molecular weight monomers, or other small molecular weight components (e.g., processing aids or impurities) present in these polymeric materials. Minimal exposure to absorbent core raw materials of diapers may also occur under various conditions. One route of exposure is via the skin, where small molecular weight components of direct/indirect skin contact materials might be absorbed into the skin. These monomers or small molecular weight compounds may reach the skin via the phenomenon of reflux in the diaper and, depending on the material and molecular weight, may also be absorbed through the skin [3]. The SAP in diapers comprises one of two main components that make up the absorbent core of a diaper, the other being absorbent fluff pulp, comprised of cellulose fibers. The most important function of SAP is to take up urine and lock it away, which in turn renders the urine non-available to the skin, thus reducing potential incidences of diaper rash. Typically, SAP is made by polymerization of acrylic acid (> Fig. 82.6) in an aqueous solution. During polymerization, the carboxylic acid
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. Table 82.1 Identification of Weight-of-Evidence No-Expected-Sensitization-Induction-Level (WoE NESIL) for citral (International Fragrance Association, 2006 [9], Lalko J, Api AM 2008 [13], Scientific Committee on Cosmetic Products and non-Food Products Intended for Consumers, 1999 [18]) NOEL-HRIPT (induction) (mg/cm2)
Name of IFRA standard LLNA weighted raw limit (skin contact mean EC3 values material CAS # products) (mg/cm2) Citral
5392– NA 40–5
5.7% (1,414)
1,400
NOEL-MAX (induction) (mg/cm2) NA
LOEL (induction) Potency WoE (mg/cm2) classification NESIL 3876
Weak
1,400
LLNA: Local Lymph Node Assay; NOEL: No Observed Effect Level; HRIPT: Human Repeat Insult Patch Test; LOEL: Lowest Observed Effect Level
. Table 82.2 Application of QRA for citral in perfumes used in adult care disposable diaper products Citral WoE NESIL
1,400 mg/cm2
SAFa
100 (10 ¥ 1 ¥ 10)
AEL
1,400/100 = 14 mg/cm2
CEL
0.00024 mg/cm2
AEL/CEL
14/0.00024 = 60,000
Risk assessment for citral
Acceptable, since AEL > CEL
a
SAF: Inter-individual variability = 10; this is based upon wellestablished principles of general toxicology and is meant to provide protection for susceptible sup-populations. Vehicle matrix = 1; matrix is very different from the experimental test conditions under which the WOE NESIL was determined. However, it is not expected to be more irritating, and the diaper matrix is an inert material and not skin impactful. Use considerations = 10; In this case, the skin areas being considered are the buttocks, groin, lower stomach, and upper thighs. Skin integrity may be compromised in some cases. Mucus membrane exposure may occur, along with somewhat occlusive diaper wearing conditions
. Figure 82.6 Acrylic acid
groups are partially neutralized (75%) with sodium hydroxide. Therefore, a major component of SAP is the neutralized, salt form of acrylic acid, namely sodium acrylate, and a very small component is pure acrylic acid. The large molecular weight polyacrylates are
generally not a cause for toxicological concern, because they are not bioavailable. Free acrylic acid monomers are expected to be available, although in very limited quantities. Furthermore, under physiological conditions in a diaper, most of the carboxylic groups will be present as the salt form, i.e., sodium acrylate, which does not penetrate the skin as easily as does the acidic form. A 2002 CIR report on the ‘‘Safety of Acrylates Copolymer and 33 Related Cosmetic Ingredients’’ [4] indicates that linear polymers of acrylic acid may contain unreacted starting materials and catalysts, with residual monomer concentrations typically between 10 ppm and 1,000 ppm, with an upper limit of 1,500 ppm. SAP raw material suppliers for disposable diapers are currently required to maintain an upper specification limit of 500 ppm for free acrylic acid monomers (P&G specification to suppliers). Thus, the presence of unreacted acrylic acid monomers in SAP is limited but likely, and the main route of exposure to this residual acrylic acid is dermal, via the phenomenon of reflux.
Acrylic Acid – Hazard Characterization Acrylic acid has been evaluated for safety as part of the European Risk Assessment Program (EEC 793/93) [7], and as part of the CIR report on the ‘‘Final Report on the Safety Assessment of Acrylates Copolymer and 33 Related Cosmetic Ingredients’’ [4]. It has also been evaluated by the US EPA [19]. The hazard profile of acrylic acid as described in these references is summarized in > Table 82.3. At low levels, however, acrylic acid is not irritating. Irritation or corrosivity is concentration-dependent. Acrylic acid at 1% in acetone shows signs of minimal
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. Table 82.3 Hazard profile of acrylic acid Acute toxicity
Irritation – oral and dermal.
Irritation/corrosion Corrosion – dermal, eyes, and respiratory tract. Sensitization
Not sensitizing.
Repeated dose toxicity
Irritation, unspecific.
Mutagenicity
Non-mutagenic – Acrylic acid is non-mutagenic in Salmonella and CHO cells, but positive in the mouse lymphoma assay and in the in vitro chromosomal aberration test. However, in vivo acrylic acid did not produce mutagenic effects in either rat bone marrow cells or mouse germ cells after oral administration. Based on these results and considering data from structurally-related acrylic compounds, it is concluded that acrylic acid is not mutagenic in vivo.
Carcinogenicity
Non-carcinogenic – Studies on animals indicate that acrylic acid is not carcinogenic.
Reproductive toxicity
Not a reproductive toxin – In oral reproductive toxicity studies (rats), no effects on reproductive function (fertility) were observed, but some signs of postnatal developmental toxicity (retarded body weight gain of the pups) were seen following exposure of the parental generation. No gross abnormalities were observed in the offspring. No prenatal developmental toxicity was observed (rats and rabbits, inhalation).
irritation, and is well tolerated by animals in dermal carcinogenicity studies.
Systemic Risk Assessment for Acrylic Acid ● The US EPA Integrated Risk Information System (IRIS) Oral Reference dose (RfD) for acrylic acid [19] is 0.5 mg/kg/day, which is based on a two-generation reproductive study in rats, with supporting data from a chronic drinking water study in rats, developmental studies by the inhalation route in rats and rabbits, and an inhalation/dermal/oral./i.v bioavailability study in rats and mice. It is based on a No Observed Adverse Effect Level of 53 mg/kg/day and an uncertainty factor of 100, which includes a factor of 10 for interspecies extrapolation and a factor of 10 to protect sensitive individuals. This RfD represents a daily exposure level that is considered to be safe for a lifetime of exposure, including sensitive subpopulations. ● A default reflux value of 1.6% is recommended for exposure calculations for materials that are not in direct skin contact, and this value may be used for acrylic acid, as a conservative approach.
Exposure Assessment of Acrylic Acid The QRA for acrylic acid in diaper cores is summarized in > Table 82.4.
It is noted that this exposure assessment is highly conservative, and assumes that 100% of the ingredient is represented as free acrylic acid in the entire diaper, and that this chemical is available for reflux, and fully penetrates the skin for each of the three diapers used, on a daily basis. Based on the risk assessment and exposure evaluation, and a margin of safety (MOS) that is greater than 1, it can be concluded that for systemic toxicological endpoints, residual acrylic acid that may be present in the SAP core of adult diapers does not present any systemic human safety risk.
Threshold of Toxicological Concern (TTC) The absence of chemical-specific safety data for some chemicals found at very low levels in diapers may be encountered during the process of safety evaluation. Many of the materials used in the construction of adult care disposable diapers are complex materials that may contain many different chemicals or may have low-level impurities. For example, low levels of unreacted monomers may be present in a high molecular weight polymer, or a low level of a solvent may be present in an adhesive. Traditional risk assessment approaches are generally used to confirm safety, based on evaluating the potential for these chemicals to migrate out of the diaper and reach the skin. However, there may be very low residual levels of chemicals/contaminants that do not have a robust safety dataset. To confirm safety for such low-level residuals, an
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Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly
. Table 82.4 Application of QRA to available acrylic acid in diaper absorbent cores
care disposable diaper is shown below. Total exposure to the adhesive itself is determined to be 192 mg/day. ðCp F Rf =100 Rw=100 Ab=100 ED=100Þ
Acrylic acid RfD of acrylic acid (U.S. EPA)
0.5 mg/kg/day
Total amt of SAP/pad
22 g
Amt of extractable monomer (RM specs)a
500 ppm (conservative)
Number of diapers/day
3
Total available acrylic acid/day
500 ppm ¥ 22 g = 11 mg/day ¥ 3 = 33 mg/day
Weight of adult
60 kg
Total available acrylic acid/kg body weight
33/60 = 0.55 mg/kg/day
Reflux factor
1.6% = 0.016
Dermal absorption
100% (conservative)
Potential consumer exposure
0.55 mg/kg/day ¥ 0.016 ¥ 1 = 0.009 mg/kg/day
Margin of safety (MOS) = RfD/Consumer exposure
0.5/0.009 mg/kg/day = 55
a
500 ppm is the maximum limit of available acrylic acid monomers that suppliers of SAP are currently required to meet, for use in adult diapers
assessment based on the concept of threshold of toxicological concern (TTC) can be used. This approach is based on the fundamental premise that there is an exposure to chemicals below which adverse effects will be negligible or absent. This TTC framework provides a conservative estimate of an acceptable chronic exposure, in the absence of chemical-specific data. Although originally developed by the US FDA [20] to support low-level exposures to indirect food additives (e.g., packaging materials), this framework has since been expanded for application to materials that may be used in consumer and personal care products [2, 12]. The TTC approach has been described in the literature in some detail [10]. For a chemical that does not have structural alerts for genotoxicity, an acceptable exposure limit is generally accepted to be 1.5 mg/day for an adult.
Exposure Assessment of Adhesive Contaminant An example of the application of TTC to determine an acceptable level of a residual level of a chemical contaminant in an adhesive used in the construction of an adult
Cp
Amount of adhesive in the diaper in g = 4.0 = 4,000 mg (Maximum level. This can range from 1 g to 4 g/diaper)
F
Number of diapers used per day = 3
Rf
Release factor = 100 (very conservative) Rw Reflux Factor = 1.6%
Ab Absorption of the adhesive through skin = (default 100%)
Exposure = 4,000 * 3 * 1 * 0.016 * 1 * 1 = 192 mg/day Assuming a body weight of 60 kg, this exposure is equivalent to 192 mg/day divided by 60 kg = 3.2 mg/kg/ day (3,200 mg/kg/day). Therefore, an acceptable upper limit of the contaminant in this adhesive can be established: Acceptable Exposure to Contaminant 1:5 ug=day ¼ Exposure to entire Adhesive 192 mg=kg=day 0:0000078 ¼ 8ppm In this example, it can be established that a level of up to 8 ppm of a contaminant (without structural alerts for genotoxicity) in the adhesive can be supported using the TTC method. Similarly, if the exact level of the potential adhesive contaminant is known in advance, the TTC method can be used to confirm its safety. In this case, any level below 8 ppm would be considered to be acceptable using this method. It is noted that the TTC method is highly conservative, and if the exposure is determined to be above a TTC-based limit, then generally there are opportunities to refine the assessment with more realistic assumptions before determining that the level of the contaminant is unacceptably high.
Conclusion The conduct of scientifically sound risk assessments for establishing safety of raw materials used in the manufacture of personal care products, such as adult care disposable diapers, is achieved by using established methods of QRA. QRA is currently used to support the safe introduction of new products and their ingredients into the market for almost all classes of personal care products.
Dermal Safety Evaluation: Use of Disposable Diaper Products in the Elderly
Kosemund et al. [10] have previously described details of this method, with reference to baby diapers. Being complex, multilayered products, adult care disposable diapers are composed of various raw materials that may or may not directly contact the skin. Most of the components of adult care disposable diapers are large molecular weight, inert polymers that have low bioavailability, and are therefore not absorbed through the skin (even though there may be direct skin contact). On the other hand, exposure evaluation of low molecular weight ingredients, such as lotion components, perfume ingredients, residual contaminants, residual monomers, or residual process aids are relevant to ensure overall safety of the raw material, and of the diaper as a whole. Skin exposure to deeper diaper materials may be possible via the phenomenon of reflux, where an aqueous vehicle such as urine may help to carry these materials toward the surface of the diaper, and hence toward the skin. This is particularly relevant when diapers are worn all night long, with a very high urine load. Reflux of a diaper is typically determined by one of two currently existing methods, and these methods provide a worst-case estimation of the amount of chemical that may be carried toward the skin. For the purposes of evaluation of exposure to adult disposable diaper raw materials, various parameters such as consumer usage habits (number of diapers used per day), surface area of the diaper, topsheet transfer factor, weight of the individual, and reflux factor are taken into account. These parameters are the foundation for characterizing exposure to a particular diaper raw material. Each of these is evaluated in detail and, depending on the parameter, conservative default assumptions are made or the actual values of the parameter are used for risk assessment. Default assumptions may be replaced with actual values: a process that helps to refine the overall exposure assessment. (See also chapter by Bramante, M.) Familiarity with both the product and the consumer is also required for a robust assessment of safety. Disposable diapers today have substantial holding and performance capacity and, as such, skin health is generally substantially better than when using cloth diapers. However, broken skin, especially in geriatric patients, can be a serious clinical concern. As such, the use of a 100% dermal penetration factor is a highly conservative assumption but, as mentioned earlier, a more realistic or exact skin penetration factor is also a usable option, whenever it is available. Two examples are used to demonstrate the principles of QRA for adult diapers: citral, a common ingredient in perfumes, is evaluated for its sensitizing potential, and acrylic acid, a constituent ingredient of SAP, is evaluated for its systemic toxicity potential.
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QRA of citral for allergen potency demonstrated that a higher AEL of citral versus the calculated CEL did not pose an increased risk for sensitization when it is used as a perfume constituent in an adult disposable diaper. The wide MOS of the AEL versus the CEL (60,000) provides overwhelming safety assurance that use of citral in diaper products does not pose a risk of sensitization. Similarly, QRA for acrylic acid showed that it enjoys a wide MOS (55) versus actual exposure when residual monomers of acrylic acid are evaluated for systemic toxicity potential. The safety of low residual levels of chemicals or contaminants whose toxicological profiles are not known or do not exist can be confirmed by using the concept of threshold of toxicological concern (TTC). Use of TTC is based on the fundamental premise that there is an exposure to contaminants or chemicals below which adverse effects will be negligible or absent. The TTC method can be used to drive the establishment of safe levels of residuals or contaminants that may be present in diaper raw materials. Despite variations in diaper usage habits and practices across various countries (and tiers of consumers), and despite the diversity of different diaper raw materials used to construct a diaper, the consistent use of the QRA method for the evaluation of diaper raw materials has demonstrated the robustness of the method. The use of QRA helps to ensure that all raw materials that are used in adult disposable diapers globally are safe. Thus, the safe introduction of new materials and cutting edge technologies in adult disposable diapers are ensured by this process. This allows consumer goods companies to be able to innovate with newer and emerging materials, and provide the latest diaper designs and technologies to consumers around the world.
Cross-references > Safety
Evaluation in the Elderly via Dermatological
Exposure
Acknowledgment The authors wish to thank Mr. Yukio Heki, Principal Scientist, Procter & Gamble Japan K.K. for use of > Figs. 82.2 and > 82.3, and for his valuable insights on adult diaper types and usage habits among incontinent individuals in Japan.
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References 1. Api AM, Basketter DA, Cadby PA, Cano M-F, Ellis G, Gerberick GF, Griem P, McNamee PM, Ryan CA, Safford R. Dermal sensitization quantitative risk assessment for fragrance ingredients. Regul Toxicol Pharmacol. 2008 Oct;52(1):3–23. 2. Blackburn KL, Stickney JA, Carlson-Lynch HL, Mc Ginnis PM, Chappell L, Felter SP. Application of the threshold of toxicological concern approach to ingredients in personal and household care products. Regul Toxicol Pharmacol. 2005;43:249–259. 3. Bos JD, Meinardi MHM. The 500-dalton rule for the skin penetration of chemical compounds and drugs. Exp Dermatol. 2000;9: 165–169. 4. Fiume MZ. Final report on the safety of assessment of acrylates copolymer and 33 related cosmetic ingredients. Int J Toxicol. 2002;21(S3):1–50. 5. Herrlein MK. 1996. European Patent EP0797967 A1. 6. Hopper LD, Oehme FW. Chemical risk assessment: a review. Vet Hum Toxicol. 1989;31(6):543–554. 7. Institute for Health and Consumer Protection, European Chemicals Bureau. Acrylic acid European Union Risk Assessment Report, 28: EUR 19836. Luxembourg: Office for Official Publications of the European Communities, 2002. 8. International Programme on Chemical Safety, 1999. Environmental Health Criteria 210: Principles for the Assessment of Risks to Human Health from Exposure to Chemicals. Accessed at http://www. inchem.org/documents/ehc/ehc/ehc210.htm, April 2007. 9. International Fragrance Association, 2006. IFRA standard, 40th Amendment: Citral. Accessed at http://www.ifraorg.org/Home/ Publications/Download-the-IFRA-Amendments/page.aspx/134, May 2008. 10. Kosemund K, Schlatter H, Ochsenhirt J, Krause E, Marsman D, Erasala G. Safety evaluation of superabsorbent baby diapers. Regul Toxicol Pharmacol. 2009;53:81–89. 11. Kroes R, Renwick AG, Cheeseman M, et al. Structure-based thresholds of toxicological concern (TTC): guidance for application to
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substances present at low levels in the diet. Food Chem Toxicol. 2004;42:65–83. Kroes R, Renwick AG, Feron V, Galli CL, Gioney M, Greim H, Guy RH, Lhuguenot JC, Vande Sandt JJ. Application of the thershold of toxicological concern to the safety evaluation of cosmetic ingredients. Food chem Toxicol. 2007;45(12):2533–2562. Lalko J, Api AM. Citral: identifying a threshold for induction of dermal sensitization. Regul Toxicol Pharmacol. 2008;52 (1):62–73. Accessed at via Science Direct, May 2008. National Academy of Science. Risk Assessment in the Federal government. National Research Council. Washington: National Academy Press, 1983. National Association for Continence, 1999. http://www.nafc.org/ Organisation for Economic Co-operation and Development (OECD), 2007. About Chemicals Hazard/Risk Assessment. Accessed at http:// www.oecd.org/about/0,2337,en_2649_34373_1_1_1_1_1,00.html, May 2008. QRA Expert Group (Api AM, Basketter DA, Cadby PA, Cano M-F, Ellis G, Gerberick GF, Griem P, McNamee PM, Ryan CA, Safford R), 2006. Dermal Sensitization Quantitative Risk Assessment (QRA) for Fragrance Ingredients, Technical Dossier. Accessed at http:// www.rifm.org/doc/QRA_Technical%20Dossier%20FINAL%20REV %202006%206%2022_1.pdf, May 2008. Scientific Committee on Cosmetic Products and non-Food Products Intended for Consumers, 1999. Opinion Concerning Fragrance Allergy in Consumers, SCCNFP/0017/98 Final. US Environmental Protection Agency, 1994. Acrylic acid Integrated Risk Information Systems (IRIS). Accessed at http://cfpub.epa. gov/ncea/iris/index.cfm?fuseaction=iris.showQuickView&substan ce_nmbr = 0002, May 2008. US Food and Drug Administration. Food additives: threshold of regulation of substances used in food-contact articles: final rule. Fed Regist. 1995;60:36582–36596.
Wrinkles
86 Facial Wrinkling: The Marquee Clinical Sign of Aging Skin Greg G. Hillebrand
Introduction If aging skin were a Broadway production, then facial wrinkling would get top billing among the large cast of benign clinical signs appearing in the show. There are several types of facial wrinkles classified with consideration to their location, pattern, histology and etiology [1, 2]. None have been studied more, nor rise as high in concern from a cosmetic point of view, as the lines and grooves associated with facial expression. The principal actors in this wrinkle cast of characters are the ‘‘crow’s feet’’ around the eye, the transverse forehead lines, and the glabellar frown lines. The nasolabial folds might also be included with these players. These kinds of wrinkles were classified as Type 3 by Pie´rard [2]. They can be either temporary or persistent. Temporary lines form in the skin during the process of muscle contraction and disappear when the facial muscles relax providing the human face with the unique ability to express emotion. On the other hand, persistent wrinkles are visible at rest without muscle contraction. Indeed, relative to the supporting cast of aging skin clinical signs like sallowness, large pores, surface roughness and age-related dispigmentation, facial wrinkling is a dynamic skin appearance feature exhibiting a tremendous range of variation across the myriad of facial expressions. There are seven basic emotions: fear, anger, happiness, contempt, surprise, disgust, and sadness. Each manifests as characteristic facial expressions such as smiling or frowning that are universal in cultures around the world [3]. These expressions are produced by 43 unique facial muscles which can combine to produce over 10,000 facial movements, thereby providing infinite variation on each emotional expression. Compared to skeletal muscles elsewhere in the body whose function is to move bone, facial muscle physiology is unique in that its primary function is to move skin (an exception would be the masseter needed for chewing). One particularly important facial muscle is the orbicularis oculi around the orbit of the eye. When this muscle contracts, for example during a smiling
expression, the skin around the eye is thrown into folds which radiate perpendicular to the direction of muscle contraction, producing the characteristic, an often disdained, ‘‘crow’s feet’’ wrinkles. In 1897, Mark Twain wrote: ‘‘Wrinkles should merely indicate where smiles have been’’ [4].
Histological Aspects The age-related changes in temporary and persistent facial skin wrinkling occur very slowly over a person’s lifetime [5–8]. Certain host and environmental factors like hormonal changes, sun exposure, and smoking, are well known to accelerate the onset and rate of skin aging, including the acceleration of facial wrinkling [5, 6, 8–10]. The histological effects of chronic sun exposure [11] include the loss and fragmentation of dermal collagen [12, 13], deposition of large amounts of poorly functional elastin in the upper dermis [14],overproduction and abnormally located dermal glycosaminoglycans [15] and changes in stratum corneum keratin intermediary filaments [16]. These molecular changes result in a decline in the skin’s elastic properties which is believed to be fundamental in wrinkle formation [17–22]. Kligman and coworkers studied the histology of persistent wrinkles [1]. While the skin showed many of the common histological features appropriate for the subject’s age and body site, they did not observe any remarkable differences in the epidermal or dermal structure between the wrinkle and the surrounding skin, a finding confirmed by Bosset et al. [23] and Pie´rard and Lapiere who did find differences in the hypodermal septae [24]. For extremely deep facial wrinkles which have persisted for decades, the fold can chronically shadow the underlying skin, protecting it from further photodamage and lessening the degree of elastosis in the fold relative to the surrounding skin [25, 26], a phenomenon modeled in hairless mice [27].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_86, # Springer-Verlag Berlin Heidelberg 2010
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Facial Wrinkling: The Marquee Clinical Sign of Aging Skin
Mechanical Aspects Kligman explained a wrinkle as a simple configurational change and proposed an ‘‘old glove’’ model for wrinkle formation: ‘‘Smooth when new, the fabric develops grooves at sites of long-sustained stress.’’ Muscle contraction, like that during everyday facial expression, forces the skin to repetitively fold along the same groove. With time, this repeated mechanical stress causes that grove or temporary wrinkle to etch in as a permanent or persistent wrinkle [1]. Persistent wrinkle formation would then have an obligatory mechanical component as born out by the lack of persistent wrinkles on the dorsal forearms, which can show severe signs of skin photodamage, yet no wrinkles. In these areas, muscle function is to move bones, not skin, and therefore the prerequisite temporary wrinkling never occurs. Ultraviolet (UV)-induced skin damage, then, is not the cause, but an accelerator of facial wrinkling when combined with repeated mechanical stress. Damage to the epidermal and dermal molecular architecture reduces the skin’s ability to accommodate that mechanical stress. Based on this model, the age of onset and rate of persistent facial wrinkling is dependent on both the cumulative amount of mechanical stress (frequency of temporary wrinkling) in combination with the decline in skin elasticity caused by, for example, cumulative sun exposure. It is generally assumed that the age-related deterioration in the elastic properties of the skin is directly or indirectly responsible for the formation of skin wrinkles [19, 28]. However, the temporal relationship between the loss in mechanical integrity and visible wrinkle formation is sequential in nature. Fujimura et al. used the Cutometer (Courage and Khazaka, Ko¨ln, Germany) to measure skin elasticity parameters in the crow’s feet area on 90 healthy female volunteers living in Tokyo, Japan (ages 18–76 years). They also used 3D surface replicas to measure wrinkling in the same area. They found that the age-related loss in skin elasticity actually precedes the formation of visible wrinkles by about 20 years [22]. The loss of skin mechanical properties starts very early in life, earlier in fact than one might expect, perhaps even at birth [29]. While the loss in elasticity appears to be similar between males and females [30], this loss is significantly faster on sun-exposed vs. sun-protected skin sites [31]. Almost all of the observational research on aging skin is based on point-in-time or cross-sectional surveys. Cross-sectional surveys need to be of sufficient base size and designed without the potential for selection bias in order to make robust conclusions regarding the agerelated changes in skin parameters [32]. In a study of 450 normal healthy Chinese women, balanced across
. Figure 86.1 Mean loss of skin elasticity (% change in R7 relative to the 10–19 age group mean) in the upper inner arm and cheek by age group (n = 450 total; n = 75 per decade)
ages 10–70, skin elasticity was measured on the sunexposed upper cheek and the sun-protected upper inner arm. The skin elasticity parameter, R7 (Ur/Uf), which is independent of skin thickness, was measured using the Cutometer MPA 580 equipped with a 2-mm probe. The pressure value was set to 350 mbar and one measuring cycle (10 s) was performed, with a 5-s Suction Time/OnTime and a 5-s Relaxation Time/Off-Time. Three measurements were taken at each site for each subject and the average of these three measurements was used for statistical analysis. The change in skin elasticity with age on the cheek and upper inner arm is shown in > Fig. 86.1. For both the cheek and upper inner arm, the decline in skin elasticity was rapid with an early age of onset, at least as early as the teenage years. The decline was significantly faster on the sun-exposed cheek compared to the sunprotected upper inner arm indicative of chronic UVinduced skin damage. The loss in elastic recovery in the oldest age group (60–70) was substantial relative to the youngest age group (10–19); the older age groups showed only 70% of the cheek skin elastic recover of the younger age groups. These data speak to the importance of practicing good skin care habits from early childhood in order to best retain the skin’s youthful elastic qualities and thereby delay the onset and lessen the severity of wrinkling as well as other aging skin issues later in life.
Clinical Aspects Everyday facial expression imposes mechanical stress at specific sites in the skin. With age, the skin’s molecular
Facial Wrinkling: The Marquee Clinical Sign of Aging Skin
architecture deteriorates and there is the concomitant loss of elasticity, making the skin less able to rebound from the repeated mechanical stress. Despite the importance of facial expression in the etiology of facial wrinkling, most of the facial wrinkling clinical research over the past 40 or more years has employed methods or protocols that measure facial wrinkling only in a relaxed or neutral state. For example, traditional methods for measuring clinical facial wrinkling usually employ subjective visual grading based on photonumeric scales, skin replicas, or 2D or 3D imaging with associated quantitative image analysis. In nearly all cases, this research is carried out with study subjects showing a relaxed facial expression. As an initial foray into this area in the late 1990s, standardized facial images of women were captured with both a neutral and smiling expression in different ethnic populations (Caucasians, Indians, Hispanics, African Americans, and Asians) and geographies around the world (North and South American, Europe, and Asia) [33]. A smiling expression was chosen because it accentuates the crow’s feet wrinkles around the eyes, the area of most concern for many people. Images were captured using a facial imaging booth consisting of a high resolution digital
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camera combined with standardized illumination and fixed head positioning (e.g., the VISIA 3.0 Complexion Analysis System, Canfield Scientific, Inc., Fairfield, NJ, USA). Subjects were prepared for image capture using standard procedures for face wash, equilibrated to ambient conditions (20 min) and use of matt black head and shoulder apparel. Image analysis (IA) software was employed to objectively quantify facial wrinkling on both the left and right sides of the face to yield an average wrinkle severity for each subject. The same exact area of the face was analyzed for both smiling and non-smiling images and the image analysis overlays were reviewed to confirm the accuracy of skin feature detection. Wrinkle severity is expressed as the fraction of total pixels in the region of interest that are wrinkle pixels, i.e., the wrinkle area fraction. > Figure 86.2 shows a typical subject’s image, with and without smiling. Persistent wrinkling is gleaned from the non-smiling images. Temporary wrinkling is gleaned from the smiling images. In total, there are over 4,000 women in this image database each with a neutral and smiling expression. In Beijing, China, facial wrinkling was assessed in 450 normal healthy Chinese women ranging in age from 10 to 70 (n = 75 per decade). The age-group means
. Figure 86.2 Example images of a Chinese subject without (top left) and with (bottom left) a smiling expression. The region of interest with wrinkle image analysis overlay (green) in shown to the right of each image
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Facial Wrinkling: The Marquee Clinical Sign of Aging Skin
for both temporary and persistent facial wrinkling in this sample population are shown in > Fig. 86.3. In youth (ages 10–19), both temporary and persistent facial wrinkles are largely absent. The skin of a child is simply too tight to form a groove during facial expression. However, in early adult life (typically the twenties), as skin laxity increases, the first signs of wrinkling show as temporary lines during facial expression; persistent wrinkles are still largely absent. Thereafter, and depending on the person’s history of sun exposure and other factors, persistent facial wrinkles will start to appear [34]. While these data speak of crow’s feet wrinkles, it is certain that similar age-related changes in temporary and persistent wrinkling apply to other Type 3 wrinkles like the transverse forehead lines and glabellar frown lines. It is noteworthy that no two person’s facial wrinkle pattern is alike. Each individual has their own unique facial ‘‘wrinkleprint’’, as unique as is their own fingerprints. How is it that wrinkles form at a particular location with such a unique and defined pattern? The answer lies in the disciplines of physics, material science and anatomy. Skin is a multilayered tissue with an outer stratum corneum (10–20 mm), a living epidermis (50–100 mm), a dermis (1–3 mm) and hypodermis composed primarily of adipocytes. The skin is connected to the underlying musculature via connective tissue and the muscles are attached to the skull bone. Computational models are being developed with the aim of predicting how skin reacts to compression forces such as those brought on by muscle contraction. Magnenat-Thalmann et al. used a three-layer computational model to help better understand the pivotal role of the stratum corneum
mechanical properties in the development of fine wrinkles [35]. Kuwazuru et al. used a five-layer skin computational model to explain changes in wrinkling with age [36]. These models do not consider facial anatomy such as facial muscle size or skull bone shape that impact the forces applied to the skin at a given facial site. The importance of facial anatomy in wrinkle formation became apparent when standardized imaging was used to quantify and compare facial wrinkling in monozygotic (‘‘identical’’) twins. Left, front and right view images of 69 sets of identical twins (male and female, ages 18–66) were captured and used to quantify the severity of facial wrinkling around the eye and on the cheek using image analysis. Since each twin pair has an identical chromosomal DNA sequence and since most twins are raised in the same households and exposed to similar environments, similar severities of wrinkling between twin A and twin B were expected. This is exactly what was found (> Fig. 86.4). There is an excellent correlation between twin A and twin B for the severity of persistent wrinkling. If each person’s facial skin ‘‘wrinkleprint’’ developed in a random fashion, then it is expected that the wrinkle pattern of twin A would be dissimilar to that of twin B. The wrinkle patterns of side-by-side images of sets of monozygotic twins were compared. There was a striking similarity in the wrinkleprints for each twin. > Figure 86.5a shows front view facial images of male identical twins. The wrinkle pattern on the forehead is remarkably similar. > Figure 86.5b shows left oblique view facial images of two female identical twins with a smiling expression. The number, length and location of the crow’s feet wrinkles are strikingly
. Figure 86.3 Mean ( standard error) facial wrinkle area fraction by age group with and without smiling in the Chinese female population (n = 450 total; n = 75 per decade)
. Figure 86.4 Correlation between twin pairs for severity of facial wrinkling (r = 0.89)
Facial Wrinkling: The Marquee Clinical Sign of Aging Skin
similar. An individual’s pattern of facial wrinkling is likely dependent on the morphology of the skull and muscles which influence exactly where on the face expression causes skin buckling. Since identical twins have such similar skull and muscle morphology, they have very similar wrinkle patterns. By following the change in pattern of facial wrinkling on the same individuals with and without facial expression over a long period of time, the question raised was: do the temporary wrinkles of youth progress into the persistent wrinkles of adulthood [37]. This 8-year longitudinal survey started in Los Angeles, CA in 1999/ 2000 [33]. A total of 1,437 women were enrolled. In 2008, at the same study location, 122 of these same women
. Figure 86.5 Standardized images of monozygotic twins. (a) Front view neutral expression image of male twin pair. Note the striking similarity in the pattern of transverse forehead lines. (b) Left oblique view smiling expression image to female twin pair. Note the striking similarity in pattern of crow’s feet wrinkling around the eye
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participated in a follow-up survey (mean age standard deviation [SD]; 40.4 15.7 at baseline and 48.8 15.7 at 8 years). They were of Caucasian (n = 60), African American (n = 20), Asian (n = 22), or Latino (n = 20) heritage. None of the subjects moved away from the Los Angeles area nor reported having had surgical, laser, filler, botulinum toxin or other cosmetic procedures between their baseline and follow-up visits deemed to affect facial wrinkling. Each subject cleansed her face with a commercial facial cleanser and sat quietly for 15 min prior to imaging. Standardized left oblique view facial images were captured using the same imaging rig at both baseline and 8 years, thereby minimizing camera and lighting variance between visits [38]. Two images were collected at each visit, one with a neutral relaxed expression (to measure persistent wrinkling) and one with a smiling expression (to measure temporary wrinkling), a total of four images per subject. To track the progression of individual lines and wrinkles on each person, each subject’s pattern of wrinkling around the eyes and on the cheek with and without facial expression at baseline was compared to the pattern at 8 years. It was consistently found that the subjects’ unique pattern of persistent facial wrinkling observed with a neutral expression at 8 years was predicted by the pattern of temporary wrinkling observed with a smiling expression at baseline (> Fig. 86.6). Thus, repeated mechanical flexure along the same skin grove causes temporary lines to eventually etch in as persistent wrinkles. An identical twin case study showing that long-term prevention of forehead muscle contraction, via regular treatment with botulinum toxin A, prevents the imprinting of forehead and glabellar frown lines further demonstrates the key role skin flexure and repetitive mechanical stress plays in the formation and progression of facial wrinkling [39]. With respect to the influence of age on the rate of wrinkling, the study found that subjects who were in their forties at baseline showed a significantly faster rate of wrinkling over the 8-year period compared to women in the other age groups at baseline [37]. Others have also found an increased rate of skin wrinkling during middle age for women [28, 36], but not necessarily men [40]. The relationship between menopausal status and rate of wrinkling was examined as a possible explanation for the observed faster rate of wrinkling for the middle-aged women in the study. Being menopausal was not associated with a higher rate of wrinkling. In fact, the postmenopausal women in this study (who tended to be in their fifties and sixties) had a very low rate of wrinkling. It was the women who had entered menopause
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. Figure 86.6 Progression of temporary into persistent wrinkling. At baseline (left column), only a few shallow wrinkles are evident in this subject’s neutral image which are better observed in the zoomed images without (middle row) and with (bottom row) wrinkle image analysis overlays. The baseline expression image (middle column) shows substantial temporary wrinkles around the eye which are not evident in the baseline neutral image. Eight years later (right column), the pattern of persistent wrinkles (individually numbered) in the neutral image can be traced back to the pattern of temporary wrinkles in the baseline expression image
between baseline and 8 years who showed the highest rate of wrinkling (+95% increase). This suggests that the change in hormonal status, rather than hormonal status per se, is the important determinant in accelerating skin wrinkling. The observation that hormone replacement therapy did not improve skin wrinkling in women who were 5 years postmenopausal is consistent with this view [41].
Aging Simulation People are naturally curious about how they will look 5, 10, 15, or more years in the future and how certain
lifestyles or treatment interventions, like cessation of smoking or use of daily sun protection, will impact their future appearance. The depiction of a person’s face in the future is of tremendous utility in the fields of cosmetics, dermatology, plastic surgery, missing person identification and entertainment. With each person having their own aging skin ‘‘destiny’’, and personal ‘‘wrinkleprint’’, the challenge for facial aging simulation is to accurately predict and visualize when and to what extent an individual’s unique future wrinkle pattern will develop. Current image-based computer simulation models typically apply population averages or attempt to blend features from an old person’s face onto that of a young person to yield a future image that in most cases will bear little
Facial Wrinkling: The Marquee Clinical Sign of Aging Skin
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. Figure 86.7 Image-based method for aging simulation. The wrinkles observed with a smiling expression are morphed onto the neutral image to yield a third image simulating future wrinkling unique for the individual. Left: original neutral image. Center: original smiling image. Right: aging simulation image
resemblance to what that particular person will actually look like [42]. An image-based method for aging simulation was developed that takes advantage of the fact that temporary wrinkles observed with facial expression are predictive of future persistent wrinkles [43]. By capturing images of a person with a neutral and, e.g., a smiling expression and then morphing the temporary wrinkles from the smiling expression image onto the neutral image, the resulting image will simulate future persistent wrinkling unique in pattern and severity to that particular individual. The spatial mapping computed through elastic registration defines the morphing of the face from the neutral to the smile state and determines how to transfer the smile wrinkles onto the neutral image. The elastic registration procedure ensures that the smile wrinkles, when transferred onto the neutral image, overwrite the correct position on the neutral image. This preserves the natural look of the face while creating a realistic prediction of the wrinkle aging process. > Figure 86.7 illustrates the simulation method.
Conclusion Facial wrinkling is a dynamic skin appearance feature. Temporary wrinkles are observed during facial expression and disappear when the muscles relax. Persistent wrinkles are apparent with a relaxed expression. Longitudinal studies confirm that persistent wrinkles evolve directly from temporary wrinkles. The rate of wrinkling is dependent on both host and environmental factors. There is acceleration in the rate of wrinkling during middle age, at least in females. Twins studies suggest that each person’s unique pattern of wrinkling, or wrinkleprint, is dependent not
only on the skin itself, but also on that person’s unique skull and muscle morphology. Protecting the skin from acute and chronic sun damage and practicing regular skin care from early childhood, will help preserve the skin’s youthful elasticity so as to better withstand mechanical stress thereby delaying the age of onset and slowing down the rate of wrinkling over a lifetime. Mark Twain was right.
References 1. Kligman AM, Zheng P, Lavker RM. The anatomy and pathogenesis of wrinkles. Br J Dermatol. 1985;113:37–42. 2. Pie´rard GE, Uhoda I, Pie´rard-Franchimont C. From skin microrelief to wrinkles. An area ripe for investigation. J Cosmet Dermatol. 2003;2:21–28. 3. Ekman P. The Face of Man: Expressions of Universal Emotions in a New Guinea Village. New York: Garland, 1980. 4. Twain M. [Samuel Langhorne Clemens] in Following the Equator. 1897. 5. Hillebrand GG, Levine M, Miyamoto K. The age-dependent changes in skin condition in ethnic populations from around the world. In: Berardesca E, Leveque JL, Maibach HI (eds) Ethnic Hair and Skin, 1st ed. New York: Infoma Healthcare, 2007, pp. 105–122. 6. Akiba S, Shinkura R, Miyamoto K, et al. Influence of chronic UV exposure and lifestyle on facial skin photo-aging — results from a pilot study. J Epidemiol. 1999;9,S136–S142. 7. Batisse D, Bazin R, Baldeweck T, et al. Influence of age on the wrinkling capacities of skin. Skin Res Technol. 2002;8:148–154. 8. Hillebrand GG, Schnell B, Miyamoto K, et al. Age dependent changes in skin condition in Japanese females living in northern versus southern Japan. IFSCC Mag. 2001;4:89–96. 9. Rexbye H, Petersen I, Johansens M, et al. Influence of environmental factors on facial ageing. Age Ageing. 2006;35:110–115. 10. Helfrich YR, Yu L, Ofori A, et al. Effect of smoking on aging of photoprotected skin: evidence gathered using a new photonumeric scale. Arch Dermatol. 2007;143:397–402. 11. Watson REB, Griffiths CEM. Pathogenic aspects of cutaneous photoaging. J Cosmet Dermatol. 2005;4:230–236.
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12. Lavker R, Zheng PS, Dong G. Aged skin: a study by light, transmission electron, and scanning electron microscopy. J Invest Dermatol. 1987;88:44–51. 13. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and therapy. Br J Dermatol. 2007;157:874–887. 14. Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol. 2008;7:S12–S16. 15. Bernstein EF, Underhill CB, Hahn PJ, et al. Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans. Br J Dermatol. 1996;135:255–262. 16. Sano T, Fujimura T, Kawada H, et al. Keratin alterations could be an early event of wrinkle formation. J Dermatol Sci. 2009;53:65–84. 17. Imokawa G. Recent advances in characterizing biological mechanisms underlying UV-induced wrinkles: a pivotal role of fibroblastderived elastase. Arch Dermatol Res. 2008;300:S7–S20. 18. Imayama S, Braverman IM. A hypothetical explanation for the aging skin. Chronological alteration of the three-dimensional arrangement of collagen and elastic fibers in connective tissue. Am J Pathol. 1989; 134:1019–1025. 19. Lee JY, Kim KY, Seo JY, et al. Loss of elastic fibers causes skin wrinkles in sun-damaged human skin. J Dermatol Sci. 2008;50:99–107. 20. Tsukahara K, Takema Y, Moriwaki, et al. Selective inhibition of skin fibroblast elastase elicits a concentration-dependent prevention of ultraviolet B-induced wrinkle formation. J Invest Dermatol. 2001; 117:671–677. 21. Tsuji N, Moriwaki, Suzuki Y, et al. The role of elastases secreted by fibroblasts in wrinkle formation: implication through selective inhibition of elastase activity. Photochem Photobiol. 2001;74: 283–290. 22. Fujimura T, Haketa K, Hotta M, Kitahara T. Loss of skin elasticity precedes to rapid increase of wrinkle levels. J Dermatol Sci. 2007; 47:233–239. 23. Bosset S, Barre P, Chalon A, et al. Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles. Eur J Dermatol. 2002;12:247–252. 24. Pie´rard GE, Lapie`re C. The microanatomical basis of facial frown lines. Arch Dermatol. 1989;125:1090–1092. 25. Tsuji T, Yorifuji T, Hayashi Y, Hamada T. Light and scanning electron microscopic studies on wrinkles in aged person’s skin. Br J Dermatol. 1986;114:329–335. 26. Contet-Audonneau JL, Jeanmaire C, Pauly G. A histological study of human wrinkle structures: comparison between sun-exposed areas of the face, with or without wrinkles, and sun-protected areas. Br J Dermatol. 1999;140:1038–1047. 27. Takema Y, Fujimura T, Ohsu H, Imokawa G. Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin. Exp Dermatol. 1996;5:145–149. 28. Akazaki S, Nakagawa H, Kazama H, et al. Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis. Br J Dermatol. 2002;147:689–695.
29. Escoffier C, de Rigal J, Rochefore A, vasselet R, Lefeque JL, Agache PG. Age-related mechanical properties of human skin: an in vivo study. J Invest Dermatol. 1989;93:353–357. 30. Cua AB, Wilhelm, Maibach HI. Elastic properties of skin: relation to age, sex, and anatomical region. Arch Dermtol Res. 1990;282: 283–288. 31. Jemed GBE, Selvaag E, Agren M, Wulf HC. Measurement of the mechanical properties of skin with ballistometer and suction cup. Skin Res Technol. 2001;7:122–126. 32. Hillebrand GG, Wickett RR. Epidemiology of skin barrier function: host and environmental factors. In: Walters K, Roberts M (eds) Dermatological and Cosmeceutical Development. 2007, pp. 129–156. 33. Hillebrand GG, Levine MJ, Miyamoto K. The age-dependent changes in skin condition in African Americans, Caucasians, East Asians, Indian Asians and Latinos. IFSCC Mag. 2001;4:259–266. 34. Miyamoto K, Hillebrand GG. The influence of facial expression on the age-dependent changes in facial wrinkling. J Soc Cos Sci. 2007;58:206–207. 35. Magnenat-Thalmann NK, Kalra P, Leveque JL, et al. A computational skin model: fold and wrinkle formation. IEEE Trans Inf Technol Biomed. 2002;6:317–323. 36. Kuwazuru O, Saothong J, Yoshikawa. Mechanical approach to aging and wrinkling of human facial skin based on the multistage buckling theory. Med Eng Phys. 2008;30:516–522. 37. Hillebrand G, Yoshii T., Yan X, Progression of temporary into persistent facial wrinkling: An 8-year longitudinal study. J Am Acad Derm. 2009;60:AB1. 38. Miyamoto K, Hillebrand GG. The Beauty Imaging System: for the objective evaluation of skin condition. J Cosmet Sci. 2002;53:62–65. 39. Binder WJ. Long-term effects of botulinum toxin type A (Botox) on facial lines. Arch Facial Plast Surg. 2006;8:426–431. 40. Koehler MJ, Ko¨nig K, Elsner P, et al. In vivo assessment of human skin aging by multiphoton laser scanning tomography. Opt Lett. 2006;31:2879–2881. 41. Phillips TJ, Symons J, Menon S. HT Study Group. Does hormone therapy improve age-related skin changes in postmenopausal women? A randomized, double-blind, double-dummy, placebocontrolled multicenter study assessing the effects of norethindrone acetate and ethinyle estradiol in the improvement of mild to moderate age-related skin changes in postmenopausal women. J Am Acad Dermatol. 2008;59:397–404. 42. Hysert PE, Mirand AL, Giovino GA, Cummings KM and Kuo CL. ‘‘At Face Value’’: age progression software provides personalised demonstration of the effects of smoking on appearance. Tobacco Control. 2003;12:238–240. 43. Hillebrand GG, Demirli R. Method and apparatus for realistic simulation of wrinkle aging and de-aging. 2009; U.S. Patent Application 20090028380A1.
Wound Healing
85 Impaired Wound Repair and Delayed Angiogenesis Matthew J. Ranzer . Luisa A. DiPietro
Introduction Initially, skin was thought of as a simple barrier to protect the internal organs from the outside world, but now it is understood to be incredibly more complex than that. Skin serves multiple functions including the regulation of water loss, thermoregulation, protection from ultraviolet (UV) radiation and entry of microorganisms, and is an integral part of the immune system [1]. Skin ages via two processes: intrinsic aging and extrinsic aging. Intrinsic aging is seen in sun-protected areas of skin and is subject to the same generalized aging conditions as any other cell or organ system. Extrinsic aging occurs in sun-exposed areas and is the cumulative effect of intrinsic aging plus the environmental exposure to the aging process. The biggest extrinsic factor affecting skin aging is UV radiation encountered from sun exposure, which is also termed photoaging [2]. As skin ages, it becomes progressively atrophied, dry, and rough, with alterations in pigmentation, decreased turgor, and increased wrinkling. This leads to a progressive loss of function, leaving the aged skin with a decreased ability to regulate homeostasis and more vulnerable to the environment [3]. Traumatic injuries are the fifth leading cause of death for persons over the age of 65 in the USA, and it is estimated that there will be well over 50 million people over the age of 65 by the year 2030 [4, 5]. Understanding the impact of aging on skin wound healing will be vital in dealing with this growing population in the future. Wound healing is a complex process involving multiple concurrent stages, dozens of cell types, and hundreds of mediators. As research on wound repair progresses, there is an increased understanding of how each of these components changes over time within an individual wound, between different wound conditions, and from wounds of differently aged individuals. Many studies dating back almost a century show that wounds from older individuals do not heal as well as those from younger individuals [6]. More recent studies have provided information on how aging affects the individual components of wound healing including the inflammatory response, deposition
of the wound matrix, and angiogenesis [7–9]. In short, the age of an individual has as profound an effect on the process of wound healing as nearly any other identifiable condition or disease may have.
Age-Related Changes in the Components of Skin Before addressing the issue of how aging affects the wound-healing process, the changes in the milieu in which this wound-healing process occurs must be examined. Skin is a multilayered organ, with each layer’s constituents optimized for its function. The outermost layer is the epidermis and is largely composed of squamous epithelial cells called keratinocytes and a smaller population of pigment-producing cells called melanocytes. The epidermis functions as a barrier against moisture loss and water entry. With age, the thickness of the epidermis does not change although the density of melanocytes decreases and the dermal–epidermal junction becomes flattened, giving the appearance of atrophy and cellular heterogeneity [10]. The dermis is composed of multiple cell types, structures, and fibers. Surrounding the hair follicles, sweat glands, and other intradermal glands are various fibers collectively referred to as the extracellular matrix (ECM). The ECM is composed of types I and III collagen, elastin, and glycosaminoglycans. The dermis is divided into two layers, the superficial papillary dermis and the deep reticular dermis. The papillary dermis maintains contact with the epidermis through the formation of papillary ridges. It is these ridges that become flattened with age, resulting in decreased surface contact and the previously mentioned appearance of atrophy, as well as decreased resistance to shear forces with lateral tension in the elderly skin [10, 11]. There is also a decrease in the cellular component of the dermis including fibroblasts, mast cells, macrophages, and other immunologically important cells including Langerhans’ cells in the epidermis and dendritic cells in the dermis [9, 10].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_85, # Springer-Verlag Berlin Heidelberg 2010
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The primary structural proteins in the dermis are collagen, fibronectin, and elastic fibers. Interestingly, while there is a decrease in both the number and diameter of elastin fibers in the papillary dermis with age, in the reticular dermis the opposite is true with an increase in the number and diameter of the elastin fibers [10]. Fibronectin has several functions including regulation of inflammation, cell adhesion, and migration, and is closely associated with fibroblast production of collagen [12]. In isolated cell culture, some studies show an increase in fibronectin synthesis with age, but other in vivo studies show an age-related decrease in fibronectin expression with reduced levels of collagen [12, 13]. Collagen, the major protein found in the dermis, may be reduced in quantity with aging skin, but there still exists some controversy on this point, as some studies show a decrease with age, others describe an increase in collagen with age, and yet others show no change in collagen content with age [12, 14–16]. While there is controversy on the quantity, there is certainly a change in the quality of the collagen in aged skin. Collagen in young skin is typically described as rope-like bundles of dense collagen I fibers, arranged in a lattice or basket weave pattern. In aged skin, the collagen is coarser, with individual bundles being primarily straight, loosely woven fibers, and with an increase in density of the collagen network [10–12]. Dermal appendages including hair follicles and sweat glands are affected by the aging process as well. Hair follicle numbers are diminished with age, but their structure is largely unchanged save for a small decrease in the number of surrounding melanocytes [17]. Sebaceous glands, which produce a waxy substance that coats hair shafts and reduces water evaporation, also decline with age [18]. Sweat glands are also reduced in number and function with age [19]. The microvascular blood flow to skin is decreased with aging as well. There is as much as a 40% reduction in cutaneous blood flow at 70 years of age compared to the skin of a 20-year-old individual [20]. There is also thinning of blood vessel walls and basement membranes, with decreased numbers of perivascular cells, which may promote extravasation of plasma into the interstitial spaces [21]. Lymphatic drainage in the elderly is also reduced, leading to greater edema and increasing the likelihood for ulcers [22]. In short, aging skin has decreased potential for replication and migration at baseline. There is a decrease in the ECM components and its architecture, resulting in decreased tensile strength. There is a reduction in dermal skin appendages including sweat glands and hair follicles [17, 18]. There is also a reduction in the nutrient supply in
the form of decreased microcirculation. Lastly, there is a greater tendency toward fluid accumulation due to increased permeability of the vasculature coupled with a decrease in lymphatic drainage. > Table 85.1 below lists the age-related changes in human skin.
Normal Wound Healing Generally, wound healing is thought of as occurring in three or four overlapping phases (> Fig. 85.1) [23]. The first phase, which is not always included, is hemostasis. After a wound has occurred, the body will attempt to stop bleeding by constricting vasculature, depositing platelets, and activating the clotting cascade. Endothelial cells normally line blood vessels, shielding platelets, and clotting factors from exposure to underlying collagen and basement membrane, and secrete inhibitors of platelet aggregation and clotting factors [24]. Once exposed to these normally hidden tissues, platelets are activated and aggregate via a combination of factors including ADP, von Willebrands factor (VWF), collagen, and thromboxane [25]. Following activation, they secrete cytosolic proteins and alpha-granules, which contain numerous mediators of clotting and inflammation including transforming growth factor (TGF)-b, TGF-a, platelet derived growth factor (PDGF), CD-40 ligand, and P-selectin [12, 25]. This leads to the release of fibrin, intracellular granules, and exposure of normally covered extracellular domains, all of which act as potent stimulators for inflammatory cells.
. Table 85.1 Summary of the changes occurring in human skin with age Clinical Atrophy
Histological Flattening of the dermal– epidermal junction
Drying
↑ Turnover time
Roughness
↓ Fibroblasts, mast cells, and macrophages
Alterations in pigmentation
↓ Collagen content
Sagging
Disorganized collagen and elastin
Wrinkling
↓ Microcirculation
Benign and malignant tumors
↓ Skin appendages ↓ Lymphatic drainage
↑, increased; ↓, decreased
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85
. Figure 85.1 The four overlapping phases of wound healing
The second phase of wound healing, the inflammatory phase, occurs from the time of injury through 1 week. This phase is dominated by inflammatory cells beginning with neutrophils which fight invading pathogens and degrade damaged tissues. Macrophages follow, removing debris and apoptotic cells, and coordinating the interaction of other cell types. Many studies have examined the inflammatory phase of wound healing in great detail. Early studies characterizing the inflammatory cell invasion show the sequential infiltration of neutrophils, macrophages, and lymphocytes in the healing wound [26]. Subsequent studies reveal that although the first leukocyte to arrive in the wound is the neutrophil, it is not a necessary component for wound healing to occur [27]. Unlike neutrophils, the macrophage is a critical mediator of tissue repair [28]. Macrophages are recruited to wounds within a few days of injury by various chemoattractants, including chemokines [29, 30]. Macrophages perform dual functions in the wound. Not only do they engulf and phagocytose wound debris, providing a clean bed for migrating proliferative cells to lay down new matrix and blood vessels; they also produce some of the angiogenic and fibrogenic growth factors that recruit and promote the cells involved in the growth phase of repair [31–33]. Finally, as neutrophil and macrophage populations decline in the wound, T lymphocytes become the dominant leukocyte in the later stages of inflammation [34]. It should be noted that the overall role of inflammation in wound healing is not completely understood.
Inflammation is not seen as necessary and in some cases may actually be detrimental in some forms of wound healing. Fetal wound healing, which is typically scarless through the first two trimesters, has a significant reduction in neutrophils and macrophages in both number and function [35]. Additionally, when inflammation is induced in fetal wounds the skin heals with scars [36]. Intestinal healing in the fetus also occurs in the absence of inflammation but still forms scars [37]. Inflammation has been shown to be entirely dispensable in some systems, such as the fetus, calling into question the true function of each cellular element. The proliferative phase is dominated by the replacement of missing tissues and occurs from shortly after injury through 3 weeks. Keratinocytes, fibroblasts, and endothelial cells proliferate and migrate in the wound laying down new collagen, ECM, blood vessels, and epithelial covering. The final stage of wound healing, remodeling, begins at 2 weeks. During remodeling, the excessive blood vessels initially laid down to support inflammation now regress toward normal vascular density, immature collagen is resorbed and replaced with mature collagen, and tissue is continually modified to approximate normal structure.
Age-Related Alterations in Hemostasis As stated previously, following injury, collagen is exposed and platelets adhere to the newly exposed collagen.
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During aging, platelet adherence to collagen is enhanced which may be one mechanism that facilitates activation of platelets in the elderly [38]. There is also an increase in the platelet release of alpha-granules containing TGF-a, TGF-b, and PDGF with age [39]. Not only are platelets affected by aging, but the clotting cascade is also altered by the aging process. Some of the changes in coagulation that are documented include elevated serum concentrations of activated clotting factors, fibrin breakdown products including d-dimers, and inhibitors of clot destroying enzymes like plasminogen activator inhibitor-1. There are also complimentary decreases in the activity of coagulation inhibitors such as antithrombin III and activated protein C [13, 40–42]. With this tendency toward increased coagulation comes an increase in some of the inflammatory mediators, but also a decrease in the some of the stimulants for chemotaxis [13, 43, 44]. This presents a mixed composition for inflammation, with some aspects increased and others decreased. These changes are summarized in > Table 85.2 below.
Age-Related Alterations in the Inflammatory Phase There are many conflicting studies regarding changes seen during the inflammatory phase of wound healing in the aged skin. There are reports of acute inflammatory reactions being slower and less intense in aging skin, equivalent in young and old skin, and faster and more intense in older skin [45–47]. Complicating matters, some aspects of wound healing appear to be enhanced while others are markedly decreased. There is plenty of evidence to suggest that neutrophil functions are impaired by aging, but their role in wound healing is controversial [48]. Neutrophils isolated from elderly humans have a decreased respiratory burst, diminished capability to phagocytose, and diminished . Table 85.2 Summary of age-related changes in hemostasis ↑ Platelet adherence to collagen
↓ Inhibitors of coagulation
↑ Platelet aggregation
↑ Inhibitors of clot lysis inhibitors
↑ Release of alpha-granules
↑ Concentrations of active clotting factors
↑ Concentrations of active clotting factors ↑, increased; ↓, decreased
chemotactic ability [49–51]. However, studies demonstrate no difference in wound debridement, cellularity, or connective tissue formation in the wounds of control and neutropenic animals [27]. Although neutrophils may play a role as a first line of defense against bacterial invasion, they do not appear to play a role in the proliferative phase of repair. More recent studies confirm that the role of neutrophils in uncontaminated wounds is probably minimal or even perhaps detrimental [52, 53]. The macrophage however, seems to be required for wound healing to occur normally and age-related changes in this cell type are likely to be important to healing outcomes [28]. In one series of experiments, the role of macrophages in young, middle aged, and elderly mice was deciphered. Middle aged and elderly mice showed a delay in wound closure compared to young mice. When young mice received intraperitoneal (ip) injections of rabbit antimacrophage serum, their healing was delayed, similar to that of untreated aged mice. Wound repair was accelerated in aged mice that received ip injections of macrophages harvested from young mice but not from old mice [54, 55]. There are many possible explanations for these effects including decreased numbers, diminished chemotaxis, or impaired cytokine production in the elderly macrophages. Whether macrophage populations are diminished with age is uncertain. Some studies suggest that hematopoetic stem cells do have a limited life span; there is a marked hypocellularity in the bone marrow of elderly humans; and CD68 positive cells (markers of macrophage populations) are decreased with age, but others demonstrate the opposite with increased macrophage population in the bone marrow and similar numbers and composition of macrophages in young and old mice [56–60]. Macrophage invasion into wounds is decreased in middle aged and elderly skin in some studies but not in others [8, 9]. The ability of macrophages to phagocytose is diminished with age. In one study, macrophages obtained from elderly mice ingested fewer particles than macrophages obtained from young mice [9]. Many other studies corroborate this general understanding that macrophage phagocytosis is impaired in aging skin [9, 61, 62]. The mechanisms to explain this decrease in macrophage function are not clear however. Glucose utilization in macrophages is decreased with age; several cell surface receptors required for macrophage activation and recognition are decreased including MHC class II. However, the Fcg receptor, which is critical for macrophage phagocytosis, does not show a decrease in number or function [9, 12, 63]. Several of the signal transduction pathways in macrophages obtained from aged individuals including
Impaired Wound Repair and Delayed Angiogenesis
the MAP kinases ERK, p38, and JNK are deleteriously affected and may partially explain the discrepancy [64]. The ability of the macrophage to coordinate the arrival and interaction of other cells participating in the wound repair process is also diminished with age. The production of interleukin(IL)-1 and IL-6 is decreased in the aging macrophage, along with the production of VEGF, and TNF-a [12, 15, 64, 65]. As is the case with much of the above information, conflicting reports showing increases in each of these cytokines are reported in the literature [12, 58, 64]. Despite the conflicting data, the following can be said of the aging macrophage with a fair degree of validity: phagocytic activity, cytokine (including TNF-a, VEGF, and FGF-2) and chemokine (including MIP-1a and CCL5) production, infiltration, and antigen presentation are all decreased [15, 58, 62, 63, 66]. As these are all vital components of the wound healing process, the age-related functional deficits displayed by macrophages probably contribute significantly to the healing impairment seen in aging skin. Many of these deficits involve cytokines that have influence beyond the inflammatory stage of wound healing and extend into the proliferative phase as well. These deficits during the proliferative phase lead to identifiable changes in wound healing and are summarized in > Table 85.3.
Age-Related Changes During the Proliferative Phase Many age-related alterations in wound healing are described. Clinical and laboratory studies describe delays in re-epithelialization, decreases in collagen synthesis, and organization in wounds of aged humans and rats [67–70]. Additional studies establish decreases in wound-breaking strength and increases in wound disruption [71, 72]. Results from excisional wound studies on mice continue to confirm prior reports describing age-related delays in reepithelialization [15, 69]. In one study (> Fig. 85.2), aged
. Table 85.3 Summary of age-related changes during the inflammatory phase of healing ↓ Macrophage function
↓ Neutrophil function
↑ Secretion of inflammatory mediators
↓ Vascular permeability
↓ Secretion of growth factors
↓ Infiltration of macrophages and lymphocytes
↑, increased; ↓, decreased
85
mice show a significant delay in terms of time to complete closure, as well as the portion of the wound bed reepithelialized [15]. This study correlates with other studies showing delayed epithelialization in aged humans and mice [12, 69, 73]. Many studies show that the proliferative capacity of keratinocytes decreases with age [74, 75]. Others show that there is a decline in the rate of normal keratinocyte turnover in aged skin [76]. Additional studies describe a decrease in keratinocyte migration in aged skin as well. Hypoxia is a potent stimulus for keratinocytes from young persons to migrate, but the opposite effect is seen in keratinocytes from aged individuals [77]. This decrease in hypoxia associated keratinocyte migration in aged skin is partially related to a decrease in MMP production. MMP-1 and MMP-9, which are both associated with keratinocyte migration in wounds, are upregulated in young keratinocytes but downregulated in aged keratinocytes [77, 78]. Combining these results with prior studies, there is an implication that there is a baseline decrease in the proliferation and migration of keratinocytes in aged skin. Once an injury occurs, the normal keratinocyte response to proliferate and to migrate across the wound is impaired. This impairment results in a delay of wound closure, increasing the chance for infection or chronic wound development. The mechanism behind this impairment is still not fully understood, but may be partially related to decreases in keratinocyte proliferation capacity,
. Figure 85.2 Representation of the time course of excisional wound re-epithelialization in young and aged mice. Young mice have smaller wound areas across all healing times and reach complete closure before aged mice (Swift et al. [15])
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and migration capacity, and may be mediated through age-related decreases in specific cytokine production. Not only are keratinocytes impaired by aging, but the major proliferative cell in the dermis, the fibroblast, displays significant age-related impairments as well. It is generally accepted that there is a decrease in number, size, and proliferation of fibroblasts in skin with age [12, 61, 79]. Some studies show a decrease in the in vitro life span of fibroblasts from aged human donors [80]. Others document a decrease in migration and proliferation of explanted rat fibroblasts with age [81]. Many studies evaluating the migratory deficiencies of aged fibroblasts demonstrate a decline in motility for fibroblasts independent of chemotactic stimulus and a decline in fibronectin associated migration [12, 82, 83]. These functional declines are due to multiple factors. Injection of senescent fibroblast mRNA into young fibroblasts impairs their ability to synthesize DNA [84]. Synthetic machinery is also decreased, with aged fibroblasts tending to have poorly developed endoplasmic reticulum [85]. Additionally, a great number of studies examine the decreased responsiveness of fibroblasts to a wide variety of cell signaling molecules and cytokines. Human fibroblasts have an age-associated decrease in mitogenic response to epidermal growth factor (EGF), insulin, dexamethsone, and transferrin [86]. They also show decreased responsiveness to FGF-7, also known as keratinocyte growth factor (KGF) [87]. Fibroblasts also require higher concentrations of PDGF to stimulate proliferation in aged individuals [12]. TGF-b1 responses are also found to be diminished in fibroblasts derived from aged individuals [88]. These studies suggest that fibroblasts may become desensitized to these stimuli with age. For some of these decreased responses, possible mechanisms have been elucidated. For instance, one study shows striking differences in EGF receptor (EGFR) number, affinity, and rate of EGF/EGFR internalization in earlypassage dermal fibroblasts derived from newborn versus young adult versus old adult donors, and another shows a decline in insulin receptor numbers in aged fibroblasts [89]. TGF-b receptor types I and II are decreased in hypoxic, but not normoxic conditions in aged fibroblasts from human donors, and downstream phosphorylation is also decreased [88]. Some of these receptors are not only important for proliferation and migration of fibroblasts, but are also involved in the production of fibroblast derived cytokines and fibrogenesis in fibroblasts. Fibroblast growth factor receptor (FGFR) expression is downregulated with age and the production of many FGFs are also found to be decreased. Some of these decreases include FGF-2, FGF-7, VEGF, and TGF-b1 [15, 90]. This reduction in synthetic ability of fibroblasts
is not limited to cytokines alone, as declines in fibronectin and collagen production are also described. As stated previously, conflicting studies exist regarding whether there is a change in collagen production with aging [12, 14–16]. Several studies show no age-related changes in collagen production [12, 15, 69, 91]. Studies that show an age associated increase in collagen production include increased production in cultures of fibroblasts from rats and pigs serially cultured to mimic aging and decreased type I collagen mRNA production from TGF-b stimulated human fibroblasts. Despite this conflicting information, many studies demonstrate a clear decrease in collagen production with age. Some of the studies showing this decrease in production include an increase in the ratio of immature type III collagen (with a decrease in the proportion of mature type I collagen) with age; reduced collagen production after age 30 in humans; delayed collagen content (but ultimately similar final levels) in aged mice; and reduced mRNA production of type I collagen with age [8, 15, 92, 93]. > Tables 85.4 and > 85.5 describe the age-related changes seen during the proliferative and remodeling phases of wound healing.
Age-Related Changes During Remodeling During the remodeling phase of healing, both collagen degradation and synthesis occur along with the . Table 85.4 Summary of age-related changes seen during the proliferative phase of healing ↓ Collagen deposition
↓ Proliferation of keratinocytes, fibroblasts
↓ Migration of keratinocytes, fibroblasts
! Re-epithelialization
↓ Receptor numbers and response ↑, increased; ↓, decreased; !, delayed
. Table 85.5 Summary of age-related changes seen during wound remodeling ! Wound strength
↑ Collagen degradation
↓ TIMP
↓ Wound strength
↓ Lysyl oxidase (LOX) crosslinking ↑, increased; ↓, decreased; !, delayed
Impaired Wound Repair and Delayed Angiogenesis
maturation of collagen structure, and the dermal architecture moves closer to the original normal structure. In aging, levels of collagen degradation in wounds appear to increase. The enzymes that are most active in collagen degradation are the matrix metalloproteinases (MMPs), a family of proteases that have various functions including, acting as intermediary signaling molecules, but are primarily thought to be active as proteolytic degradation enzymes. The MMPs are secreted by various cells including keratinocytes and fibroblasts, and collectively are known to degrade collagen, gelatin, and other ECM components [78]. Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs and their presence helps balance the degree of collagen synthesis and breakdown in wound healing. It should come as no surprise that debate exists on whether MMPs and TIMPs are increased or decreased with age and evidence exists in favor of each [12, 44, 61, 70, 77, 78, 94, 95]. However, the preponderance of evidence currently points toward increased MMP levels and decreased TIMP levels in aging skin. As new research continues to examine this process, this notion will likely evolve. Additionally, alterations in protease balance are likely not to be uniform and depending on the cell types, substrates, and wound conditions being examined, it is quite possible that divergent effects will be seen. It is possible that collagen degradation by MMPs may be enhanced while receptor mediated interactions with MMPs may be reduced. Divergent effects may also occur over early wound time points versus later wound time points as wound healing is incredibly dynamic requiring appropriate increases and decreases in each element at appropriate times. The end result of the remodeling phase is a durable dermis, one measure of which is the strength of the wound. Studies demonstrate a decrease in tensile strength in older individuals in a variety of settings including intestinal anastomoses, cutaneous wounds, and abdominal incisions [71, 72]. Tensile strength of wounds is not solely dependant on the amount of collagen present, but also relies upon the degree of crosslinking of the collagen fibers and the overall architecture. Collagen can be crosslinked by two mechanisms. In enzymatic crosslinking, collagen is crosslinked by a posttranslational modification via the enzymatic activity of lysyl oxidase (LOX). This enzyme crosslinks collagen in a specific pattern and maintains an association with the collagen fibrils preventing nonspecific crosslinking [96]. Enzymatic crosslinking occurs in normal and wounded skin and contributes to improved collagen architecture and tissue strength. The second method of collagen crosslinking is via a nonspecific chemical modification by crosslink oxidation or nonenzymatic glycosylation [97, 98].
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Both enzymatic and nonspecific crosslinking are described to be altered with aging. In regard to enzymatic crosslinking, alterations in LOX activity are described with aging. An age associated decrease in LOX activity in skin from elderly monkeys has been described [99]. LOX mRNA levels were also found to be decreased in skin from aged rats in several studies [100, 101]. Despite this described decrease in LOX activity, increases in collagen insolubility were reported with higher intra and intermolecular crosslinking in older subjects [96, 101, 102]. This apparent disparity between decreased LOX activity and increased collagen crosslinking may actually go hand in hand. As previously stated, while LOX does crosslink collagen, it also protects it from nonspecific crosslinking. Some studies show that the specific LOX derived crosslinks decrease with age and the nonspecific chemical and glycosylation crosslinks increase [96]. This increase in nonspecific collagen crosslinks may explain some of the physical changes described in elderly skin including coarse collagen structure, stiffer ECM, and decreased chemotaxis of inflammatory and proliferative cells such as endothelial cells.
Age-Related Changes in Angiogenesis While some controversy exists as to whether wound angiogenesis is increased or decreased with age, the preponderance of evidence points toward an overall decrease in angiogenesis in aging skin [8, 13, 103, 104]. Many of the previously described changes in cytokines, proliferation, and structural proteins also affect angiogenesis. Studies on both excisional wounds and subcutaneous implant models in aged animals show a delay in wound capillary ingrowth [105, 106]. This delay may be a function of impaired migration because of the altered collagen crosslinking mentioned above, cellular senescence of endothelial cells, or may be due to decreases in growth factor expression. Many studies have delineated the perturbations caused by altered levels of growth factors. VEGF, PDGF, TGF-b1, and FGF have all been found to impact angiogenesis through their influence on endothelial cell functions and age associated impairments of these growth factors can have profound effects on wound angiogenesis [15, 107–109]. Senescent HUVEC cells fail to migrate in response to FGF and the phosphorylation of FGF receptor1 substrates is impaired [107]. A decrease in capillary density, along with a decrease in the production of the pro-angiogenic stimuli FGF-2 and VEGF has been demonstrated in excisional wounds from aged mice. In addition, the in vivo response to a defined level of proangiogenic stimulus, implanted subcutaneously, decreases in aged animals [15]. Other studies demonstrate similar
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impairments including decreased perfusion and capillary density with decreased VEGF levels in aged animals and a concomitant increase in subsequent vascular density with recombinant VEGF supplementation [110]. Other studies show a reduction in sprouting of vessels in aged mice with a rise to levels approaching that of young mice when supplemented with IGF-1, VEGF, TGF-b1, or bFGF [111]. An increase in anti-angiogenic factors, such as members of the thrombospondin (TSP) family, is found in wounds of aged animals, and may provide a second layer of inhibition on endothelial functions beyond the simple deficiency of stimulatory growth factors. TSP is known to inhibit neovascularization through a variety of mechanisms including limiting vessel density, inducing apoptosis in endothelial cells, and decreasing the response of endothelial cells to various stimulatory signals including VEGF [112]. In one study, TSP-2 expression was increased in fibroblasts from the wounds of aged mice; TSP knockout mice have increased angiogenesis compared to wild-type counterparts [7]. Several studies describe an increase in TSP levels with age [7, 13, 113]. SPARC (also termed osteonectin), is a multifunctional glycoprotein that modulates cellular-ECM interaction, inhibits cellular proliferation, and regulates the activity of growth factors. SPARC can bind directly to VEGF, inhibit VEGF-receptor interaction, and prevent VEGFinduced phosphorylation of VEGF receptor-1 [114]. SPARC has been shown to inhibit the proliferative and migratory effects of FGF and VEGF on endothelial cells [115, 116]. SPARC expression increases with age in several models both in vivo and in vitro [116, 117]. Specifically, SPARC increases with age in human periodontal ligament cells’ in murine wounds using a sponge implant model, and in fibroblasts and endothelial cells obtained from the skin of young and old human donors. This age-related increase in SPARC could contribute to the decrease in VEGF and angiogenesis that is observed in aging. The regulation of SPARC itself is not completely understood, although some studies show that TGF-b1 and PDGF increase the expression of SPARC, while bFGF decreases the expression of SPARC [118, 119]. While there are probably many reasons for the decrease in VEGF expression and endothelial cell migration in aging skin and wounds, the increase in inhibitors of angiogenesis like TSP-1 and SPARC provide additional means of inhibition, beyond mere cellular senescence in wounds from aged individuals. Regardless of the underlying mechanism, endothelial function and angiogenesis is impaired in aging skin (> Table 85.6). Whether due to an age-related increase in angiogenic inhibitors; a decrease in growth factors like
. Table 85.6 Summary of age-related changes seen in angiogenesis ! Capillary ingrowth
↓ Migration and proliferation of endothelial cells
↑ Inhibitors of angiogenesis
↓ Angiogenic cytokines
! and ↓ Vascular density ↑, increased; ↓, decreased; !, delayed
VEGF, EGF, or FGF, a decrease in endothelial migration and proliferation, or an impairment of downstream receptor signaling, the end result is that angiogenesis in wounds from aged individuals is impaired. A significant decrease in angiogenesis can often negatively impact healing. However, the influence of the age-related angiogenic impairment on the healing capacity of any particular wound probably varies due to individual wound conditions.
Conclusion Even though angiogenesis and wound healing may be delayed in an aged individual, if the person is otherwise healthy, and the wound is in optimal condition, no functional detriment might be observed. The wound will close, no infection will set in, and the individual will be none the wiser that it took a few extra hours to close, or that there were fewer blood vessels, or that the strength of the skin across the wound will take an extra few weeks to achieve maximal strength. The real impact of the impairment in wound healing and angiogenesis is not on the healthy aged individual, but on the aged individual with baseline impairments: a lower extremity laceration on an elderly woman with peripheral vascular disease, or a man with diabetes, for example. For these individuals who already have a baseline deficiency in the maximal potential for wound healing, a slight reduction in angiogenesis or a slight delay in wound closure, cytokines, or growth factor activity could mean the difference between an infection and a clean wound, a chronic ulcer or a healthy scar.
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Part 4
Toxicological/ Safety and General Considerations
Safety Evaluation for the Elderly Population
79 Irritant Contact Dermatitis Florian Seyfarth . Peter Elsner
Introduction Both irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD) are the results of the exposure to external agents, which cause inflammation of the skin. Both show a similar histopathological and immunohistochemical pattern [1]. The clinical picture of ICD differs in dependence from the duration of disease. Acute forms feature erythema, edema, vesicles (in part coalescing), bullae, oozing, and, in severe cases, after contact to corrosive substances, necrosis and/or ulceration. Chronic forms are characterized by erythema, lichenification, excoriations, scaling, and hyperkeratosis. In acute ICD, intraepidermally located vesicles or bullae dominate the histological picture, surrounded by a variably pronounced spongiosis. An inflammatory infiltrate may be present, consisting of mononucleated cells. Parakeratosis of the stratum corneum is sometimes described. The upper dermis is characterized by edematous alterations. In contrast, chronic ICD shows acanthosis, marked hyper- and parakeratosis, and slight spongiosis. Vesicles are absent. The inflammatory infiltrate generally has a perivascular distribution in the upper dermis [2]. Since morphological criteria are insufficient for differentiation of ICD from ACD, one has to rely on anamnestic and exposure data, patch test results, and the location of the dermatitis. ICD is often found on the hands; in older incontinent patients, it can also be found in the perineal region.
Pathophysiology of ICD in Aged Skin For a long time, the pathophysiology of the irritant contact dermatitis (ICD) was considered to be very different from allergic contact dermatitis. While ACD is associated with hapten-specific lymphocytes, ICD was explained as a completely nonimmunological reaction to exogenous irritants [3]. Utilizing the scientific progress of the last few years, immune response to irritants was investigated, which led to a more complex view on ICD [4]. Exposure to irritants causes an unspecific impairment
of keratinocytes and leads to expression of integrin receptors and the intracellular adhesion molecule 1 (ICAM-1) [3, 4]. Consequently, production of proinflammatory cytokines (especially interleukin-6, interleukin-8, interleukin-2, tumor necrosis factor a, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin 1b becomes enhanced [5, 6]. Interestingly, the same cytokines, which are also found in allergic contact dermatitis, are detected in ICD. Additionally, activation of CCL21, a chemokine of lymphatic tissues, was described [4]. Irritation and the activation of the inflammation cascade are linked to the damage of the stratum corneum lipid barrier, which is associated with loss of cohesion of corneocytes and desquamation causing an increased transepidermal water loss (TEWL) [7]. Thus, TEWL is used as a marker for irritation and epidermal barrier disruption. The basal TEWL decreases with age, suggesting a lower irritability of aged skin. In this context, Thune et al. showed a TEWL decrease in aged patients suffering from dry skin (median age: 65 years) compared with a younger control group (median age: 29 years), while the hydration showed no significant differences between the groups [8]. This was confirmed by Cua et al. [9], who found a lower TEWL increase and attenuated patch test results (‘‘visual score’’) after irritation with SLS in elderly subjects. Schnichels and Elsner performed an acute irritation by patch testing with SLS in an older (50–70 years) and a younger (20–40 years) age group. The baseline TEWL level before exposure was generally lower in the old age group. After acute irritation, twice as many older persons showed no erythema compared with the young group, which presented more intense clinical signs and a more increased TEWL after irritation [7]. However, when irritation by SLS is terminated, TEWL decreases faster to normal values in younger persons, suggesting faster barrier recovery in this age group. In another study, Elsner et al. [10] compared the reactivity of premenopausal women with postmenopausal women by SLS irritation on the forearm. The older group showed a slower and less intense reaction than the younger women (visual score, TEWL).
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_79, # Springer-Verlag Berlin Heidelberg 2010
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Coenraads et al. [11] found less susceptibility in aged people for croton oil, but not for thymoquinone and crotonaldehyde. An age-dependence of irritation is only to be seen if very young people are compared with very old people. Within the subgroup of adult individuals younger than 50, there is no relation between age and severity of irritation [12, 13]. On the other hand, children are more susceptible to irritation. For example, there is more irritation after exposure to DMSO and SLS in children [9], while older people show a milder, but persistent skin reaction [14]. The most studies presented here draw their conclusions from SLS exposure and determination of the TEWL, which may be a point of criticism. On the one side, it is difficult to determine if the study results are transferable to other irritants than SLS, on the other side, some authors consider that TEWL decrease in aged skin does not reflect less irritability. Thune et al. [8] attributed this effect to a generally decreased hydration of aged skin, possibly due to decreased ceramide levels [7]. Other studies could not affirm decreased water content due to age [9, 15–18]. Ghadially et al. interpreted the decreased TEWL in the older skin as a result of reduced sweating rates, decreased microcirculation, and decreased temperature in old subjects [19]. Starting from the observation of decreased permeability of lipophilic drugs in aged skin, the authors emphasized a decrease of the epidermal lipid content in old people. This hypothesis is supported by electron microscopic analysis, which revealed a decreased number of lamellar bodies in the stratum granulosum – stratum corneum interface, as well as a reduced lipid content in a mouse model. Additionally, tape stripping experiments resulted in a faster occurring TEWL increase in old subjects compared with young ones. Finally, the time which was needed for normalization of TEWL after acetone exposure was longer in older test persons. The authors conclude that ‘‘the aged epidermal permeability barrier is both easier to perturb and slower to repair’’ [19]. Investigations of recent years provided some information about the influence of age on regulation of the epidermal barrier after disruption (> Fig. 79.1) [20]. Different biochemical pathways have been elucidated, which are enhanced after barrier disruption and affect its stabilization. The epidermal barrier is represented mainly by the hydrophobic stratum corneum lipid structures (very-long-chain saturated fatty acids, cholesterol, and ceramides in equimolar proportion) and the upper layer of nucleated keratinocytes. Lipid synthesis and the proliferation of keratinocytes are enhanced after activation of
the interleukin-1a cascade due to barrier disruption, which is accompanied by an increase of interleukin-1 receptor type 2 (T2 IL-1 R) and the interleukin-1 receptor antagonist (IL-1 ra) [21]. Interestingly, concentrations of interleukin-1a, T2 interleukin-1 R, and interleukin-1 ra in old mice show a reduced increase after irritation, compared with young mice. Moreover, aged interleukin-1a knock-out mice show a notably impaired barrier reconstitution compared with the wild type [21]. Another important molecule more expressed after barrier disruption is the transcription factor SREBP2 (sterol regulatory element-binding protein 2), which induces expression of b-hydroxy-b methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl coenzyme A carboxylase. Both enzymes play a key role in the synthesis of longchain fatty acids and cholesterol, and thus, in barrier recovery. The expression of the enzyme serine palmitoyl transferase is directly enhanced by barrier disruption and facilitates synthesis of ceramides, which – for their part – stimulate proliferation of keratinocytes. In aged skin, there are lower levels of all three enzymes for lipid synthesis to be found, supporting the thesis of a reduced barrier recovery in age. Next to cytokines and lipid synthesis, nerve growth factor (NGF) and epidermal growth factors (EGF) are upregulated after disruption. The EGF subtype amphiregulin shows elevated levels especially in aged skin [20].
Irritants Studies in occupational dermatology emphasize wet work as a crucial influencing factor on ICD. Wet work thereby is characterized by a repetitive exposure to water and detergents, especially under occlusive conditions. Hereunder, water increases erythema, pH, the cutaneous blood flow, TEWL, and the permeability to low-molecular-weight irritants [22]. Additionally, the frictional coefficient as a predictor of skin vulnerability also increases. Consecutively, there is a higher risk for pressure ulcers and increased susceptibility for bacterial infection [22]. Important chemical irritants, arranged in order of frequency, are detergents (e.g., soap), solvents, oil, dusts and fibers, and, last but not least, acids and alkalis. Important physical irritants are heat, sweating, friction, pressure, vibration, UV irradiation, and occlusion [4]. Occlusion increases the signs of inflammation, pH, TEWL, stratum corneum hydration, skin surface temperature, and skin permeability [22]. Furthermore, lipid organization and
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. Figure 79.1 The influence of age on epidermal barrier recovery Elias PR, Ghadially R [20]. IL-1 ra: IL-1 receptor antagonist; SREBP2: sterol regulatory element-binding protein 2; NGF: nerve growth factor; EGF: epidermal growth factor
metabolism, as well as cellular function (e.g., DNA synthesis, mitosis) are inhibited [22]. Other common exogenous factors on irritation are the temperature of detergents [23] and climatic conditions, especially dry air [3, 4].
Predisposing Factors for ICD ICD is not only dependent on the irritant, but there is also a high number of endogenous factors predisposing for ICD, such as concomitant atopic eczema [4, 24–28] or any other concomitant dermatitis [29]. Although experimental studies concerning irritability in atopic and non-atopic skin did not support this hypothesis unanimously [30, 31], determination of an atypical filaggrin gene as a marker of atopy is also assumed as predictor for ICD [32]. The protein filaggrin is involved in the formation of the
skin barrier. Independent from atopic diathesis, tumor necrosis factor a polymorphism was recommended as a genetic marker for ICD [4]. Other factors are structural properties of the skin, a deficient hardening phenomenon of the skin, and an increased sensitivity to UV irradiation [3], especially in subjects with skin type I [33]. Concerning sex, the influence on irritation is ambiguous [9]. While experimental studies did not support a correlation between sex and irritability [34, 35], epidemiological studies emphasize a higher incidence of ICD in women [25, 36, 37]. Additionally, skin irritation depends on the body area exposed to the irritant. The most sensitive areas are the face, the upper back, and the retroauricular as well as the genital skin [3]. Some authors showed a higher sensitivity in the face compared to the back. Within the face, chin and nasolabial folds are the most vulnerable regions [4]. Several investigations with ammoniumhydroxide [38], dimethylsulfoxide (DMSO), and SLS [9] showed a higher
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sensitivity at the forearms and hands compared to the legs and feet.
Clinical Types of ICD The term irritant contact dermatitis includes various subtypes, each one with its own age distribution [39]. The most common type is cumulative irritant contact dermatitis [4], which is important in occupational dermatology and is found mainly in young adults [40]. The disease is caused by repetitive irritation of the skin over years with mild symptoms, which are often neglected by the patient. Thus, irritation continues unnoticed, until a threshold is reached and a severe ICD develops [3]. The clinical picture is characterized by dryness, erythema, lichenification, and hyperkeratosis (‘‘xerotic dermatitis’’). The prognosis is poor. Meding et al. found that after 15 years follow-up, 44% of patients still had hand eczema within the past year [41]. Veien et al. confirm these findings reporting a persistent or intermittent hand eczema 5 years after initial diagnose in 65% of all cases [42]. For persistent ICD in spite of changing the work place, the term post-occupational dermatitis was introduced [43]. The so-called non-erythematous ICD, which shows changes in skin-physiological parameters without any visible inflammation, and the irritant reaction can be regarded as a pre-stage of cumulative ICD. Non-erythematous ICD is also seen after exposure to cocoamidopropyl betaine, coconut dieethanolamide, etc. [44]. The irritant reaction displays only one or few clinical signs, for example, dryness, scaling, redness, vesicles, pustules, and erosions. These reactions mostly appear after a period of intense contact to water, especially in young professionals. Their ability for an intrinsic skin hardening determines whether the irritant reaction disappears or a cumulative ICD develops. Acute ICD develops within minutes or hours after accidental contact to potent irritants. Thereafter, the symptoms such as burning, itching, algesia, and formation of erythema, edema, bullae, or necrosis rise quickly. After the symptoms have exceeded a maximum and the irritant stimuli were eradicated, the process of healing starts and is marked by a good prognosis. The clinical course of delayed acute ICD is different from acute ICD, whereas the clinical picture is identical. Inflammation becomes visible not before 8–24 h after contact to the irritants (e.g., anthralin: dithranol, benzalkonium chloride, and tretinoin).
Traumatic ICD develops after traumata like burns or severe acute ICD and displays erythema, vesicles/papulovesicles, and scaling instead of healing of the trauma. It resembles nummular dermatitis [45]. Another type of ICD is pustular dermatitis due to contact to metals, tars, oils, chlorinated agents, and naphthalene. Sensory irritation, characterized by stinging, burning, tightness, itching, or painful sensations occurring immediately or delayed after contact to mainly cosmetic products, seems to be a problem mostly in middle-aged, Caucasian and Asian women [46]. In contrast, aged people seldom show this form of ICD, which is addressed to the lower content of nerve fibers in aged skin [47]. Asteatotic irritant dermatitis (synonyms: exsiccation eczematoid, winter eczema, or eczema craquelae, > Fig. 79.2) is found mainly in older patients [48] with a maximum in the winter depending on a decreased air moisture [49]. Another contributing factor is misbehavior in personal care (frequent bathing/showering, extensive usage of soaps, and cleansing products), which is assumed to be a common problem in older people [50]. Intensive care practices learned in the youth are missed to become adapted to the aged skin with its special properties (decreased stratum corneum hydration, decreased levels of stratum corneum lipids, and ceramides) [51–53]. Patients . Figure 79.2 Exsiccation eczema (courtesy: Department of Dermatology, Jena)
Irritant Contact Dermatitis
with asteatotic irritant dermatitis show dry skin with ichthyosiform scaling and fissuring, especially on the extremities. TEWL and pH are increased [8], and keratinosomes are disturbed [54]. The most compromising symptom is intensive itching, wherefore this disease has to be considered as differential diagnose in patients with pruritus senilis (see > Table 79.1). Interestingly, xerosis cutis induced by HMG-CoA reductase inhibitors mimics asteatotic irritant dermatitis [55]. For treatment of ICD, avoiding irritants, topical corticosteroids, moisturizers, rich oil-based creams, cold compresses and cold water for washing, UV radiation, and training programs are recommended [3].
Perineal and Genital ICD in the Aged Skin While occupational cumulative irritant contact dermatitis is especially a problem in younger people [40], older persons show another distribution of clinical subtypes of ICD. The perineal or ‘‘incontinence’’ dermatitis is typical for older individuals. In the USA, 48.4% of the women older than 50 years suffer from urinary incontinence, 15.2% from fecal incontinence, and 9.4% from both [22]. The clinical picture is characterized by an initially mild and sometimes pruritic erythema, which becomes complicated by the development of small vesicles and erosions, with a tendency to superinfection (Staphylococcus aureus, candidiasis, tinea). In severe cases, pressure ulcers develop. Depending on the type of incontinence, the disease begins in the perianal (fecal incontinence) or vulvar region (urine incontinence) [22], which is more irritable and permeable than skin from body areas [56]. Perineal ICD is related to typical irritants, primarily to urine or stool as chemical irritants. . Table 79.1 List of differential diagnosis of generalized pruritus Elewski BE et al. [74] Pruritus Xerosis cutis/ asteatotic ICD
Malign tumors (lymphomas/ leukemia, etc.)
Diabetes mellitus
Polycythemia
Hepatic and biliary diseases
Hypothyreosis, hyperthyreosis
Nephrological diseases
Dermatitis herpetiformis
Iron deficiency
Psychogeneous
Drugs
Parasite infestation
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Urine, as cause of a humid environment under occlusive conditions in the perineal region, is an equivalent to wet work in the younger patient, who has to wear gloves permanently on his hands, which provokes ICD of the hands. Additionally, urine is characterized by a high amount of ammonia, which causes a skin pH increase to 8. Thus, synthesis of lipid barrier components and keratinization is inhibited and repair functions of the skin are impaired [22]. Next to urine, there is a high amount of other potential vulvar irritants like sweat, secretions, cosmetics, disinfectants, vaginal douches (especially in younger women [57]), lubricants, spermicides, antifungal creams and other topical medicaments, depilatory cream, and semen [58]. Physical irritants might be sanitary pads, tampon strings, tight clothing, synthetic underwear, toilet paper, overzealous cleansing, scrubbing, shaving, plucking hair, and prolonged sitting [58]. Occlusion caused by the use of diapers is an essential contributing factor in perineal ICD [22], especially in older individuals, and also in neonates. Vulvar ICD is generally the most frequent vulvar disease with a high impact on genital pruritus [58]. In contrast, genital allergic contact dermatitis against ‘‘applied medicaments, contraceptives, lubricants, or feminine hygiene deodorant spray’’ seems to be less frequent [59]. Relevant irritants in feces are lipases and proteases. Andersen et al. performed occlusive irritation tests with fecal enzymes and different bile mixtures on dorsal skin of individuals aged between 21 and 66 years. The influence on visual score, TEWL, and pH was investigated. After 21 days, ‘‘severe skin erythema and epidermal barrier disruption’’ were observed [60]. Furthermore, specific endogenous factors in aged people play an important role in pathogenesis of perineal ICD, for example, lower immune function, inadequate care, impaired cognition, and less mobility [22]. Farage and Maibach reviewed characteristics of vulvar skin in aged women [61]. A decrease of vaginal secretion, a reduction of lubrication, and a higher susceptibility to infective agents, for example, enteric organisms was concluded. Contrary to the statement that aged vulvar skin ‘‘is intrinsically less hydrated, less elastic, more permeable, and more susceptible to irritation’’ [61], no significant differences accrued from skin-physiological studies (water barrier function, friction coefficient) of vulvar skin from premenopausal and postmenopausal women [17]. On the other hand, 47% of postmenopausal women suffer from vaginal dryness [62], which is interpreted as manifestation of atrophic vulvovaginitis [63]. This disease is characterized by dryness, itching, and dyspareunia, and depends on low estrogen levels, causing vaginal atrophy, increase of pH, and enforcement of susceptibility to microbial pathogens [63].
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Compared with senile female perineal and genital ICD, this genital ICD is rarely investigated in elderly men. In Fritsch’s Textbook of Dermatology [64], the term ‘‘Reinlichkeitsbalanitis’’ (balanitis of neatness) is used and stated to be coincident with high age. Birley et al. report irritant balanitis according to excessive washing as most frequent subtype in nonspecific balanitis [65], while other authors assume an infectious balanitis as most important [66].
Occupational Dermatology in Aged Employees The knowledge about the influence of age on occupational hand dermatitis is limited, since generally few epidemiological data are available concerning occupational dermatology [67]. Thus, the influence of age may be underestimated, which is problematic, especially within the scope of an aging society and an increase of aged workers. However, age-adapted training programs on the basis of secondary and tertiary prevention of occupational ICD are still missing. In general, incidences of hand ICD and atopic hand dermatitis are assumed to be higher than allergic contact dermatitis, whereas nonoccupational hand dermatitis is more frequent than occupational ICD [25, 36, 68]. In 1990, Meding estimated a point prevalence of 5.4% for hand eczema (hereof 35% ICD, 19% ACD, 22% atopic eczema). Risk factors were history of childhood eczema, female sex, occupational exposure, a history of asthma and/or hayfever, and a service occupation (multiple logistic regression analysis) [25]. However, the age as influencing factor was not considered. The few epidemiological data concerning age are ambiguous. Soder et al. detected a decrease of quality of life, especially in older patients with occupational hand dermatitis [69]. While older works point out a positive influence of age on development of occupational ICD [70, 71], Diepgen and Kanerva emphasize that neither sex nor age are risk factors for the manifestation of this disease [67]. Thus, conditions at the workplace, atopic diathesis, and xerosis cutis are the leading risk factors [67]. Another study by Diepgen and Coenraads deals with the mean age of employees at the beginning of their occupational skin diseases [40]. According to these data, beginning of occupational dermatitis is associated with an early age (haircutter: 19 years, food worker: 22 years, medical personal: 24 years, metal worker: 33 years) [40]. In contrast, the mean age of construction
and cement workers with occupational ICD is relatively high (39 years) [72, 73].
Conclusion Irritant contact dermatitis is an underestimated problem in aged people. Epidemiological data are scarce. In spite of this, immunological studies have contributed to the preliminary understanding of the pathophysiological processes in ICD of the aged. The clinical presentation of ICD in older people is more discrete than in younger, with the TEWL increase being less pronounced. On the other hand, older skin regenerates more slowly. Although occupational hand dermatitis manifests itself mainly in young professionals, the requirements of older patients have to be studied more intensively, considering the increasing percentage of aged employees due to the demographic change. The demographic change will also cause an increased need for sufficient geriatric care, which has to regard the irritant potential of personal hygiene products especially in aged people. Thereby, perineal ICD claims specific attention, since it has to be considered as preliminary stage of decubital ulcers.
Cross-references > Bioengineering > Cutaneous
Methods and Skin Aging Effects and Sensitive Skin with Incontinence
in the Aged > Susceptibility
to Irritation in the Elderly: New
Techniques
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45. Lammintausta K, Maibach H. In: Adams RM (ed) Occupational Skin Disease. Philadelphia: W.B. Saunders, 1990, pp. 1–15. 46. Farage MA, Katsarou A, Maibach HI. Sensory, clinical and physiological factors in sensitive skin: a review. Contact Dermatitis. 2006;55:1–14. 47. Besne I, Descombes C, Breton L. Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. 2002;138:1445–1450. 48. Simon M, Bernard D, Minondo AM, Camus C, et al. Persistence of both peripheral and non-peripheral corneodesmosomes in the upper stratum corneum of winter xerosis skin versus only peripheral in normal skin. J Invest Dermatol. 2001;116:23–30. 49. Melnik B, Braun-Falco O. The value of oil baths for adjuvant basic therapy of inflammatory dermatoses with dry, barrier-disrupted skin. Hautarzt. 1996;47:665–672. 50. Effendy I, Kerscher M. Haut und Alter. Stuttgart/New York: Thieme, 2005. 51. Raab WP. The skin surface and stratum corneum. Br J Dermatol. 1990;122(35):37–41. 52. Rogers J, Harding C, Mayo A, Banks J, Rawlings A. Stratum corneum lipids: the effect of ageing and the seasons. Arch Dermatol Res. 1996;288:765–770. 53. Jin K, Higaki Y, Takagi Y, Higuchi K, et al. Analysis of betaglucocerebrosidase and ceramidase activities in atopic and aged dry skin. Acta Derm Venereol. 1994;74:337–340. 54. Tezuka T. Electron-microscopic changes in xerosis senilis epidermis. Its abnormal membrane-coating granule formation. Dermatologica. 1983;166:57–61. 55. Krasovec M, Elsner P, Burg G. Generalized eczematous skin rash possibly due to HMG-coa reductase inhibitors. Dermatology. 1993;186:248–252. 56. Elsner P, Maibach HI. The effect of prolonged drying on transepidermal water loss, capacitance and ph of human vulvar and forearm skin. Acta Derm Venereol. 1990;70:105–109. 57. Brotman RM, Klebanoff MA, Nansel T, Zhang J, et al. Why do women douche? A longitudinal study with two analytic approaches. Ann Epidemiol. 2008;18:65–73. 58. Welsh B, Howard A, Cook K. Vulval itch. Aust Fam Physician. 2004;33:505–510. 59. Buechner SA. Common skin disorders of the penis. BJU Int. 2002;90:498–506.
60. Andersen PH, Bucher AP, Saeed I, Lee PC, et al. Faecal enzymes: in vivo human skin irritation. Contact Dermatitis. 1994; 30:152–158. 61. Farage M, Maibach H. Lifetime changes in the vulva and vagina. Arch Gynecol Obstet. 2006;273:195–202. 62. Dennerstein L, Dudley EC, Hopper JL, Guthrie JR, Burger HG. A prospective population-based study of menopausal symptoms. Obstet Gynecol. 2000;96:351–358. 63. Van Voorhis BJ. Genitourinary symptoms in the menopausal transition. Am J Med. 2005;118(12B):47–53. 64. Fritsch P. Dermatologie, Venerologie. Heidelberg: Springer, 2004. 65. Birley HD, Walker MM, Luzzi GA, Bell R, et al. Clinical features and management of recurrent balanitis; association with atopy and genital washing. Genitourin Med. 1993;69:400–403. 66. Edwards S. Balanitis and balanoposthitis: a review. Genitourin Med. 1996;72:155–159. 67. Diepgen TL, Kanerva L. Occupational skin diseases. Eur J Dermatol. 2006;16:324–330. 68. Ku¨hner-Piplack B. [Klinik und Differentialdiagnose des Handekzems. Eine retrospektive Studie am Krankengut der Universita¨tshautklinik Heidelberg 1982–1985.] Thesis. Universita¨t Heidelberg, Heidelberg, 1987. 69. Soder S, Diepgen TL, Radulescu M, Apfelbacher CJ, et al. Occupational skin diseases in cleaning and kitchen employees: course and quality of life after measures of secondary individual prevention. J Dtsch Dermatol Ges. 2007;5:670–676. 70. Coenraads PJ, Nater JP, van der Lende R. Prevalence of eczema and other dermatoses of the hands and arms in the Netherlands. Association with age and occupation. Clin Exp Dermatol. 1983; 8:495–503. 71. Varigos GA, Dunt DR. Occupational dermatitis. An epidemiological study in the rubber and cement industries. Contact Dermatitis. 1981;7:105–110. 72. Bock M, Schmidt A, Bruckner T, Diepgen TL. Occupational skin disease in the construction industry. Br J Dermatol. 2003;149: 1165–1171. 73. Conde-Salazar L, Guimaraens D, Villegas C, Romero A, Gonzalez MA. Occupational allergic contact dermatitis in construction workers. Contact Dermatitis. 1995;33:226–230. 74. Elewski BE, Hughey LC, Parsons ME. [Dermatologische Differentialdiagnose]. Mu¨nchen: Elsevier Urban & Fischer Verlag, 2007.
81 Safety Evaluation in the Elderly via Dermatological Exposure Mario Bramante
Introduction The chapter discusses the relevance of advanced age in the human safety risk assessment of substances that may come in contact with the skin through use of consumer products for personal use. Susceptibility of the elderly to chemical toxicity is reviewed in the context of current risk assessment practices for the general population, and default assumptions for human variability. Consumer products for personal use, ranging from solid manufactured items such as absorbent hygiene devices to cosmetics, may contain a broad range of substances that are not formulated to penetrate inside the human body to have any active function beyond the skin, nor to have a pharmacological action. Avoidance of hazardous constituents is either enforced by regulations (e.g., for cosmetics) or a basic expectation, in recognition of the close contact of these products with the consumer, and their wide accessibility by the general population. Because of these characteristics, consumer products tend to have a low potential for toxicity, confirmation of good skin compatibility being often the key focus of their safety evaluations The proportion of elderly people in the general population continues to grow rapidly [1] driven by a steep increase in life expectancy. This trend has heightened the interest of researchers, risk assessors, and safety decision makers in better understanding age-related changes, and their potential impact on chemical toxicity. Most of the knowledge on elderly-related physiological changes is derived from medical research on pharmaceutical agents, driven by the need to optimize drug dosage regimens, to account for concomitant illnesses and co-medications that are frequent in the aged patient. Already in 1989, guidelines for the study of drugs to be used by the elderly were published by the U.S. Food and Drug Administration, and today’s international guidelines on drug development strongly recommend inclusion of older adults in clinical trials [2]. Beyond pharmaceuticals, regulatory guidelines for risk assessment of chemicals, stress the importance to
identify sensitive subpopulations and characterize their specific risk, age being recognized an important variable [3, 4]. In 2002, the U.S. Environmental Protection Agency announced a coordinated effort to study and prioritize environmental health threats to the elderly [5]. Physiological changes associated with aging can alter the processes of absorption, distribution, metabolism, and elimination of substances [6], and the aged skin progressively looses structural integrity and physiological function [7]. The significance of these changes for risk assessment is discussed in the following paragraphs.
Elderly Skin Susceptibility to Irritation A number of skin structures decline with age with a progressive reduction in skin thickness, dermal vascularization, number of hair follicles, epidermal turnover rate, and sensory nerve endings, the latter associated with an increase in pain threshold [8]. Alterations in collagen and elastin organization produce a less stretchable and resilient dermis with a reduced resistance to shearing forces [9]. As aged skin tends to be drier and easier to crack due to loss of elasticity, mechanical damage is more likely to occur. A diminished repair capacity prolongs wound healing, general skin recovery from damage, and restoration of skin barrier [10]. Despite the thin and fragile appearance of older skin, the inflammatory response is delayed and less intense [11], and older subjects react to skin irritants less sharply and more slowly than younger individuals [12, 13]. The reduction in basal trans epidermal water loss (TEWL) observed in the elderly indicates that skin barrier is not compromised, and is indicative of a decreased susceptibility to irritants with advancing age [14]. Several studies based on in vivo human test methods could be quoted in support of a decreased sensitivity of aged skin to irritants. A human 4-h patch test, developed as a valid and predictable alternative to animal testing [15], was used in a series of investigations on
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_81, # Springer-Verlag Berlin Heidelberg 2010
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population differences in acute skin irritation response to common chemical irritants [13]. These investigations demonstrated a significantly reduced irritation in the oldest 56–74 age cluster, with only directional reduction for weaker irritants. Milder reactions with advancing age were also reported in clinical studies performed with a variety of substances like dimethyl sulfoxide, histamine, ethyl nicotinate, chloroform-methanol, and lactic acid [16], with fewer studies reporting no significant differences. Compared to younger individuals, irritant response to sodium lauryl sulphate (SLS), evaluated via TEWL and visual scores, decreases in the elderly on various body sites [12]. For example, TEWL value of upper arm and abdomen was three to four times lower in the aged. The forearm skin of premenopausal women also produced more erythema when exposed to 0.1% SLS solution as compared to postmenopausal women [17]. Overall, the body of evidence supports that aged skin is generally less susceptible to irritant insults: test methods and irritation risk assessment practices that are adequate for the general adult population would also cover the elderly. In addition to chemical irritation, depending on the nature of the product being assessed, mechanical irritation could be an additional endpoint to consider due to higher susceptibility of aged skin to damage caused by friction and shearing forces. Simulated in-use clinical testing or specific test methods can be used to evaluate the mechanical irritation potential of a whole product [18].
Elderly Skin Susceptibility to Sensitization Allergic contact dermatitis differs from skin irritation in its requirement for initial recognition by the immune system of allergenic, low molecular weight molecules (induction). A secondary exposure to the allergen can elicit a dermatitis response with clinical manifestations very similar to irritant dermatitis. The attention of safety assessors is primarily devoted towards the prevention of the onset of a sensitization via induction. The induction of sensitization is recognized to be a threshold phenomenon which depends on allergens’ intrinsic allergenic potency, amount of allergen on exposed skin surface, and duration of exposure. The quantitative skin sensitization risk assessment approach is based on the determination of a no observed adverse effect level (NOAEL) for the contact allergen in humans, specifically the unexpected sensitization induction level (NESIL) determined as dose per unit area, and is compared to the estimated human skin exposure to that allergen [19].
Accordingly to this approach, three areas of variability (also referred as uncertainty factors) have to be properly weighted and accounted for in risk assessment. These areas of variability cover for interindividual susceptibility to a given allergen (genetic variability and other population differences), for the effect of a vehicle or product matrix (e.g., concomitant presence of irritants or skin penetration enhancers could lower the nonsensitizing exposure threshold) and for skin-site exposure considerations (dermal integrity, site of body exposed, effect of occlusion) [20]. Age-related susceptibility is included in the 10-folds interindividual variability factor, which is considered appropriate to cover for human population differences in susceptibility to sensitizers. An overall decline in immune function in the aged is reported as compared to young individuals, including a reduction in epidermal Langerhans cells [21] that are necessary during the induction phase of the immune response, the critical initial step in the onset of allergic contact dermatitis. This is suggestive of a lower susceptibility of the aged subject to develop an allergic contact dermatitis. Most studies on skin sensitization as a function of age are retrospective, and describe the incidences of elicitation responses through diagnostic patch testing. These studies are not considered useful for evaluation of age-susceptibility to the development of a new sensitization as no insight is provided e.g., on the impact of age on the NESIL. There are few studies available on population differences in susceptibility to the induction of a skin sensitization. Ability to acquire a sensitivity to poison ivy [22], and the capacity of potent sensitizer 2,4-dinitrochlorobenzene of developing an allergic contact dermatitis was reported to decrease with increasing age. In a study involving 116 elderly subjects, 69% of individuals over 70 years of age developed a sensitization to 2,4-dinitrochlorobenzene versus 96% in the population under 70 years [23]. In another study, about only one fourth of population greater than 65 years became sensitized by 2,4-dinitrochlorobenzene as compared to subjects aged 20–40 years [24]. Other studies failed to demonstrate a difference in the ability to sensitize the adult and the elderly. Exposure of naı¨ve subjects to 2,4-dinitrochlorobenzene resulted in no significant differences in the incidence of sensitization in 3 age cohorts, 21–59 years, 60–79 years and greater than 80 years [25]. Kwangsukstith and Maibach reviewed the effect of age and gender on induction and elicitation of contact dermatitis [26]. They concluded that in the elderly there is an agedependent decrease of delayed hypersensitivity reactions, a decreased ability to be sensitized to new allergens, and a reduced elicitation response in presensitized individuals.
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In a subsequent review by Robinson on population differences and their implication for skin irritation and sensitization, the author concluded that standard risk assessment procedures and safety testing can be considered relatively conservative to cover age, gender, and racial differences [27], recognizing that there is very little agerelated difference in sensitization susceptibility. In conclusion, the elderly appears to be less or at maximum equally susceptible to sensitization than younger ages. Risk assessments incorporating default human population variability assumptions are adequate to protect the elderly population. The likelihood for predisposed individuals to acquire a sensitization from exposure to a contact allergen is dependent on occasions and level of exposure to a much larger extent than any age-related population difference.
Risk Assessment Approach for Systemic Toxicity The risk assessment process for substances with a threshold for toxicity was described by the U.S. National Academy of Sciences in 1983 [28] (> Fig. 81.1). The approach has been applied widely by regulatory agencies and institutions to assess health endpoints related to exposure to chemicals present in food, air, or drinking water, sometimes with slight modifications and different terminology [29, 30]. The initial step of risk assessment (hazard identification) evaluates the inherent toxicity of a substance, and whether it can cause an adverse health effect, given the relevant route of exposure, considering all potential safety endpoints. The second step is a dose-response evaluation to assess the relationship between the dose of a chemical and the incidence and severity of an adverse
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health effect in the exposure population. The third step is the accurate and robust assessment of exposure to the substance taking into account all relevant exposure scenarios and routes. Finally, the risk characterization and final assessment step integrates hazard, dose-response, and exposure considerations into advise suitable for use in decision-making or risk management. All relevant areas of data extrapolations and uncertainty are accounted for including e.g., interspecies variability, allowing extrapolations from animal to man, and human variability, to account for sensitive individuals of the population. The term margin-of-safety (MOS) is commonly used to compare estimated human exposure to a risk value for which the risk of causing adverse effects in humans is considered to be none-to-minimal such as a reference dose (RfD, used, e.g., by the U.S. Environmental Protection Agency to describe an acceptable daily exposure to an environmental substance), or an acceptable daily intake (ADI, typically used by the WHO/FAO for chemicals in food). An essentially similar sequence of steps is followed for nonthreshold effects (such as cancer from a genotoxic mechanism), where according to the current paradigm, low level of exposure or even a single molecule could hypothetically increase the probability of genetic mutation. For nonthreshold effects, however, the outcome of risk assessment is the quantification of the risk to human health associated with a particular level of exposure.
Human Variability in Susceptibility to Chemical Toxicity It is an integrant part of risk assessment to account for human population variability in susceptibility to toxicants, including potential for age differences. Risk assessors
. Figure 81.1 Schematic representation of quantitative risk assessment process for substances with threshold toxicity
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and health agencies worldwide, for many years have adopted a standard default uncertainty factor (or safety factor) of 100-fold to derive acceptable exposure levels for compounds with threshold toxicity. This uncertainty factor comprises a 10-fold factor for interspecies differences and a 10-fold factor for human variability, which can be further subdivided into a 100.5 (3.16) factor for toxicokinetic (absorption, distribution, metabolism, elimination), and in another 100.5 (3.16) factor for toxicodynamic (how the target tissue responds to a given target tissue dose) [30]. This subdivision allows for chemical-specific toxicokinetic and mechanistic data to be considered allowing for substance-specific risk assessment refinement [31] (> Fig. 81.2). Renwick and Lazarus [32] developed a model to evaluate impact of human variability on risk assessment of chemicals based on toxicokinetics data from 60 therapeutic drugs metabolized and eliminated through a variety of
pathways, and on toxicodynamic data from 49 different effects. Based on the model, they concluded that the general 3.16 toxicokinetic uncertainty factor would cover on average the 99.1% of total human population, although no specific break out was made for the elderly. When accounting for combined toxicokinetics and toxicodynamics, the default cumulative 10-fold uncertainty factor for human variability would cover on average more than >99.9% of human population. It was acknowledged by the authors that genetic polymorphisms can influence the universal validity of a 3.16 folds kinetic uncertainty factor. From a risk assessment impact point of view, polymorphisms may pose a greater risk when genetic mutations inactivate or reduce the activity of an enzyme involved in detoxification. Subjects with these mutations are defined poor metabolizers, if enzymatic activity is reduced, or nonphenotyped if completely missing. A greater risk is also associated to polymorphic metabolic
. Figure 81.2 Use of uncertainty factors in risk assessment and approaches for their refinement. The 100-fold uncertainty factor can be considered the product of two separate 10-fold factors for interspecies differences and human variability. These 10-fold factors can be further subdivided into toxicokinetic and toxicodynamic differences (World Health Organization [30]). (a) If data are available on population variability on the major route of elimination or on a mode of action for a chemical, default kinetic, and dynamics values could be replaced by categorical factors such as pathway-related factors (Dorne JLCM, Walton K, Renwick AG [33]). (b) If chemical specific data on toxicokinetic or toxicodynamic are available for a compound under assessment, such data can be used instead of defaults World Health Organization [31]
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pathways when multiple copies of a gene overexpress an enzyme responsible for bioactivation of a parent compound into a toxic metabolite (fast metabolizers). For many compounds, however, the presence of alternative or multiple pathways of elimination would not necessarily invalidate the validity of default uncertainty factors for human variability.
Dermal Absorption in the Elderly Dermally applied substances that penetrate the skin barrier and become systemically bioavailable can potentially cause an adverse health effect if a sufficiently high concentration reaches the relevant target organ. Default risk assessments often start with the assumption of 100% dermal penetration, but there are many factors influencing the actual penetrant fraction of the substance. Effectiveness of skin barrier function is the key factor influencing skin permeability, which largely depends on integrity and hydration of stratum corneum and its lipid content, and the hydrophilicity of the substance in question. Aged skin is drier, thinner, and has lower lipid content as compared to the young [34]. Although reduction in both water and lipids may suggest compromising of the skin barrier, the decline in TEWL with age [14, 35] signals that barrier function is not impaired, and reduced skin hydration indicates a lower dermal penetration potential for hydrophilic compounds in the aged human skin. Furthermore, the reduced cutaneous vascularization in the skin of older individuals also suggests a poorer absorption capability due to reduced removal of permeable compounds from dermal compartment by impaired microcirculation [34]. An in vivo percutaneous penetration study with both lipophilic (testosterone, estradiol) and hydrophilic (hydrocortisone, benzoic acid) compounds, indeed demonstrated a reduced absorption of hydrophilic substances in older skin, while no age relationship was found for the two lipid-soluble compounds [34]. Other studies have confirmed the slower percutaneous absorption in the aged of hydrocortisone and benzoic acid, and no age difference for estradiol and testosterone [36]. Hydrocortisone and testosterone penetration through both vulva and forearm skin were also directionally reduced in postmenopausal versus premenopausal women, with significant reduction only for hydrocortisone applied to the vulva of postmenopausal women [37]. Significantly, lower skin permeation in the older was also reported for watersoluble compounds methyl nicotinate, methyl salicyclic
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acid, caffeine [34], and tetrachlorosalicylanilide [38]. The extent of the reduction for water-soluble compounds ranged from 36 to 52% in the older ages (greater than 65 years) versus the young adults (22–40 years) [34]. These data are consistent with outcomes from previous studies, which imply that the barrier function of human skin in vivo increases with increasing age [39], with a general decrease in skin permeability especially for compounds with low solubility in lipids. Highly lipidsoluble chemicals may still dissolve readily in the stratum corneum of aged skin, even if lipid medium is reduced to the same extent of younger skin.
Distribution, Metabolism and Elimination in the Elderly Body weight, total body water, and lean body mass decrease with advancing age. The average percent body fat in young adults is about 1/3 and 1/5 of total body mass in women and men respectively. Between the ages of 25 and 65–70 years body fat percentage increases on average by +48% in women and by +79% in men [40]. By the age of 70 years, body fat reaches approximately 36 and 48% of total body weight of males and females respectively [41]. The consequence of these changes is that hydrophilic compounds have smaller volumes of distribution, resulting in higher serum levels, while lipophilic compounds have higher volumes of distribution with increasing age. The kidney is the main route of excretion of watersoluble compounds, and for the water-soluble metabolites of lipophilic compounds. Renal mass, blood flow, tubular excretory capacity, glomerular filtration rate, and general function decline with age. Glomerular filtration rate, quantitatively the most important parameter for renal excretory function, declines by approximately 30% between 30 and 80 years in many elderly subjects [42]. Decreased renal function can result in a prolongation of the half-life of metabolites. Liver is by far the most important organ for metabolism of drugs and xenobiotics. Advancing age is associated with a 20–40% reduction in liver size [43] and liver blood flow [44] that may reduce hepatic first-pass metabolism and clearance of substances. Alteration of hepatic structure and enzymatic functions with aging, however, is moderate: in the elderly healthy person, routine tests for liver function involving the metabolism and elimination of specific dyes, radioisotopes, and protein synthesis do not show significant differences between 50–69 years and 70–89 years [45–47]. Studies on human liver tissue showed that mono-oxygenase activities are maintained
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even in advanced old age [48]. In general, Phase I metabolic function (hydrolysis, reduction, oxidation) is reduced in the elderly while Phase II functions (glucoronidation, sulfation, acetylation, methylation, conjugation with glutathione or amino acids) are not affected by aging [49]. The cytochrome P450 family is the primary enzyme system involved in Phase I metabolic reactions. In an investigation of microsomal enzyme activity of human liver, a reduction of about 30% of CYP-450-linked drug metabolism was reported after 70 years of age [50], while other studies found no significant differences in the level, and activity of human liver microsomal enzymes [51]. See > Table 81.1 for a summary of kinetic differences in the elderly.
Toxicokinetics Variability in the Elderly and Significance for Risk Assessment Age-related human variability in toxicokinetics and toxicodynamics is embedded in default human variability factor in standard risk assessments.
. Table 81.1 Toxicokinetics in the elderly: Difference in absorption, distribution, metabolism, and elimination in the older individual versus the younger adult Dermal absorption in the elderly Water-soluble substances: reduced absorption vs. young adults Lipid-soluble substances: no difference vs. younger adults Skin barrier function increases with increasing age Distribution in the elderly Water-soluble substances: Smaller volumes of distribution, higher serum levels Lipid-soluble substances: Higher volumes of distribution Reduction in total body weight Metabolism in the elderly Decrease in liver size and blood flow Generally reduced Phase I metabolic functions Generally unaffected Phase II metabolic functions Elimination in the elderly Decline in glomerular filtration rate (by 30%) Decline in renal mass, renal blood flow, tubular excretory capacity
Clewell et al. [52] conducted a comprehensive review on age- and gender-related differences in physiological, biochemical, and kinetic parameters based on data sets from 70 substances with different physicochemical characteristics. A few differences were found between young adults and the elderly in estimates of target tissue exposure, related to the declined capacities of some enzyme systems and renal elimination. Clinical data for specific therapeutic drugs have been used to characterize human variability in pharmacokinetics within a therapeutic class [53, 54]. The resulting chemical-specific kinetic factors ranged from 1.2 to 3.2 for unimodal population distribution, and a kinetic factor of 3.0 was specifically calculated for the elderly. The adequacy of default uncertainty factor to cover the human variability in toxicokinetics, including the elderly, was the subject of a recent review by Dorne and Renwick [33]. The authors conducted a meta-analysis of metabolism and pharmacokinetic data for probe substrates of a number of phase I and phase II metabolic and renal excretion pathways, including seven major isozymes of the cytochrome P450 superfamily (CYP) that constitutes the major oxidative hepatic system for metabolism of xenobiotics. Despite the impairment of hepatic and renal function in the elderly subpopulation, the default 3.16 kinetic factor was found to be adequate for most monomorphic pathways, except for renal excretion and CYP3A4 metabolism, for which specific pathway-related kinetic factors for the elderly were calculated as 4.2 and 4.9, respectively, covering the ninetyninth percentile of the elderly population. Uncertainty factors were above default kinetic factor for nonphenotyped elderly subjects for CYP2D6 and CYP2C19 metabolism (8.4 and 4.3 pathway-related kinetic factor respectively) and for slow acetylators (7.6 factor). The majority of kinetics data have been obtained with pharmaceuticals, which tend to have physiochemical and biochemical properties that may differ from many compounds of relevance for consumer product assessments, such as environmental contaminants. While several chemicals of toxicological concern are lipohilic, pharmaceuticals tend to be water soluble and can be preferentially metabolized by specific enzyme sub-systems, such as the CYP3A4, which are less relevant e.g., for environmental substances [55]. In a study aimed to investigate age- and genderrelated differences in physiological and biochemical processes that affect tissue dosimetry for the purpose of development of a predictive physiologically-based pharmacokinetic life-stage model, two water-soluble chemicals (isopropanol and nicotine), and four lipophilic
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chemicals (vinyl chloride, methylene chloride, perchloroethylene and 2,3,7,8-terachlorodibenyo-p-dioxin) were analyzed [55]. The choice of substances reflected the need to better represent the variety of physiochemical, biochemical, and mode-of action properties of nonpharmaceuticals, including CYP2E1 metabolism which is relevant for many environmental toxicants. The model was based on a number of simplifications and approximations, and authors cautioned that model predictions should be regarded as reasonable expectations not as validated predictive extrapolations. Nonetheless, their results were based on reasonable descriptions of agedependent physiological and biochemical processes, and provide useful insights on the potential differences in toxicokinetics across life stages, and how these differences may impact internal dose metrics. Overall, the results of the simulations indicated that variation of toxicokinetic dose metrics associated with aging are relatively modest. In the 75 year old, the average internal daily dose was slightly higher as compared to the 25-year old only for perchloroethylene (+20%), for its metabolite trichloroacetic acid (+40%) and for the reactive metabolite of methylene chloride (+30%). For all other compounds and their metabolites, the average internal daily dose was generally comparable or slightly lower in the 75 year old as compared to the 25 years old (17% as maximum reduction). In all cases, the magnitude of the resulting human toxicokinetic factor was significantly lower than default kinetic factor of 3.16 used in standard assessments, reaching at maximum a factor of 1.4 for trichloroacetic acid. In the same study, blood concentrations of isopropanol and of its metabolite acetone were assessed for cross-route internal exposure comparison [55]. Specifically for the dermal exposure route, higher peak concentrations of isopropanol in arterial blood were predicted by the model to occur in advanced age, with an approximate increase of +25% by the age of 75 years vs. the age of 25 years. Blood concentration of isopropanol metabolite acetone did not vary much between these two ages. Again the magnitude of the resulting human toxicokinetic factor was 1.1, significantly lower than default value of 3.16 (> Table 81.2).
Exposure Considerations for the Elderly A robust exposure assessment should be based on accurate, product-specific data on frequency, extent, and duration of an exposure as well as on proper characterization of anatomical site in contact with the substance, skin conditions (occlusion or compromission), and dimensions of skin-contact area.
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Skin permeability potential differs by anatomical site and can be amplified by occlusion, abraded, or otherwise damaged skin. The prevalence of these conditions may increase with older age and in particular sub-groups (e.g., perineal dermatitis in subjects with fecal or urine incontinence, occluded skin and pressure sores of immobilized patients). Age-related patterns may exist in type of product used as well as in frequency and amount of product used per exposure event, collectively referred as habits and practices. Products like incontinence absorbent aids or antiwinkles treatments are predominantly used by the senior population. Elderly subjects, especially women, may use hair products such as dyes, permanent waves, and others more regularly because of the hair graying and loss [56]. Cosmetics and toiletry products may be equally appealing, for different reasons, to the younger as well as the older who wants to protect his fragile and dry skin. On the other side of the spectrum, a number of products may not be used in the elderly, or not used to the same extent of younger ages. Self-reported oral hygiene habits among institutionalized elderly subjects identified inadequate oral hygiene practices, with 26% of dentate population reporting no tooth brushing [57] hence, virtually no exposure to common oral care products (dentifrices, toothbrushes) should be expected in a quarter of the surveyed population, without accounting for prevalence of denture wearers. The amount of published data on habits and practices for consumer products is limited. Existing data does not include detailed segmentations for age, and the elderly tend to be inadequately represented in the surveyed populations. Building on an internal database of habits and practices, an experimental probabilistic exposure assessment model was developed for various consumer products. The model was used to investigate exposure variability and its dependency from age, gender, ethnic, geographic origin among other factors (unpublished data). Hair-care data analysis, for example, revealed that frequency of hair washing significantly decreases with advancing age, with a parallel reduction in the use of shampoo and conditioners and related exposures. The magnitude of exposure reduction to these products for the population older than 65 years, versus teenagers, was bigger than default uncertainty factor for human kinetics. This example illustrates the importance of accurate exposure estimate in risk assessments for the elderly: for shampoos and conditioners, use of exposure data obtained from the general population inclusive of younger consumers would lead to a significant overestimation of the actual risk for the elderly.
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. Table 81.2 Studies on toxicokinetic variability in the elderly and correlated toxicokinetic factors Data source Default uncertainty/safety factor for human variability in toxicokinetics, elderly implicitly included (World Health Organization [30])
Derived toxicokinetic factor 3.16 ! Default factor for human variability
Study objective: To characterize interindividual differences and 3.0 ! Chemical-specific adjustment factor for elderly derive acceptable daily intake values. Data type: patients, accounting for interindividual differents in kinetic Pharmacokinetic clinical data on therapeutic for drugs in one therapeutic class agents representative of various classes (antidepressants, ACEinhibitors, nonsteroidal anti-inflammatory drugs, cholesterol lowering agents, antibiotics) (Silverman et al. [53], Naumann et al. [54]) Study objective: To derive pathway-related uncertainty factors associated with variability in kinetics. Data type: Meta-analysis of metabolism and kinetic data for pharmaceutical probe substances, including elderly-specific data for seven Phase I (CYP1A2, CYP2A6, CYP2C9, CYP2E1, CYP3A4, Alcohol dehydrogenase, hydrolis), and three Phase II enzymatic monomorphic pathways (glucuronidation, glycine conjugation, sulfate conjugation) plus renal excretion pathway. Analysis considered polymorphism for some pathways (nonphenotyped subjects for CYP2C19 and CYP2D6, fast metabolizers for CYP2D6, fast and slow acetylators) (Dorne JLCM, Walton K, Renwick AG [33]) Study objective: To develop a life-stage, physiologically based pharmacokinetic life-stage model. Data type: Chemical-specific adjustment factor based on metabolism data of two watersoluble (isopropanol and nicotine) and four lipid-soluble (vinyl chloride, methylene chloride, perchloroethylene and 2,3,7,8-terachlorodibenyo-p-dioxin) probe substances selected to represent differences in physicochemical properties and in metabolic characteristics of environmental chemicals (Clewell et al. [55])
Beyond exposure assessment, a proper characterization of exposed elderly population is important for risk management considerations. The aged population undergoes progressive sensory deficits (in vision, hearing, taste, smell), decreased short-term memory, and other cognitive changes which can make the older adult more inclined to err when using pharmaceuticals, cleaning agents, and personal care products [58]. These changes may reduce the ability to recognize and interpret warnings, cues and product instructions that might render risk reduction measures ineffective. Proper consideration should be given to these aspects when designing products for the elderly, including their packaging and use instructions.
Monomorphic Pathways: (ninetyninth population) 1.5–3.16 ! Elderly-specific factor for 9 pathways; 4.9 ! Elderly-specific factor for CYP3A4 metabolism; 4.2 ! Elderly-specific factor for renal excretion Polymorphic Pathways: (ninetyninth population) 2.3–2.9 ! Elderly-specific factor for fast metabolizers; 4.3–8.4 ! Elderly specific factors for nonphenotyped; 7.6 ! Elderly-specific factor for slow acetylators;
0.83–1.4 ! Range of elderly-related chemical-specific adjustment factors; 1.1 ! Elderly-related, isopropanol-specific adjustment factor, specific to the dermal exposure route
Conclusion This review evaluated the adequacy of current risk assessment practices and default human variability factors when assessing consumer product substances for the elderly. Research has started to elucidate the variability of human response to toxic insults and the specific risk for susceptible populations. Risk assessment approaches are under constant refinement to incorporate emerging knowledge into more accurate risk predictions and models. Some of these refinement options are based on inclusion in the assessment of chemical-specific metabolism and toxicokinetic data. However, available data are limited to a restricted number of substances, mainly
Safety Evaluation in the Elderly via Dermatological Exposure
therapeutic agents. This limits routine applicability of refined approaches to common constituents of consumer products and use of default uncertainty factors to account for human variability in the elderly remains an important option for routine assessments. The risk assessment for consumer products is an iterative process that progresses via successive refinements. An initial assessment is often based on a number of worst-case assumptions, upper estimates, or extreme product uses such as 100% dermal penetration, complete release of a substance from a product, highest theoretical frequency of product use that, when taken together, lead to a significant overestimation of the real risk. In the risk characterization step, an expert professional estimates if the available data are adequate to protect the most sensitive individuals of a target population at the light of latest knowledge and evolution of risk-assessment concepts. This includes considerations for special subpopulation that might not be adequately covered by default variability factors, such as genetic polymorphism in the elderly. If the outcome of an initial assessment is favorable, then the assessment can be stopped at this stage. If the outcome of risk characterization is that a substance is of potential concern in the most sensitive subpopulation, further refinement of the default assessment parameters is needed that may require additional data to reduce the level of conservatism. A number of age-related differences have been highlighted in the chapter, and their relevance for the safety evaluation of substances in the elderly has been reviewed in the light of current risk assessment practices. Dermatological effects, specifically skin irritation and skin sensitization, are often the most relevant toxicological endpoints in the risk assessment of substances contained in consumer products. Available evidence suggests that the elderly does not constitute a specific risk population for skin irritation or the induction of contact sensitization. Current safety assessment approaches, builtin conservatism, and test methods, appear to adequately cover the older segment of the population. Because of the increased susceptibility of elderly skin to mechanical insults and diminished repair capacity, mechanical irritation may be an additional endpoint to consider, depending on the type of product being assessed. Elderly skin does not constitute an easier barrier for substances to cross then younger skin. Dermal penetration in the elderly is either reduced for hydrophilic compounds or unchanged for lipophilic ones, which also have higher volumes of distribution in the aged body. Despite specific physiological and functional differences have
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been highlighted in the elderly that affect the systemic distribution, metabolism and excretion of substances, these tend to be relatively modest in their contribution to the final risk level when default uncertainty factors for human variability in sensitivity to chemicals is incorporated in risk assessment. Default uncertainty factors appear to be over-conservative for the majority of the population, but generally protective for the elderly. Regardless of age, further research is needed to better understand the contribution of genetic polymorphism in individual susceptibility to chemicals. However, older age does not seem to be an important aggravating factor. The limited data available mainly for drugs on genetic polymorphism indicate that elderly specific, kinetics-driven susceptibility to toxicants might be increased by a factor of 2–3 versus the human default factor applied in standard risk assessments. This is a considerable difference, e.g., in a risk assessment of a therapeutic agent with a low therapeutic index, but is a modest factor in the context of an initial, favorable consumer product risk assessment built on conservative assumptions and high-end use scenarios. In fact, it might be sufficient to refine those initial conservative assumptions, e.g., via generation of more accurate exposure data, to demonstrate an adequate margin of safety also for those highly susceptible individuals. Methods for refinement of an assessment via incorporation of chemical-specific kinetic or dynamic data are also available that could be used if needed. A proper characterization of the exposed population and a precise estimate of the dose of substance reaching the skin are particularly important aspects in the context of the exposure assessment step, with exposure being a potential source of significant variability in final risk estimate. Therefore, it should be considered that older age may determine the product of choice (ranging from almost no use to exclusive use), and that it can influence how products are used, how often and how much.
Cross-references > Dermal
Safety Evaluation: Use of Disposable Diaper Products in the Elderly > Susceptibility to Irritation in the Elderly: New Techniques
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23. Waldorf D, Willkens R, Decker J. Impaired delayed hypersensitivity in an aging population. JAMA. 1986;203:111–114. 24. Girard JP, Paychere M, Cuevas M, Fernandes B. Cell-mediated immunity in an aging population. Clin Exp Immunol. 1977;27:85–91. 25. Schwartz M. Eczematous sensitization in various age groups. J Allergy. 1952;24:143–148. 26. Kwangsukstith C, Maibach HI. Effect of age and sex on the induction and elicitation of allergic contact dermatitis. Contact Dermatitis. 1995;33:289–298. 27. Robinson MK. Population differences in skin structure and physiology and the susceptibility to irritants and allergic contact dermatitis: implications for skin safety testing and risk assessment. Contact Dermatitis. 1999;41:65–79. 28. National Academy of Sciences. Risk Assessment in the Federal Government. Washington: National Research Council. National Academy Press, 1983. 29. U.S. Environmental Protection Agency. A review of the reference dose and reference concentration process. EPA/630/P-02/002F., 2002. 30. World Health Organization. International Program on Chemical Safety: Principles for the assessment of risks to human health from exposure to chemicals. Environmental Health Criteria 210. Geneva: World Health Organization, 1999. 31. World Health Organization. International Program on Chemical Safety: Chemical-specific adjustment factors for interspecies differences and human variability: Guidance document for use data in dose/concentration-response assessment. Geneva: World Health Organization, 2005 http://whqlibdoc.who.int/publications/2005/ 9241546786_eng.pdf 32. Renwick AG, Lazarous NR. Human variability and noncancer risk assessment. An Analysis of the default uncertainty factor. Regul Toxicol Pharmacol. 1998;27:3–20. 33. Dorne JLCM, Walton K, Renwick AG. Human variability in xenobiotic metabolism and pathway-related uncertainty factors for chemical risk assessment: a review. Food Chem Toxicol. 2005;43:203–216. 34. Roskos KV, Maibach HI, Guy RH. The effect of aging on percutaneous absorption in man. J Pharmacokinet Biopharm. 1989;17 (6):617–30. 35. Roskos KV, Maibach HI. Percutaneous absorption and age-implications for therapy. Drugs Aging. 1992;2:432–449. 36. Ghadially R. Aging and epidermal permeability barrier: implications for contact dermatitis. Am J Contact Dermatitis. 1998;9:162–69. 37. Oriba HA, Bucks DA, Maibach HI. Percutaneous absorption of hydrocortisone and testosterone on the vulva and forearm: effect of the menopause and site. Br J Dermatol. 1996;134:229–233. 38. Grove GL. Physiologic changes in older skin. Clin Geriatr Med. 1989;5:115–125. 39. Christophers E, Kligman AM. Percutaneous absorption in aged skin. In: Montagna W (ed) Advances in Biology of Skin VI. New York: Pergamon Press, 1965, pp. 163–175. 40. Mayersohn M. Pharmacokinetics in the elderly. Environ Health Perspect. 1994;112(S11):119–124. 41. DeVane CL. Metabolism and pharmacokinetics of selective serotonin reuptake inhibitors. Vellular Mol Neurobiol. 1999;19(4):443–466. 42. Lindeman RD, Tobin J, Shock NW. Longitudinal studies on the rate of decline in renal function with age. J Am Geriatr Soc. 1985;33 (4):278–285. 43. Woodhouse KW, James OF. Hepatic drug metabolism and ageing. Br Med Bull. 1990;46(1):22–35. 44. Wynne HA, Goudevenos J, Rawlins MD, James OF, Adams PC, Woodhouse KW. Hepatic drug clearance: the effect of age using
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80 Susceptibility to Irritation in the Elderly: New Techniques Miranda A. Farage . Kenneth W. Miller . G. Frank Gerberick . Cindy A. Ryan . Howard I. Maibach
Introduction Numerous changes in the structure of the skin occur as the skin ages [1]; these structural changes modulate cutaneous function in a variety of ways [2]. A critical role of the skin is to protect the vulnerable internal tissues from a multitude of potentially harmful exogenous agents [3]. Exactly how, and to what extent, structural changes in the aging skin influence its ability to act as a barrier to exogenous agents is a matter of ongoing research [4]. Definitive differences in skin structure and physiology between younger and older subjects are difficult to establish owing to interpersonal variation in populations and methodological approaches and statistical difficulties inherent in sampling. One factor that can influence a substance’s skin irritation potential is its ability to penetrate the skin. Both skin barrier and the capacity of the microvasculature play a role in percutaneous absorption which is, by definition, a functional concept describing the process by which external substances (including both toxins and drugs) pass from the skin surface into the internal physiological milieu [5]. The first step in percutaneous absorption is the penetration of the chemical agent [3], dependent primarily upon the competency and hydration of the stratum corneum (SC) barrier [3, 4]. The SC is a two-component structure consisting of lipid-depleted corneocytes embedded in a continuous, lipid-enriched, extracellular matrix [6]. The barrier thickness varies substantially at different anatomical sites, about 15 stacked cell layers (roughly 10 mm) at most body sites, although areas such as the face and genitalia can be less than 10 cell layers, while the soles of the feet may be as many as 50 [7]. The structure of the SC prohibits permeability through organization into a lamellar membrane structure, the composition of the lipid component (including very long-chain fatty acids but no polar lipids) and the extracellular location of the lipid fraction, as well as a critical balance (1:1:1 in molar ratio, although ceramides are about 50% by weight) [8] of three key
lipids: ceramides, cholesterol and free fatty acid, which [6] form a barrier to diffusion [9]. Stratum corneum competence determines the diffusion of compounds across the skin, and is dependent on functional biogenesis of corneocytes as well as the proper synthesis and processing of intracellular lipids [9]. Penetration is the limiting step in the formation of cutaneous reactions to chemicals and is always by passive diffusion [9]. A penetrating chemical may, however, cause structural and functional damage to the barrier, resulting in facilitation of increased penetration by the offending agent [10]. Some well characterized skin irritants such as sodium lauryl sulfate, hydrochloric acid, and nonanoic acid are capable of causing such types of damage. The second step in percutaneous absorption is diffusion through the viable epidermis and the dermis [11], a large sink (compared to the SC) where compounds are significantly diluted and may undergo significant changes in composition and structure [9]. The third step is the successful uptake of the compound by microcirculation, so that it comes in contact with internal physiology [3].
Factors in Skin Irritation in the Elderly Percutaneous Absorption Structural alterations that occur as the skin ages have the potential to alter the process of percutaneous absorption. The aged epidermis displays several functional abnormalities possibly related to impaired percutaneous absorption, including altered drug permeability [6], a decreased tendency to exhibit irritant contact dermatitis [12], and decreased immunocompetence [13, 14]. A definitive understanding of the effects of age on percutaneous absorption is elusive. Animal data are conflicting, and in vitro experiments have demonstrated little difference between younger and older skin [15]. Percutaneous penetration would appear to be largely impaired in older patients as compared with younger patients, as most compounds studied have been observed to be less
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_80, # Springer-Verlag Berlin Heidelberg 2010
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. Table 80.1 Cutaneous absorption in the elderly Percutaneous absorption
Test parameters
Definition of aged population
Effect of age
Reference
Water soluble-dye flourescein
In vitro, forearm
61–90 years
Increased diffusion
Tagami H. (1971) [11]
Tetrachlorosalicylanilide (1.5% in ethylene glycol
In vivo, forearm
62–82 years
Greater time for penetration
Tagami H. (2008) [7]
Testosterone
In vitro
>75 years
Increased absorption
Roskos KV et al. (1986) [20]
Estradiol
In vitro
>75 years
Decreased absorption
Roskos KV et al. (1986) [20]
Hydrocortisone
In vitro
>75 years
Decreased absorption
Roskos KV et al. (1986) [20]
Benzoic acid
In vitro
>75 years
Decreased absorption
Roskos KV et al. (1986) [20]
Methyl nicotinate
In vitro
64–86 years
No difference
Roskos KV et al. (1990) [65]
Testosterone 1.0 mCi/cm2
In vivo, forearm
Menopause (mean age 55)
No difference
Oriba HA et al. (1996) [17]
Fluorescein
In vitro
Cadavers 67–78 years Decreased penetration
Christopher E, Kligman A. (1965) [66]
Testosterone
In vivo, back
71–82 years
Decreased absorption
Christopher E, Kligman A. (1965) [66]
Sodium chloride
In vivo
71–82 years
Reduced intradermal clearance rate
Christopher E, Kligman A. (1965) [66]
efficiently absorbed by aged individuals (> Table 80.1). One study investigated the percutaneous absorption of six different compounds as follows: testosterone, estradiol, hydrocortisone, benzoic acid, acetylsalicylic acid, and caffeine. Testosterone and estradiol absorption in patients older than 65 were slightly suppressed (16.6 2.5 vs 19.0 4.4) and (5.4 0.4 vs 7.1 1.1), respectively, neither significant) as compared with subjects aged 22–40 years, while benzoic acid, acetylsalicylic acid, hydrocortisone, and caffeine absorption were significantly decreased [16]. To date, no absorption of specific compounds appears to depend on the chemical composition of the penetrant, no tendency towards a general increase in absorption in aged skin has been observed, despite the presupposition that older skin may display diminished barrier function [17]. It has been suggested that observed differences in absorption of different compounds may relate to the hydrophobic or hydrophilic nature of the penetrant itself. The biophysical characteristics of aged skin (reduced hydration levels and reduced lipid content) would predict facilitated penetration of hydrophobic compounds and relatively poor penetration of hydrophilic compounds [15], and a striking relationship between the hydrophobicity of a compound and its permeability coefficient has been observed [9]. In the evaluation of six compounds above, lipophilic compounds exhibited the
same absorption in younger versus older subjects, while hydrophilic compounds (hydrocortisone, benzoic acid, acetylsalicylic acid and caffeine) exhibited decreased absorption in older subjects [3], with relative absorption of each compound consistent to its permeability coefficient [15].
Barrier Function Many components of healthy barrier function are known to decline with age (> Table 80.2), casting suspicion on the efficacy of barrier function in the elderly. Stratum corneum thickness remains constant with age [18], but a global decrease in lipid content (as much as 68%) leads to alterations in lamellar bilayer morphology [19]. Waterholding capacity, which is a function of the ceramide fraction of the intercellular lipid component of the SC, is diminished in aged skin [12]. An additional ageassociated decrease in the sterol ester and triglyceride fraction of the SC lipids [3] contributes to the decrease in water-binding capacity, producing decreased hydration of the SC in aged individuals. It is known that skin barrier function is impaired in many dermatoses, including psoriasis, atopic dermatitis, and contact dermatitis [7, 9]. Characteristics of aged skin with the potential to influence barrier function are displayed in > Table 80.2.
Susceptibility to Irritation in the Elderly: New Techniques
80
. Table 80.2 Components of barrier function in the aged Skin parameter
Effect of aging
Reference
pH of skin surface
Elevated
Summers PR, Hunn J. (2007) [67]
SC water content
Normal
Elsner P et al. (1990) [68], Thune P. (1989) [69], Wilhelm KP et al. (1991) [70]
Slightly decreased
Ghadially R et al. (1995) [19]
Decreased (4.43 0.16 g/m2 vs 6.41 0.93 g/m2 (p < 0.05)
Potts RO et al. (1984) [71]
Total lipid content of SC
Decreased 65%
Akimoto K et al. (1993) [72]
Corneocyte surface area
Increased
Le´veˆque JL et al. (2008) [73]
Delivery of secreted lipids to SC
Reduced
Ghadially R et al. (1995) [19]
Number of extracellular lamellar bilayers in the SC as visualized by electron microscopy with ruthenium tetroxide postfixation
Decreased
Ghadially R et al. (1995) [19]
Reduction in global lipid content
1/3 less by weight
Elias PM, Ghadially R. (2002) [6]
Interleukin-1alpha
Reduced
Ye J et al. (1999) [74]
Amphiregulin
Reduced
Ye J et al. (1999) [74]
Upregulation of lipid synthesis after barrier disruption
Reduced
Ghadially R et al. (1996) [75]
Mitotic response to glycosycleramide
Reduced
Marchell NL et al. (1998) [76]
Epidermal proliferation and differentiation
Significantly decreased in older individuals
Engelke M et al. (1997) [77]
Barrier function
Less stable (18 2 tape strippings required for perturbation in older skin (volar forearm) vs 31 5 in young
Ghadially R et al. (1995) [19]
Less cohesive (disrupted with fewer strippings)
Reed JL et al. (1997) [23]
Compromised 15% recovery at 24 h in older patients, 50% at 24 h in younger
Ghadially R et al. (1995) [19]
Barrier recovery
90% recovery seen at 7 days in older vs 4 days in younger TEWL
Normal
Ghadially R et al. (1995) [19]
Subnormal
Wilhelm KP et al. (1991) [70], Cua AB et al. (1990) [78], Thune P et al. (1988) [79]
Decreased significantly after age 60
Leveque JL et al. (1984) [80]
Mitotic activity and turnover time
Decreased
Baker H, Blair CP. (1968) [81]
Capacity for repair
Impaired (aged 65–75 years) compared with 18–25 years
Grove GL, Duncan S, Kligman AM. (1982) [42]
SC = Stratum corneum; TEWL = Transepidermal water loss
Transepidermal water loss (TEWL) is considered a measurement of the integrity of the horny layer, as one of the primary functions of the SC is to impede passive loss of water from the body [20]. Much research effort has
evaluated whether or not baseline TEWL is significantly affected by aging [3]. The evaporation process was demonstrated to be slower in aged skin as compared with younger skin in vitro [21].
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Susceptibility to Irritation in the Elderly: New Techniques
Although several studies have observed a decreased TEWL in older patients, most studies have observed no impact or only slight decreases [3]. Importantly, TEWL measurements have been observed to be highly variable, with 8% variation within individual by site, and 21% within the same individual from day to day, and up to 48% variation between healthy individuals [9]. The tendency for many substances to be less well absorbed in older skin has led some authors to suggest a slight enhancement of barrier function in the elderly [6, 7]. Despite the deterioration of the skin on many levels (with regard to the SC, most importantly the global loss of lipids), baseline TEWL in older patients appears to be normal [7]. Under normal conditions, barrier function is preserved, with lipid deficits compensated for by an increase in the thickness of the SC, larger corneocytes, and a decrease in the rate of desquamation [7]. Further investigation, however, has revealed that the apparently normal TEWL in the geriatric population masks substantial impairment of barrier function; obvious differences in permeability support the hypothesis that a barrier abnormality may, in fact, exist [19]. The functional pathology of the SC is revealed only after active insult [6]. In younger patients, barrier disruption stimulates the release of numerous cellular messengers, which stimulate epidermal lipid synthesis and effect rapid restoration of barrier integrity [22]. The skin of aged individuals exhibits normal baseline barrier function, but recovery of barrier activity after perturbation is markedly reduced [9]. TEWL measurements in vivo in both aged (greater than 80 years) and young (20–30 years) patients before and after disturbance of the barrier by tape stripping demonstrated a dramatic delay in recovery times in the older patients as compared with the younger. Younger subjects had recovered 50% of barrier function by 24 h, whereas older patients had only recovered 15% of barrier function; 90% recovery was achieved within 4 days in younger patients while older patients required a full week. Erosion of barrier function was shown to be correlated to lipid content of the skin, and thus likely related to a deficiency of key SC lipids in old age [19]. A similar experiment showed that delayed recovery times in the aged are extended by 20% in aged skin when characterized by superimposed photoaging [23].
Microcirculation Absorption into the blood stream depends on the integrity of the microcirculation. The microvasculature in
elderly skin appears to be compromised [24]. Histological observations show structural impairment, with a decrease in the number of dermal capillary loops and flattening of the dermoepidermal junctions, creating a decreased area for absorption [25]. In addition, functional deficits have been observed. Perfusion in the dorsum of the foot by capillary bloodflow amplitude has been found to be significantly decreased in older patients [26]. Blood flow at multiple body sites evaluated by laser Doppler velocimetry showed significant decreases in areas of high blood flow [27]. Age-associated decreases in the sensitivity of the microvasculature have been linked to autonomic control. A significant time delay in expected blood flow changes after the Valsalva maneuver (in which exhalation is performed against resistance) as well as after postural changes were observed in aged patients [28]; similar impairments in vascular response were observed after body cooling [29], cold-arm challenge [29, 30], and inspiratory gasp [29]. Clearance experiments are a more specific measure of the ability of the cutaneous microvasculature to absorb penetrants into the systemic pool [20]. There is substantial evidence that drug clearance is impaired in elderly patients; reduced drug elimination due to compromised blood flow in elderly patients is an integral pharmaceutical consideration in determining doses in the elderly, with an approximate 40% cumulative blood flow decline in a person 70 years of age [31]. The comparative permeability of aged skin, as well as the functional status of the stratum corneum and the skin’s microcirculation, impact both contact irritation and contact sensitivity in aged skin. The effects of aging on skin sensitization are reviewed elsewhere in this work [32]. This review will focus on aging and skin irritation, particularly new techniques being used to increase sensitivity in irritation testing.
Irritant Response An inflammatory reaction in the skin due to a single exposure to an offending chemical is termed acute irritant dermatitis [33]. Cumulative contact irritation, which is more common, is the result of ongoing contact with lowlevel irritants. Clinical irritation depends primarily on the irritant potential of the substance, as well as the chronological and anatomical extent of exposure [34]. Threshold concentrations in viable tissue are also dependent on the surface concentration, degree of occlusion, number of exposures, and vehicle of delivery [9]. Individual
Susceptibility to Irritation in the Elderly: New Techniques
predictors of the degree of irritation achieved include the integrity of the SC, the quality of the epidermal repair response, and the ability to mount an inflammatory response to the irritant [6]. Irritation is more commonly associated with formulations than with individual substances [34]. Certain individuals experience more intense and frequent adverse sensory effects than the normal population when exposed to certain substances, even substances that are benign in the majority of the population [35]. This phenomenon, termed ‘‘sensitive skin’’, is a subject of focused research, as subjective irritation often occurs in the absence of any objective signs [35]. Environmental factors, such as very cold or dry conditions, can also exacerbate irritation [36]. Numerous authors have observed a decreased susceptibility to cutaneous irritation in the aged. Irritation is less likely to develop, positive reactions are slower to develop, and, where irritation does occur, reactions are less intense. The aged also have a diminished capacity for recovery [12, 37, 38]. Irritant reactions are also more prolonged in older groups [32, 39]. Initial research into the effect of age on irritant response found decreased reactivity in the elderly to substances like soap [40] and croton oil [41]. Primary irritant response in both younger and older patients after application of a 1:1 solution of ammonium hydroxide in water demonstrated that although the time to raise a blister was reduced in older subjects, the time to fully fill the blister was significantly extended [42]. Further research evaluated a variety of irritants with different mechanistic actions as follows: dimethyl sulfoxide and ethyl nicotinate (nonimmunologic contact urticants), histamine (a mediator of immunological contact urticaria), chloroform-methanol (a stinging irritant), and lactic acid. All substances tested, despite having widely disparate mechanisms of irritation, produce much less irritation, quantified by visual inflammatory scores, in older patients as compared with younger [43]. Numerous authors have looked at differences in response to sodium lauryl sulfate between older and younger skin, at a variety of anatomical sites and concentrations, and have uniformly found a decrease in reactivity in older patients, including both prevalence of positivity and strength of reaction when present [44–48]. Less irritant reactivity was observed, both in visual scoring by erythema and by lesser increases in TEWL in response to application of irritant to older skin [3]. Inflammatory reactions in elderly are less intense in causing scaly, dry eczematous response; less vesicular; less erythema than in younger patients [46]. A summary of the effects of age on the irritant response is found in > Table 80.3.
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Vulvar Susceptibility to Dermatitis The unique characteristics of the epithelium of the vulvar area may make this region of the body more susceptible to irritation caused by topical medicaments and hygiene products. The vulvar epithelium is derived from two different embryological layers. The mons pubis and labia majora, derived from embryonic ectoderm, physiologically resembles exposed skin: It is keratinized and stratified, with sweat glands, sebaceous glands, and hair. However, in contrast to exposed keratinized skin, it is occluded, more hydrated than exposed tissues, and subject to more frictional stress. The keratinized areas of the vulva have been shown to be seven times more permeable than forearm skin, determined by application of radiolabeled hydrocortisone in benzene:ethanol or acetone solution [17, 48, 49]; although permeability varies with skin thickness [49]. The nonkeratinized area, from the inner third of the labia minora through the vulvar vestibule (derived from embryonic endoderm), is thinner and more loosely packed. It is also known to be significantly more permeable than keratinized skin [50, 51]. Vulvar symptoms are often related to irritant contact dermatitis [24]. In fact, prevalence rates as high as 54% for vulvar dermatitis have been reported in vulvar clinic patients [52]. Extrinsic factors often exacerbate the intrinsic susceptibility to irritation produced by friction and occlusion in the vulvar area. Personal hygiene habits, characterized by excessive cleansing routines and use of soaps and other hygiene products with surfactants, alcohols, and antiseptics, is the most common cause of contact dermatitis in the vulvar area [24]. However, it should be noted that self medication of vaginal itch with over-the-counter products is often the source of secondary sensitization to topical medicaments.
New Approaches in Skin Irritant Testing Numerous factors can influence skin testing results. Best practice in skin testing of irritant response would ensure standardization of protocols in choice of irritant; test site; environmental controls; batch volume and concentration; delivery vehicle; use of occlusive dressing; time to evaluation; assessment tools; and, in females, menstrual cycle phase. Skin testing, which evaluates consumer products, tests, by definition, the substances intended to have no irritant potential. Such testing often utilizes an approach which seeks to exaggerate exposure conditions in a controlled
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Susceptibility to Irritation in the Elderly: New Techniques
. Table 80.3 Effects of age on irritant reactions Irritant
Criteria of aged population
Characteristics of older skin as compared with younger
Reference
Aqueous solution of ammonium hydroxide (1:1)
Over 75
Initial response time faster in older Grove GL, Duncan S, subjects, time to fully tense blister slower Kligman AM. (1982) [42]
Dimethyl sulfoxide, histamine, ethyl nicotinate, chloroformmethanol, lactic acid
Over 75
Reduction in visual scores of inflammation
SLS (0.25%) With occlusion, 24 h
Over 65
Older: lower visual score, lower Cua AB et al. (1960) [78] percentage or responders TEWL lower in older (indicative of decrease in inflammatory response)
Soap
Over 50
Lower prevalence of positive tests
Bettley FR et al. (1960) [40]
SLS
Postmenopausal
Slower reaction, less intense nine visual score
Elsner P et al. (1990) [82]
SLS
Mean age 74.6
Less intensity of response (visual score, TEWL) reduction in SC water content
Cua AB et al. (1990) [44]
Decreased prevalence of positive reactions
Coenraads PJ et al. (1975) [41]
Croton oil
Grove G et al. (1981) [43]
SLS (0.1%, 0.5%, 1%) applied to forearm
N = 20 (premenopausal 32.2 mean age, postmenopausal 63.2 mean age
Less prevalence of erythema in Elsner P et al. (1990) [47] postmenopausal women (5/10 vs 9/10), lower degree of TEWL induction, smaller increase in relative capacitance
SLS (2%, 3%, 5%) applied to forearm
N = 20 (premenopausal 32.2 mean age, postmenopausal 63.2 mean age
Less prevalence of erythema in Elsner P et al. (1990) [47] postmenopausal women (6/10 vs 8/10), lower degree of TEWL induction, smaller increase in relative capacitance
SLS (2%) applied to face and neck
N = 20, younger group Less prevalence of erythema in older average age 25.2, older group, lower degree of TEWL group average 73.7
Marrakchi S, Maibach HI (2006) [45]
SC Stratum corneum; SLS Sodium lauryl sulfate; TEWL Transepidermal water loss
manner and typically proceeds through patch testing followed by visual scoring. Such testing however, is not practical for some products or anatomical areas (e.g., genital area) and may not be robust enough to detect irritation, which may nonetheless cause consumer discomfort [53].
The Behind-the-Knee (BTK) Test The behind-the knee (BTK) approach was developed specifically to test frictional and chemical irritation [54] and employs the popliteal fossa as a test site, applying samples to the back of the knee with an elastic knee band (> Fig. 80.1a–d). The BTK test more closely simulates
real-world exposures, as mechanical friction is supplied in addition to the chemical exposure provided by traditional testing [55]. The test has been validated and now been utilized in testing of more than 25 different materials, including topsheets, interlabial pads, pantiliners, tampons, lotion coatings on products such as facial tissues, and fabrics/textiles resulting in reproducible data to traditional clinical testing [53]. The BTK test offers other advantages to traditional testing in that it provides a controlled environment, in which solid products like catamenial pads and diapers may be tested under convenient and controlled conditions [56] as well as the opportunity to independently evaluate different test conditions (e.g., wet vs dry product)
Susceptibility to Irritation in the Elderly: New Techniques
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. Figure 80.1 The Behind-the-Knee (BTK) method: test material is placed horizontally and held in place behind the knee by an elastic knee band of the appropriate size (Fig. 80.1a–c). Skin irritation grading is then performed by an expert grader (Fig. 80.1d)
or do side-by-side comparisons of two different products. In addition, BTK testing can employ any healthy adult (i.e., does not require menstruating or incontinent patients), exposure can be controlled, grading is not intrusive, and is relatively inexpensive and productive of rapid results. In addition, the BTK method facilitated shorter exposure periods, allowing the traditional 24-h traditional patch test (consisting of four applications) to be shortened to a 6-h, two-application method while producing equivalent results [55]. BTK testing has demonstrated utility beyond irritation testing, in one study being used in quantifying subject exposure to a lotion component added to sanitary pads [57]. Three different lotions were evaluated side-byside in 54 healthy females by applying products (feminine pads containing lotion) to the popliteal fossa and comparing data obtained compared with that of traditional clinical testing. BTK data were found to be more
consistent than those obtained with traditional testing, as well as being obtained at a fraction of the cost [57]. BTK testing has been used in concert with other newly developed techniques, as discussed here, with additional potential to enhance the sensitivity of current testing procedures. Despite extensive testing of consumer products, which document the absence of irritant potential in manufactured products, consumers often develop product preference on the basis of perceived sensory effects that were not revealed during testing [53]. Sensory data has recently been added to BTK irritant testing with the objective of correlating subjective sensory effects produced by different irritants with the development of objective signs of irritation. BTK testing was able to detect subtle differences between two products, consistent across a variety of test conditions; in addition, subjective data gathered with regard to sensory discomfort demonstrated product preference identical to that predicted by irritation observed through BTK testing [54].
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Susceptibility to Irritation in the Elderly: New Techniques
. Figure 80.2 Modified Forearm Controlled Application Test (mFCAT)
Modified Forearm Controlled Application Test (mFCAT) Additional methodology, which combined a mechanical friction component to patch testing to simulate realworld exposures, was designed to distinguish among products intended to be extremely mild, such as facial tissues and baby wipes [53]. The modified Forearm Controlled Application Test (mFCAT), in which several products are tested on adjacent sites on the volar forearm (> Fig. 80.2), was modified by the addition of repeated wiping to simulate the use of lotion-infused facial tissues in cold sufferers. Testing evaluated seven different lotions on SLS- treated skin (added to simulate underlying irritation common in skin around the nostril in cold sufferers); each test site was wiped 400 times and irritation evaluated by visual scoring [2]. The technique was successful in demonstrating consistent product differences, supporting its benefit in skin testing in irritancy that results from more than just simple chemical exposure [58]. Visual scoring has been the foundation of skin irritation testing, producing excellent reproducibility with trained graders [53, 59]. Optimal testing, however, would have the ability to detect subclinical skin alterations, which occur before frank visibility occurs, an improvement in testing sensitivity, which would facilitate future product-development efforts and may prove instrumental in understanding the currently elusive connection between irritancy and skin sensitivity (which often occurs without objective signs). There are several investigative approaches designed to increase the sensitivity of scoring. Methods include enhancement of visual scoring, measurement of inflammatory components, and use of novel instrumentation.
. Figure 80.3 Enhancing visual grading by using Cross-Polarization Techniques such as the v600™ Instrument
Cross-Polarization One promising approach focuses on the improvement of visualization through the use of polarized light, a technique that has previously demonstrated improved visualization of various dermatological conditions. The use of cross-polarized light using the v600™ (which facilitates subsurface visualization) (> Fig. 80.3) was more effective than unaided visual grading in detecting very minor irritation (upper arm testing) and more effective than unaided scoring in the differentiation of two specific products (BTK) [60]. Results of enhanced grading with the v600 sub-surface in the BTK test were also consistent with data collected on subjective sensory effects as compared with unaided visual grading (> Fig. 80.4) [60].
Susceptibility to Irritation in the Elderly: New Techniques
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. Figure 80.4 The percentage of panelists who experienced burning sensations at the test sites is shown in this figure. With every sample application, there was a significantly higher number of individuals reporting burning sensations with Pad A compared with Pad B. In addition, a significantly higher number of individuals reported pain with Pad A during the third sample application, and the sensation of the sample sticking to the skin during the second and third application. Enhanced visual scoring (sub-clinical changes) enables detection of physiological changes that are not apparent using standard visual scoring. This current investigation confirms that sensory effects correlate with visual scoring in the BTK, and confirms that sensory effects enable the differentiation between two very similar products shown in this figure * Significantly different than pad B (p < 0.05) ** Significantly different than pad B (p 0.001)
Enhanced visualization through polarized light was recently employed in the evaluation of dryness and irritation in women with unexplained vulvovaginal pain [61]. Subsurface visualization through cross-polarized light was more sensitive in detecting genital erythema and dryness at all sites regardless of whether or not clinical symptoms were present. In addition, subsurface inflammation of specific anatomical sites was observed to be specific to women with vulvar vestibulitis syndrome, demonstrating physical signs in a condition heretofore believed to be of sensory origin [61].
Cytokine Mediators Skin irritation testing has also been recently performed through measurement of cytokine mediators of
. Figure 80.5 Infared thermographic scanner using the DermaTemp™
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Susceptibility to Irritation in the Elderly: New Techniques
inflammation. Cytokine levels were measured in uncompromised skin as well as skin experiencing diaper or heat rash, skin which was SLS-treated, and skin that was sunexposed, through the use of an adhesive tape which absorbs oils. Tape was applied to skin and then processed to quantify levels of Il-1a, Il-1RA, and IL-8. Il-1 levels were demonstrated to be higher in diaper rash, heat rash, and SLS-treated samples than in normal controls [62].
Infrared Thermographic Another approach to assessing irritation through quantification of sub-clinical inflammation involves measuring skin surface temperature via an infrared thermographic scanner (> Fig. 80.5). Skin temperature changes were consistent and proportional to visual signs of irritation, but were less consistently associated with subjective sensory effects than were with visual scoring of erythema [63].
Increasing Clinical Sensitivity for the Elderly Improved sensitivity of testing methods, with enhanced ability to detect subclinical changes in the structure and physiological function of skin, are profoundly relevant to the burgeoning population of older individual in different societies. Visible signs of irritation in older individuals are delayed and less intense than in younger people, despite the fact that older people are far more affected by skin irritation in general in the form of dryness and chronic itch. A recent, comprehensive evaluation of the results of irritant testing, which specifically utilized BTK testing and compared younger and older individuals, observed a consistent trend toward decreased erythema but increased skin irritation with age [64].
Conclusion The skin of older subjects is compromised both structurally and functionally by the intrinsic and extrinsic effects of aging, and may therefore be less effective at providing a barrier between the vulnerable internal tissues and the environment outside. Contact irritation is a potential sequelae to a comprised barrier. Surprisingly, with age, structural changes which produce attenuation of barrier function, impacting absorption, permeability, and vascular health, appear to be compensated for by increased SC thickness and a retardation of the desquamation process. The elderly thus appear to be less susceptible to irritant
responses. Traditional irritant testing, however, relies on visual scoring of erythema and other physical signs of irritation, while patients often report sensory discomfort in the absence of physical signs. Increasing the sensitivity of current skin testing methods, through intensifying exposure or by increasing sensitivity of scoring, is needed. Several new testing approaches are increasing the ability to define differences in irritant potential among even the mildest of consumer products, as well as providing some correlation between subclinical skin changes and discomfort that has been formerly considered purely sensory in nature. In addition, results from these recently developed techniques that provide correlation between objective and subjective data, suggest that although objective signs of irritation decrease with age, sensory discomfort from dry, defective, and pruritic skin increases. It is also important to recognize that the decrease in inflammatory response in the elderly has the potential to negatively affect patient health. Elderly patients are at higher risk of serious injury, for example, from burns, because early warning signs do not rapidly appear. Medicaments or other treatments may cause irritation or even skin damage, yet they are continued because objective signs of irritation are absent [12]. In addition, impairment in percutaneous absorption discourages penetration of toxins, as well as potentially therapeutic topical medicines. The reduction in clearance of penetrants also means that both toxins and medicines remain in skin tissues for a longer amount of time. An understanding of the ramifications of differences of penetration, absorption, and clearance of contact irritants in aged skin, as well as the unique presentation of contact irritation in older patients, is necessary for optimal therapeutic choices and efficacious dosing in the treatment of dermatological conditions in the aged patient [12].
Cross-references > Bioengineering > Cutaneous
Methods and Skin Aging Effects and Sensitive Skin with Incontinence
in the Aged Contact Dermatitis > Percutaneous Penetration of Chemicals and Aging Skin > Irritant
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66. Christopher E, Kligman A. Percutaneous absorption in aged skin. In: Montagna W (ed) Advances in Biology of the Skin. Oxford: Oxford University Press, 1965. 67. Summers PR, Hunn J. Unique dermatologic aspects of the postmenopausal vulva. Clin Obstet Gynecol. 2007;50:745–751. 68. Elsner P, Wilhelm D, Maibach HI. Frictional properties of human forearm and vulvar skin: influence of age and correlation with transepidermal water loss and capacitance. Dermatologica. 1990; 181:88–91. 69. Thune P. Evaluation of the hydration and the water-holding capacity in atopic skin and so-called dry skin. Acta Derm Venereol Suppl (Stockh). 1989;144:133–135. 70. Wilhelm KP, Cua AB, Maibach HI. Skin aging. Effect on transepidermal water loss, stratum corneum hydration, skin surface pH, and casual sebum content. Arch Dermatol. 1991;127:1806–1809. 71. Potts RO, Buras EMJ, Chrisman DAJ. Changes with age in the moisture content of human skin. J Invest Dermatol. 1984;82:97–100. 72. Akimoto K, Yoshikawa N, Higaki Y, et al. Quantitative analysis of stratum corneum lipids in xerosis and asteatotic eczema. J Dermatol. 1993;20:1–6. 73. Le´veˆque JL, Franc¸ois G, Sojic N, et al. A new technique to in vivo study the corneocyte features at the surface of the skin. Skin Res Technol. 2008;14:468–471. 74. Ye J, Calhoun C, Feingold K, et al. Age-related changes in the IL-1 gene family and their receptors before and after barrier abrogation. J Invest Dermatol. 1999;112:543 [Abstract 125]. 75. Ghadially R, Brown BE, Hanley K, et al. Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice. J Invest Dermatol. 1996;106:1064–1069. 76. Marchell NL, Uchida Y, Brown BE, et al. Glucosylceramides stimulate mitogenesis in aged murine epidermis. J Invest Dermatol. 1998;110:383–387. 77. Engelke M, Jensen JM, Ekanayake-Mudiyanselage S, et al. Effects of xerosis and ageing on epidermal proliferation and differentiation. Br J Dermatol. 1997;137:219–225. 78. Cua AB, Wilhelm KP, Maibach HI. Frictional properties of human skin: relation to age, sex and anatomical region, stratum corneum hydration and transepidermal water loss. Br J Dermatol. 1990;123: 473–479. 79. Thune P, Nilsen T, Hanstad IK, et al. The water barrier function of the skin in relation to the water content of stratum corneum, pH and skin lipids. The effect of alkaline soap and syndet on dry skin in elderly, non-atopic patients. Acta Derm Venereol. 1988;68:277–283. 80. Leveque JL, Corcuff P, de Rigal J, et al. In vivo studies of the evolution of physical properties of the human skin with age. Int J Dermatol. 1984;23:322–329. 81. Baker H, Blair CP. Cell replacement in the human stratum corneum in old age. Br J Dermatol. 1968;80:367–372. 82. Elsner P, Wilhelm D, Maibach HI. Sodium lauryl sulfate-induced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal women. J Am Acad Dermatol. 1990;23: 648–652.
88 The Baumann Skin Typing System Leslie S. Baumann
Introduction Since the ancient civilizations of Egypt, Greece, and Rome thousands of years ago, human beings have created cosmetic formulations to alter the function or appearance of the skin, especially facial skin. This practice has continued into current times, clearly at a vast and commercial level, and has significant implications in the topical realm of the practice of cosmetic dermatology. The contemporary cosmetic and skin care product market actually took root in 1915, courtesy of the intense rivalry between the ambitious cosmetics entrepreneurs Helena Rubinstein and Elizabeth Arden. Both women started salons that year that would ultimately expand into powerful business empires. In the subsequent years, the labels ‘‘dry,’’ ‘‘oily,’’ ‘‘combination,’’ and ‘‘sensitive’’ have been the primary ways to describe the four basic skin types, as identified by Helena Rubinstein. While these categories have held sway, remaining the stagnant standards for understanding skin type for over three-quarters of a century, the cosmetics market has expanded almost geometrically, developing into a multi-billion-dollar industry and giving rise to a new category of products called ‘‘cosmeceuticals’’ – cosmetic formulations that may alter the biological function of skin. Recent sales estimates and actual figures reveal just how popular these products have become. In November 2004, sales figures from earlier that year contributed to projections of $6.4 billion in sales of skin care cosmeceuticals in the USA, an increase of 7.3% from the previous year [1]. More recently, such lofty sales expectations have been surpassed; by spring 2006, cosmeceuticals sales in the USA had exploded to the $12 billion level [2]. The understanding or classifying of skin type has undergone comparatively few changes to coincide with the exponential growth of the skin care product market during the past century. The traditional skin-type categories have even come to be considered inadequate characterizations, especially given their limitations in assisting physicians and consumers in identifying the most suitable formulations. This is particularly significant because an increasing number of products are developed and
advertised for specific skin types, usually dry or sensitive skin. What has become gradually apparent over time is that the labels identified by Rubinstein do not fully address various other clinically observed skin features, including resistance and pigmentation or wrinkling proclivities. The innovative Baumann Skin Typing System (BSTS) classifies skin type using three of Rubinstein’s categories as poles in four dichotomous parameters that are simultaneously considered. In other words, the skin is not simply dry, oily, sensitive, or a combination of these qualities. Instead, the skin can be seen as dry or oily; sensitive or resistant; pigmented or non-pigmented; and wrinkled or unwrinkled (tight). Further, each dichotomy represents a continuum on which an individual’s prevailing skin characteristics can be identified and characterized. Simultaneously assessing the skin based on these four non-mutually exclusive parameters yields 16 potential skin-type permutations (> Table 88.1). The BSTS designations for each category of skin type are derived from a 64-item questionnaire, the Baumann Skin Type Indicator (BSTI), designed to establish skin type identifications at baseline as well as after significant life or environmental changes, as skin type is not necessarily static and can be influenced by stress and the environment [3]. The BSTS, by nature, offers specific guidance for physicians and patients/consumers in identifying the most suitable skin products, as it considers multiple concurrent cutaneous characteristics. That is to say, the questionnaire yields significant data regarding an individual’s skin type. The author follows up with a thorough discussion of the corresponding cutaneous needs associated with each of the 16 skin types. For example, a person with dry, sensitive, pigmented, wrinkled skin would require significantly different skin care products than a person with oily, resistant, non-pigmented, unwrinkled skin. Notably, individuals with sensitive skin need more assistance in anti-aging skin care. The four parameters on which the BSTI is based will guide the discussion in this chapter. Emphasis will be placed on defining the characteristics of these dichotomies and focusing on pertinent basic science. Various
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_88, # Springer-Verlag Berlin Heidelberg 2010
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. Table 88.1 The Baumann Skin Typing System (BSTS) skin type paradigm
Sensitive
Oily
Oily
Dry
Dry
Pigmented
Nonpigmented
Pigmented
Nonpigmented
OSPW
OSNW
DSPW
DSNW
Wrinkled
Sensitive
OSPT
OSNT
DSPT
DSNT
Tight
Resistant
ORPW
ORNW
DRPW
DRNW
Wrinkled
Resistant
ORPT
ORNT
DRPT
DRNT
Tight
Oily = O; Dry = D; T = Tight; W = Wrinkled; R = Resistent; S = Sensitive; P = Pigmented; N = Non-pigmented
The most recent version of the BSTI exam is accessible online to physicians, who can register at www.SkinIQ. com. Once registered, doctors can e-mail the link, with passwords, to patients so that they can self-administer the questionnaire. The score or result of the BSTI – the patient’s skin type – is e-mailed to the physician. This information can be especially beneficial in assisting doctors to identify which products and procedures are most appropriate for their patient populations. The quiz is updated often, after statisticians assess the most recent incoming data. Accordingly, new questions are developed and added to the questionnaire, and some may be deleted, so the BSTI is not always a 64-item exam. Of note, the non-identifying data culled from the site has the potential to broaden the existing knowledge of skin types and their prevalences around the world.
aspects of the 16 skin-type variations will be described in the process. Cutaneous aging is explained in the context of the wrinkled (W) to tight (T) continuum. Approaches to skin care or treatment options that follow from the BSTS will also be cited, with non-invasive, mostly topical therapies addressed.
Skin Hydration: The Oily (O) to Dry (D) Continuum Dry skin is considered to be a significantly more problematic condition than oily skin. Skin that falls in the middle of this dichotomy – skin that is adequately hydrated – is the ideal condition, of course, but one end of this spectrum is typically favored in most individuals. Oily skin is caused by the excess production of sebum; dry skin, also known as xerosis, is associated with a complex, multifactorial etiology. Oily skin is characterized by a greasy, oily quality, while dry skin is distinguished by
a dull color (usually gray white), rough texture, and an increased number of ridges [4]. The most important factors that regulate the degree of dryness/oiliness are stratum corneum lipids, sebum, natural moisturizing factor, and aquaporin. A discussion on each of these factors follows.
Stratum Corneum (SC) The role of the stratum corneum (SC), particularly its ability to maintain skin hydration, is the most significant factor in preventing or setting the stage for xerosis. The SC is composed of ceramides, cholesterol, and fatty acids, among other less active constituents. These primary constituents of the SC, when present in the appropriate amount and balance, assist in protecting the skin and keeping it watertight. The stimulation of keratinocyte lipid synthesis and keratinocyte proliferation by primary cytokines is also thought to contribute to maintaining SC equilibrium [5]. When ceramides, cholesterol, and fatty acids are improperly balanced, the SC endures a cascade of interrelated events, including a reduced capacity to maintain water and increased susceptibility to exogenous elements, thereby elevating skin surface sensitivity and ultimately leading to xerosis. Such an impairment in the SC initially leads to transepidermal water loss (TEWL). In addition, the desquamation of corneocytes is rendered abnormal, because the enzymes necessary for desmosome metabolism are inhibited by insufficient hydration [6]. Simultaneously, superficial SC desmoglein I levels remain elevated. The abnormal or compromised desquamation leads to a visible collection of keratinoctyes, manifesting in skin that appears rough and dry [7]. Increased fatty-acid levels and decreased ceramide levels cause a perturbation in the lipid bilayer of the SC, which is also linked to xerosis [8]. In addition, the lipid bilayer is susceptible to deleterious
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effects induced by exogenous factors such as ultraviolet radiation, detergents, acetone, chlorine, and prolonged water exposure or immersion. Finally, recent research suggests that local changes in pH may account for the initial cohesion and ultimate desquamation of corneocytes from the SC. These alterations are thought to selectively activate numerous extracellular proteases in a pH-dependent manner [9].
Natural Moisturizing Factor (NMF) An intracellular hygroscopic substance present only in the SC, natural moisturizing factor (NMF) is released by lamellar bodies and produced via the breakdown of the protein filaggrin. NMF plays an integral role in maintaining water within skin cells. Filaggrin, or filament-aggregating protein, consists of lactic acid, urea, citrate, and sugars. It is broken down into free amino acids, such as arginine, glutamine (glutamic acid), and histidine, by a cytosolic protease in the stratum compactum, an outer layer of the SC [10]. These water-soluble free amino acids remain in the keratinoctyes and potently bind to water molecules. Aspartate protease (cathepsin) is considered to be responsible for the rate of filaggrin decomposition and the level of NMF present [11]. Cathepsin has been shown to be susceptible to changes in external humidity, potentially causing fluctuations in NMF production. After an individual enters a low-humidity environment, NMF synthesis usually increases over the course of several days [12]. Low NMF levels are correlated with xerosis and ichthyosis vulgaris. Ultraviolet radiation as well as surfactants can suppress the development of NMF. As of now, no products or procedures have been developed that have the capacity to artificially influence or regulate NMF synthesis.
Aquaporin-3 (AQP-3) A member of a subclass of aquaporins labeled aquaglyceroporins, which transport water, glycerol, urea, and other small solutes, aquaporin-3 (AQP-3) is a water channel protein that exerts an influential role in skin hydration. Present in the kidney collecting ducts and the epidermis, as well as in the urinary, respiratory, and digestive tracts, AQP-3 is an important member in the family of homologous integral membrane proteins that selectively facilitate the transport of water and small neutral solutes across biological membranes [13]. Significantly, AQP-3 has been shown to be copiously expressed in the plasma membrane of human epidermal keratinocytes [14]. The water
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conduction function in the skin is thought to occur along an osmotic gradient beneath the SC, where high AQP-3-mediated water permeability is exhibited. In this scenario, AQP-3 water acts by clamping viable epidermal layers, thereby facilitating the hydration of skin layers below the SC. In the superficial SC, a high concentration of solutes (Na+, K+, and Cl ) and a low concentration of water (13–35%) [15] generate in the steady-state gradients of solutes and water from the skin surface to the viable epidermal keratinocytes [16–18]. The molecular mechanisms of fluid transport across epidermal keratinocyte layers and the relationship between keratinocyte fluid transport and SC hydration have not been elucidated, even though transepithelial fluid transport has been extensively studied in the kidneys and lungs. At this point, it is theorized that AQP-3 enhances transepidermal water permeability to protect the SC by preventing water evaporating from the skin surface and/or to spread water gradients throughout the epidermal keratinocyte layer [14]. In a study evaluating the functional expression of AQP-3 in human skin, researchers noted that the water permeability of human epidermal keratinocytes was inhibited by mercurials and low pH, which was consistent with AQP-3 involvement [14]. Some of the same investigators conducted a different study assessing skin phenotype in AQP-3-deficient transgenic mice, and found significantly lower water and glycerol permeability in the AQP-3-null mice, supporting previous evidence that AQP-3 functions as a plasma membrane water/glycerol transporter in the epidermis [19]. In the majority of skin areas tested in the null mice, conductance measurements revealed much lower SC water content. Epidermal cell water permeability, however, is not an important determinant of SC hydration, because water transport across AQP-3 is slower in skin than in other tissues [20]. The use of extracts of the herb Ajuga turkestanica is the only external method yet shown to enhance the activity of AQP-3 [21]. Ajuga turkestanica is currently included as an active ingredient in one high-end line of skin care products. Eventually, skin conditions mediated by excess or insufficient hydration may be treated via the pharmacologic manipulation AQP-3.
Sebum Sebum, the oily secretion of the sebaceous glands containing wax esters, sterol esters, cholesterol, di- and triglycerides, and squalene, imparts an oily quality to the skin and significantly influences the development of acne [22]. At
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normal levels, sebum, which is an important source of vitamin E, is believed to confer cutaneous protection from exogenous factors. When present in low levels, sebum is thought by some authors to play a role in the development of dry skin [23]. However, this theory has been undermined by the fact that low sebaceous gland activity has not been shown to foster xerosis. Indeed, sebum production has been found to have a more convoluted role in dry skin etiology. It has been previously speculated that sebum has no effect on epidermal permeability barrier function, mainly because skin with few sebaceous glands, as in prepubertal children, manifests normal basal barrier function [24]. Prepubertal children between 2 and 9 years old, in fact, often present with eczematous patches (pityriasis alba) on the face and trunk that do not appear with the onset of sebaceous gland activity. In addition, barrier function or SC lamellar membranes are not impacted by the pharmacologic involution of sebaceous glands with supraphysiologic isotretinoin doses [25–27]. Similarly, SC function is not interrupted with the use of ether to denude the skin. Although sebum levels do not affect barrier function, sebum may still play a role in xerosis etiology in people with dry, resistant skin (the DR type in the BSTS). Lipids from meibomian glands, which are modified sebaceous glands located in the eyes, have the capacity to stave off dryness by preventing tear evaporation [28, 29]. Likewise, sebum-derived fats may create a lipid film over the skin surface, precluding TEWL. This theory is buttressed by a study in 2003 that assessed permeability barrier homeostasis and SC hydration in asebia J1 mice with sebaceous gland hypoplasia [30]. Consistent levels of the three primary barrier lipids (ceramides, free sterols, and free fatty acids) and the persistence of normal SC extracellular membranes accounted for the normal barrier function in these sebum-deficient mice. The researchers noticed, however, that the asebia J1 mice manifested reduced SC hydration, implying that while an intact intercellular membrane bilayer system suffices for permeability barrier homeostasis, it does not necessarily lead to normal SC hydration. The investigators did note that the topical application of glycerol restored normal SC hydration. Sebaceous gland-derived triglycerides (TG) are hydrolyzed to glycerol before transport to the skin surface in normal skin. In sebum-deficient individuals, dry skin may be alleviated by replacing this glycerol. The use of glycerol has also been demonstrated to be effective in accelerating SC recovery [31]. Patients rarely complain about below-normal sebum production, but elevated sebum production, leaving an
oily film that can lead to acne, is a common complaint. The age-related trajectory of sebum production levels is well understood. During childhood, sebum levels are usually low, then rise in the middle-to-late teens, and remain relatively stable for decades, until declining in the 7th and 8th decades as endogenous androgen production falls [32]. It is important to note, however, that sebum production levels are also influenced by other factors. In particular, sebum production is influenced by an individual’s genetic background and diet, as well as the person’s stress and hormone levels. A fascinating study of 20 pairs of identical and non-identical like-sex twins was especially informative. Almost equivalent sebum excretion rates with significantly divergent acne severity was observed in the identical twins, but there were significant variations in both parameters among the non-identical twins, suggesting that both genetic factors and environmental factors affect acne development [33]. Oral retinoids have been well established as effective in reducing sebaceous gland activity, but topical retinoids have not yet been demonstrated to exhibit this capacity. Further, no other topical formulations have been demonstrated to diminish sebum production.
Skin Care Along the O–D Continuum An intact SC and skin barrier, normal levels of NMF and hyaluronic acid (HA), normal AQP-3 expression, and balanced sebum secretion together characterize an ideal cutaneous state, which, in terms of the oily–dry continuum, would fall in the middle of the spectrum. Increased sebum secretion positions an individual’s skin on the oily side of this parameter, regardless of whether or not acne is manifested. Oily skin accompanied by acne falls under the oily, sensitive (OS) categories in the BSTS framework, because skin affected by acne is distinguished by heightened sensitivity. Treatment of individuals with OS skin should be aimed at decreasing sebum levels with retinoids; eradicating or reducing skin bacteria with antibiotics, benzoyl peroxide, or other antimicrobials; and easing inflammation using anti-inflammatory agents. Treatment of individuals exhibiting oily skin without acne, which translates to the oily, resistant (OR) BSTS categories, should be focused on reducing sebum production, unless key elements of other parameters (e.g., dyspigmentation and wrinkling) are factors. Oral medications, specifically ketoconazole and retinoids, have been successfully used to suppress sebum secretion [34, 35], but such results have not yet been duplicated using topical agents. In addition,
The Baumann Skin Typing System
sebum-absorbing polymers and talcs are effective in camouflaging the sebum in OR skin. An impaired skin barrier and diminished NMF characterize xerotic skin, particularly dry skin chronically exposed to the sun. Treatment of individuals with such skin, which would fall under the dry, wrinkled (DW) categories in the BSTS, should concentrate on skin barrier repair and curtailing sun exposure, avoiding it when possible and otherwise using adequate sun protection. Harsh foaming detergents (present in laundry and dish cleansers as well as body and facial cleansers) remove hydrating lipids and NMF from the skin, and should be avoided by all patients with dry skin. Individuals with dry skin should also be advised to abstain from protracted bathing, especially in hot or chlorinated water. It is recommended that people with extremely dry skin use humidifiers in low-humidity environments, and apply moisturizers two to three times daily and after bathing. Several OTC moisturizers (e.g., occlusives, humectants, and emollients) are effective in hydrating the skin. In fact, of all the OTC topical skin care product types, moisturizers are the third most frequently recommended [36]. Moisturizers are typically packaged as water-in-oil emulsions (e.g., hand creams) and oil-in-water emulsions (e.g., creams and lotions). A brief discussion follows of the differences among moisturizer types, which is important to a practitioner’s knowledge base in terms of offering appropriate product selection recommendations to patients.
Occlusives Oily compounds that can dissolve fats, occlusive agents are incorporated into skin care formulations in order to coat the SC and prevent TEWL. In addition to inhibiting TEWL, occlusives exhibit emollient properties, and are therefore appropriate products for treating dry skin. Petrolatum and mineral oil are thought to be the most effective occlusive ingredients. Used as a skin care product since 1872, petrolatum is also considered one of the best moisturizers, as well as the gold standard by which other occlusive agents are measured [37]. Notably, petrolatum is thought to display a resistance to water vapor loss that is 170 times that of olive oil [38]. Unfortunately, many consumers deem petrolatum to be cosmetically unacceptable because of its greasy texture. Other frequently used occlusive ingredients include paraffin, squalene, silicone derivatives (dimethicone, cyclomethicone), soybean oil, grapeseed oil, propylene glycol, lanolin, lecithin, stearyl stearate, and beeswax [39, 40]. Lanolin, which is derived
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from the sebaceous secretions of sheep, warrants special mention. It contains the important SC lipid cholesterol, and can coexist with SC lipids as solids and liquids at physiologic temperatures. However, lanolin has been identified as a sensitizer by enough people to elicit changes in marketing strategies, although it has been shown to be only a weak allergen [41]. Indeed, some consumers may eschew lanolin because it contains animal products. Lanolin remains widely used, but it has become less popular for the above-stated reasons, and manufacturers have responded to changes in consumers’ preferences by labeling several products as ‘‘lanolin-free.’’ Regardless of the specific ingredients, it is important to note that no occlusive ingredients confer long-lasting benefits. Once an occlusive product is removed from the skin, TEWL returns to its previous level. Because the reduction of TEWL by more than 40% poses a risk of maceration, with increased levels of bacteria, occlusive agents are typically used in combination with humectant ingredients [42].
Propylene Glycol (PG) Functioning as both a humectant and an occlusive agent, propylene glycol (PG) is an odorless liquid that also exhibits antimicrobial and keratolytic activity. In addition, PG has been demonstrated to contribute to the cellular penetration of some drugs, such as minoxidil and steroids. Although believed to be a weak sensitizer, PG may provoke or factor into contact dermatitis by facilitating allergen penetration into the epidermis [43].
Humectants Humectants are hygroscopic, water-soluble substances. In conditions with at least 80% humidity, humectants applied to the skin exhibit the capacity to attract water from the external environment, as well as from the underlying skin layers, to the skin surface. In low-humidity conditions, however, humectants applied to the skin can absorb water from the deeper epidermis and dermis, thus contributing to TEWL and exacerbating xerosis [44]. Combining humectants with occlusive products can help prevent such results. Cosmetic moisturizers are formulated with humectants in order to prevent product evaporation and thickening, thus extending the product’s shelf life. It is especially important for patients/consumers to
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understand that these products do not provide long-lasting anti-wrinkle effects on the skin. Indeed, by drawing water into the skin, humectants engender a minor swelling of the SC, leaving a perception of smoother skin with fewer wrinkles. Notably, some humectants impart other benefits, including bacteriostatic properties [45]. Glycerin and glycerol are considered the most effective humectant ingredients found in skin care products. Alpha hydroxy acids, carboxylic acid, gelatin, honey and other sugars, panthenol, propylene glycol, sodium hyaluronate, sodium and ammonium lactate, sodium pyrrolidine, sorbitol, and urea are among other substances that function as active humectant ingredients [40]. Effective moisturizers typically incorporate occlusive as well as humectant ingredients. It is worth noting that numerous humectant formulations also display emollient activity [46].
lingered regarding the ability of urea to promote such action. In 2005, the Cosmetic Ingredient Review (CIR) Expert Panel declared that urea does indeed have the capacity to enhance the percutaneous absorption of other chemicals and, further, that urea is safe for use in cosmetic products [52]. Regarding its humectant activity, a 3-week double-blind study comparing 3% and 10% urea cream revealed the study formulations to be more effective in ameliorating clinical signs of dry skin than the vehicle control. Both creams successfully reduced scaling and enhanced hydration. The 3% cream caused the skin to appear golden or yellow and had no impact on TEWL, whereas the 10% cream reduced TEWL, although subjects reported the creams to be equally effective [53].
Glycerin
Alpha hydroxy acids (AHAs) are naturally-occurring organic acids that have been discovered to exhibit humectant and exfoliant activity. Glycolic and lactic acids, respectively derived from sugar cane and sour milk, are the AHAs most often used in moisturizing products and were the first ones to become commercially available. Citric acid, malic acid, and tartaric acid are among the other AHAs. Topical preparations that contain AHAs were demonstrated to confer significant effects on epidermal keratinization more than 30 years ago [54]. Just over a decade ago, glycolic acid was shown to act as a photoprotective agent [55]. Salicylic acid, the only beta hydroxy acid (BHA), is derived from willow bark, wintergreen leaves, and sweet birch. BHA functions as a chemical exfoliant, and is found in synthetic form in several topical formulations [56]. At the lowest levels of the SC, corneocyte cohesiveness is attacked and eroded by AHAs and BHA, influencing pH in the process, as these ingredients break down desmosomes, thus contributing to desquamation [57, 58].
Displaying hygroscopic activity comparable to that of NMF, glycerin is considered a potent humectant [4]. Using ultrastructural analyses of skin treated with highglycerin formulations, investigators have demonstrated that this humectant expands the SC by enhancing corneocyte thickness and creating greater distance between corneocyte layers [47]. In addition, after a 5-year study that compared two high-glycerin moisturizers with 16 other popular moisturizers (including petrolatum preparations) used by 394 patients with severe xerosis, researchers reported that the high-glycerin products were the most effective, rapidly restoring dry skin to normal hydration with longer-lasting results than the other products [7]. Glycerin has also been shown to stabilize and hydrate cell membranes along with the enzymes essential for desmosome degradation[7].
Urea Also known as carbamide, urea, perhaps better known as the main nitrogen-containing ingredient of urine, is an end product of mammalian protein metabolism, as well as an NMF constituent. This versatile compound exhibits humectant and mild anti-pruritic activity [48]. Urea has been included as an ingredient in several hand cream formulations since the 1940s [49]. In addition, it has been successfully used in combination with hydrocortisone, retinoic acid, and other ingredients to facilitate the cutaneous penetration of these agents [50, 51]. However, despite such findings in the mid-to-late 1980s, skepticism
Hydroxy Acids
Lactic Acid Significantly, this prominent AHA is also a component of NMF. Lactic acid was first used as part of the dermatologic armamentarium in 1943, for the treatment of ichthyosis [59]. Since then, in vitro and in vivo experiments have demonstrated that lactic acid can augment ceramide synthesis by keratinocytes [49, 60]. This moisturizing AHA ingredient has also been shown to combat signs of photoaging. Specifically, 8% L-lactic acid was found to be superior to the vehicle in a double-blind
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vehicle-controlled study, with statistically significant improvements measured in sallowness, skin coarseness, and blotchiness [61].
collagen and polypeptide ingredients have little or no effect on TEWL, but are usually labeled as moisturizers and firming creams.
Emollients
Skin Sensitivity: The Sensitive (S) to Resistant (R) Continuum
Comprised primarily of lipids and oils, emollients are included in cosmetics to hydrate, soften, and smoothen the skin. These substances fill in the gaps between desquamating corneocytes, yielding a smooth skin surface [39]. In addition, emollient formulations improve cohesion, flattening out the curled edges of individual corneocytes [4]. A smoother skin surface, in turn, lessens friction, while enhancing light refraction. Emollient ingredients are divided into classes of compounds, including those that exhibit astringent, desiccating, fatting, protective, and protein rejuvenating activity [40]. Further, several of the numerous emollients function as both humectant and occlusive moisturizers. An emollient effect is also imparted by ingredients that are considered primarily occlusive agents, such as lanolin, mineral oil, and petrolatum. Reports of adverse effects linked to moisturizers are very rare. There have been reports of allergic contact dermatitis associated with products that contain preservatives, perfumes, solubilizers, sunscreens, and some other classes of compounds. Specifically, cases of contact dermatitis involving lanolin, propylene glycol, vitamin E, and Kathon CG have been reported [62, 63].
Collagen and Polypeptide Ingredients It is important for physicians and patients to know that the preponderance of collagen ‘‘extracts’’ contained in the host of expensive moisturizers touted for the capacity to restore collagen lost due to aging have a molecular weight of 15,000–50,000 Da, but only compounds with a molecular weight of 5,000 Da or less can actually penetrate the SC [42]. In other words, these products cannot deliver on their advertised claims of replacing collagen. However, the collagen and other hydrolyzed proteins and polypeptides yield a temporary film on the epidermis that, upon drying, fills in surface depressions and other irregularities. Essentially, the film generated by these products provides a subtle stretching out of fine skin wrinkles. Using a humectant product can further enhance the fuller or somewhat plumper appearance created by collagen and polypeptide ingredients. Formulations that contain
Sensitive skin is considerably more complex and challenging to describe than is resistant skin. A robust SC that strongly protects the skin from allergens and other environmental irritants characterizes resistant skin. Individuals with resistant skin rarely experience erythema or acne. In such skin, erythema may appear if an individual is overexposed to the sun; acne may emerge as a result of stress or hormonal fluctuations. In terms of skin care product usage, resistant skin might be considered a double-edged sword. That is to say, individuals with resistant skin can use most skin care products without experiencing adverse reactions (e.g., acne, rashes, or a stinging response). However, the same qualities that shield resistant skin from adverse responses to the preponderance of topical formulations available also render many products ineffective, as people with such skin manifest an extremely high threshold for the penetration and bioefficacy of product ingredients. Consequently, individuals with resistant skin tend not to benefit from a majority of products, or may be unable to detect varying effects among cosmetic skin care formulations because most products are too weak to penetrate the potent SC of such people, and are thus rendered ineffective in achieving the intended results. Sensitive skin is an increasingly common complaint [64]. Healthy women of childbearing age comprise the majority of patients who present to a dermatologist reporting complaints of sensitive skin. While the prevalence of sensitive skin seems to be on the rise, the incidence of sensitive skin appears to decline with age, as implied by the previous demographic statement. With the overall increase in the prevalence of the sensitive skin types, the number of products marketed specifically for the treatment of sensitive skin has risen accordingly. It is important to note, however, that sensitive skin has various qualities and presentations. In fact, sensitive skin can be accurately categorized into four discrete subtypes: acne type (proclivity to develop acne, black heads, or white heads); rosacea type (propensity to experience recurrent flushing, facial redness, and hot sensations); stinging type (tendency to experience stinging or burning sensations); and allergic type (prone to manifesting erythema, pruritus, and skin flaking). Such variations in
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sensitive skin characteristics present treatment challenges to dermatologists, as products intended for sensitive skin are not designed to address these subtypes and are therefore not suitable for all sensitive skin subtypes. The four subtypes of sensitive skin do, however, share one salient quality: inflammation. Consequently, any sensitive skin treatment program must focus on reducing and eradicating inflammation. Treatment regimens for patients that present with more than one type of sensitive skin are understandably more complex, as the practitioner must carefully calibrate therapeutic options based on the relative intensity of patients’ various susceptibilities.
Acne Type Although incidence and prevalence rates are difficult to measure, acne is estimated to affect 17 million people in the USA [65], and is easily the most common skin disease, usually affecting adolescents and young adults between the ages of 11 and 30 years, affecting both genders equally. In fact, it is estimated that 80% of all people in this age group experience an acne outbreak at some point [66]. Adult women, who display a hormonal aspect to their acne, are the second largest demographic group among acne sufferers. The confluence of four primary factors has been implicated in the pathogenesis of this conspicuous and consequently stressful disorder, including: elevated sebum production; clogged pores, resulting from dead keratinocytes inside the hair follicles clinging more strongly than in those without acne (increased sebum production may also foster cellular adherence); the presence of the bacteria Propionibacterium acnes; and inflammation. Although acne can arise through various idiopathic courses, not always involving the main identified etiologic factors, the characteristic feature of this condition is the adherence of dead keratinocytes in the hair follicles due to augmented sebum production, leading to clogged follicles and the appearance of a papule or pustule. P. acnes then move into the hair follicle, mixing with the amassed sebum and dead keratinocytes. This interaction incites the release of cytokines and other inflammatory factors that initiate the inflammatory response, resulting in the development of the quintessential redness and pus. High levels of primary cytokines, chemokines, and other inflammatory markers are indeed typically present in various chronic inflammatory skin conditions such as acne [5]. Acne therapy targets the four primary etiologic factors: decreasing sebum production (with retinoids, oral contraceptives, or stress reduction), unclogging pores (with retinoids, AHAs, or BHA), eliminating
bacteria (with benzoyl peroxide, sulfur, antibiotics, or azelaic acid), and reducing inflammation.
Rosacea Type Approximately 14 million Americans, usually adults between 25 and 60 years of age, are affected by rosacea [67]. This acneiform disorder, like acne, is associated with facial redness, flushing, and papules; however, rosacea is also characterized by the formation of prominent telangiectases, the primary manifestation of this condition. Although the pathophysiology of rosacea remains to be elucidated, a recent study by Yamasaki et al. showed that rosacea symptoms can be aggravated by factors that initiate an innate immune response, particularly the release of cathelicidin antimicrobial peptides; cathelicidin was demonstrated to be expressed in unusually high levels in the facial skin of rosacea patients and to play a role in cutaneous inflammatory responses [68]. This suggests newer directions in the study of the pathogenetic pathways for this distressing condition. In the meantime, rosacea therapy focuses on topical formulations that include anti-inflammatory ingredients to decrease the dilation of the blood vessels, and behavioral adjustments, specifically the avoidance of exposure to factors known to trigger or exacerbate symptoms. The goal of medical therapy for rosacea is to diminish vascular reactivity; neutralize free radicals or reactive oxygen species (ROS); hinder immune function; and interfere with eosinophilic activity, the arachidonic acid pathway, and the degranulation of mast cells (which often colocalize to areas of eosinophil-mediated disease). Eosinophils, which are pleiotropic multifunctional leukocytes, contribute to initiating and promoting several inflammatory responses [69, 70]. In the plethora of topical rosacea formulations, the most effective anti-inflammatory ingredients (several of which are botanically derived) include aloe vera, arnica, chamomile, colloidal oatmeal, cucumber extract, feverfew, licochalcone, niacinamide, quadrinone, salicylic acid, sulfacetamide, sulfur, witch hazel, and zinc [71]. Significantly, the rosacea armamentarium includes various prescription anti-inflammatory products such as antibiotics, immune modulators, and steroids.
Stinging Type In reaction to various triggers, some people experience a stinging sensation, which is a non-allergic neural sensitivity. The stinging propensity, or ‘‘stingers,’’ can be
The Baumann Skin Typing System
identified through various available tests. In particular, the lactic acid stinging test is well regarded, and is an established method for assessing patients who report invisible and subjective cutaneous irritation. It is worth noting that the stinging response is not necessarily associated with erythema; many patients experience stinging without exhibiting redness [72]. However, rosacea patients who manifest facial flushing are more susceptible to feeling the stinging response when exposed to lactic acid [73]. Patients who are confirmed to have the stinging subtype of sensitive skin should be advised to avoid topical products containing the following ingredients: alpha hydroxy acids (particularly glycolic acid), benzoic acid, bronopol, cinnamic acid compounds, Dowicel 200, formaldehyde, lactic acid, propylene glycol, quaternary ammonium compounds, sodium lauryl sulfate, sorbic acid, urea, and vitamin C.
Allergic Type An epidemiologic survey in the UK, published in 2004, reported that 23% of women and 13.8% of men displayed adverse reactions to a personal care product (e.g., deodorants and perfumes, skin care products, hair care products, and nail cosmetics) over the course of 1 year [74]. More recently, in a 1999–2006 Brazilian study of 176 patients (154 women and 22 men) seen in a private office who complained of dermatoses resulting from cosmetics, 45% had dermatoses linked to cosmetics and 14% had skin lesions that were found to be caused by inappropriate use of cosmetics [75]. In addition, several studies have demonstrated that approximately 10% of dermatologic patients who are patch tested for 20–100 ingredients exhibit allergic sensitivity to at least one ingredient common in cosmetic products [74]. Fragrances and preservatives are the most common allergens, and women aged 20–60 years represent the demographic group that experiences the majority of these reactions [76]. Individuals who are overexposed to skin care products and patients with an impaired SC, as manifested by dry skin, reportedly have increased susceptibility to allergic reactions [77]. These findings underscore both the significance of the allergic subtype as well as the need for matching skin type and skin care products, which the BSTS facilitates.
Treatment Approaches for Sensitive Skin The principles underlying the BSTS suggest that individuals with oily, sensitive (OS) skin require oil control. A person with OS skin is also likely to need an acne or
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rosacea treatment regimen. Skin barrier repair is needed in those with dry, sensitive (DS) skin. Lessening the depth or severity of existing wrinkles and preventing the formation of new ones is the focus for patients with sensitive, wrinkled (SW) skin. Individuals with sensitive, pigmented (SP) skin usually seek the removal of pigmentary lesions, as well as a treatment regimen and/or behavioral modification that will reduce the likelihood or frequency of additional undesired pigmentary alterations.
Skin Pigmentation: the Pigmented (P) to Nonpigmented (N) Continuum This skin-type parameter refers not to skin color, but to the tendency to develop hyperpigmentations, mainly on the face or chest. Within the BSTS framework, pigmentary conditions or changes that can be ameliorated using topical formulations or minor dermatologic procedures (such as ephelides, melasma, post-inflammatory hyperpigmentation, and solar lentigos) are the focus. Congenital nevi, seborrheic keratoses, and other skin lesions that require excision or treatment beyond topical skin care are beyond the scope of the BSTS. The mechanisms of pigmentation should be clearly understood in order to prepare physicians to treat these anxiety-producing pigmentary conditions. The enzymatic breakdown of tyrosine by tyrosinase into dihydrophenylalanine (DOPA) and then dopaquinone, ultimately results in the production of the skin pigment melanin, specifically the two melanin types, eumelanin and pheomelanin [78]. Under normal circumstances, melanin is produced by melanocytes, and then transferred via melanosomes to keratinocytes. However, melanogenesis can also by induced by UV exposure. Under these circumstances, melanin production represents the cutaneous defense against the insult of UV irradiation. In this scenario, melanocytes accelerate melanin synthesis and its transfer to keratinoctyes [79], leading to skin darkening in the affected regions. Eumelanin, which is the more abundant type of melanin, regularly correlates with the visual phenotype [80]. More melanin is synthesized in individuals with darker skin as compared to people with lighter skin. The melanosomes are larger in darker-skinned individuals than in lighter-skinned people; therefore, melanosomes in darker-skinned individuals accommodate more melanin and decompose more slowly [81]. Melanocytes, each of which is typically attached to about 30 keratinocytes, load melanin in the process of transferring through melanosomes, and then link to keratinocytes. Melanosomes are surrounded by
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keratinocytes, which absorb the melanin after the activation of the protease-activated receptor (PAR)-2 [82]. A seven-membrane G-protein-coupled trypsin/tryptase receptor activated by a serine protease cleavage, PAR-2 is expressed in keratinocytes, but not in melanocytes. PAR-2 is believed to regulate melanosome transfer, and thus pigmentation, through interactions between keratinocytes and melanocytes [83]. Cutaneous pigmentation can be hindered via two primary pathways: the inhibition of tyrosinase, which prevents melanin formation, and impeding melanin transfer into keratinocytes. Hydroquinone, vitamin C, kojic acid, arbutin, mulberry extract, and licorice extract are considered effective inhibitors of tyrosinase. Soybean trypsin inhibitor (STI) and Bowman-Birk inhibitor (BBI), which are proteins contained in soy, have been found to exhibit the capacity to inhibit skin pigmentation development. STI and BBI have also been shown, in vitro and in vivo, to prevent UV-induced pigmentation [84]. Melanosome transfer into keratinocytes is influenced by STI and BBI by dint of their inhibition of the cleavage of PAR-2. The introduction of niacinamide, a vitamin B3 derivative, has also been demonstrated to impede the transfer of melanosomes to keratinocytes [85]. Soy and niacinamide, which are considered the most effective PAR-2 blockers, are the main agents for inhibiting melanin transfer to keratinocytes. There are three types of topical agents known to be effective within the two approaches to inhibiting melanin formation. In addition to the tyrosinase inhibitors and PAR-2 blockers, exfoliating agents such as AHAs, BHA, and retinoids can sufficiently accelerate cell turnover to outpace melanin synthesis. Melanin formation can also be hindered through the use of procedures such as microdermabrasion and instruments such as facial scrubs. A broad-spectrum sunscreen should also be included in any skin care regimen intended to diminish or eliminate the development of undesired dyschromias. In all cases, the most effective way to prevent pigmentary and even more harmful changes to the skin is to practice sun avoidance, within reason. A high score on the BSTI questionnaire, reflecting the ‘‘P’’ skin type, correlates with a tendency to develop unwanted pigmentary changes; a person with the ‘‘N’’ skin type does not exhibit this tendency.
Skin Aging: The Wrinkled (W) to Tight (T) Continuum Exogenous and endogenous factors play considerable roles in the complex, multifactorial process of cutaneous
aging. Traditionally, the etiologic pathways of skin aging have been considered discrete phenomena leading to similar manifestations. Extrinsic aging, which results from chronic exposure to various environmental insults, particularly UV radiation, is avoidable by nature and definition. Natural intrinsic aging is genetically driven or cellularly programmed, and is thus inevitable. Both pathways ultimately manifest in visible skin alterations, particularly wrinkles, lost elasticity, and hyperpigmentation. Interestingly, recent research findings suggest that UV radiation, the primary factor accounting for extrinsic aging, may actually alter the natural course of cellular aging. If such results are substantiated through further research, intrinsic and extrinsic aging may come to be seen as less distinct and, in fact, interrelated or interdependent processes. The following discussion will treat these processes separately, briefly considering intrinsic aging, as the primary emphasis is on extrinsic aging, which has been more extensively studied, and its implications regarding BSTS skin type. According to one current theory on aging, in recent years intrinsic aging has come to be associated with or understood in terms of the function of telomeres, specialized structures that protect the ends of chromosomes. Telomere length is known to decrease with increasing age. Charting this erosion is considered equivalent to a method of measuring chronologic aging. Indeed, this mechanism, which serves as a veritable internal aging clock, is the basis for one of the presently favored theories on aging, the telomere shortening theory [86]. According to this theory, telomerase, the enzyme that stabilizes or lengthens telomeres, is expressed in approximately 90% of all tumors, but is absent from many somatic tissues [86]. The implications of this theory are fascinating, as it is suggested that most cancer cells, unlike healthy ones, are not programmed for apoptosis, or cell death. Consequently, aging and cancer are considered to be the proverbial opposite sides of the same coin. Particularly of interest to dermatologists, the epidermis is one of the few regenerative tissues to express telomerase [87]. Given that current data does not adequately account for the safety of extending telomere length, no treatment options are yet available that target telomerase. In addition to the telomere shortening theory on aging, other theories on the etiology of cutaneous aging include cellular senescence (Hayflick limit), genetics (genes and mutations), hormone deficiency, the mtDNA theory, and oxidative stress [88]. As implied in its definition, extrinsic aging is subject to human volition and therefore preventable. In other words, individuals can control their exposure to the
The Baumann Skin Typing System
causes or risk factors of exogenous aging, which include excessive alcohol consumption, smoking, other forms of pollution, poor nutrition, and the primary culprit, solar exposure. The diverse mechanisms through which UV radiation results in damage to the skin include the development of sunburn cells, as well as pyrimidine and thymine dimers; collagenase synthesis; and the promotion of an inflammatory response. Aging and photodamage, in particular, have been associated with signaling through the p53 pathway after UV (especially UVB)-induced telomere disruption [89, 90]. Although much remains to be learned regarding the mechanisms by which UV irradiation initiates and promotes deleterious effects, UV (particularly UVA) irradiation is well known, essentially by definition, as the cause of photoaging, photocarcinogenesis, and photo-immunosuppression [91]. Insofar as UV irradiation impairs DNA and accelerates telomere shortening, this chief cause of extrinsic aging can be thought of as exerting an impact on the natural course of intrinsic aging. The primary evidence of cutaneous aging is the development of rhytides, the formation of which is initiated in the lower dermal layers. It is important to note that few skin care formulations can actually penetrate far enough into the dermis or the deeper epidermis to reverse deep wrinkles, despite the multitude of products touting such a capacity. Preventing wrinkle formation is therefore the primary focus of anti-aging skin care [92]. Accordingly, topical products are formulated to prevent the degradation or promote the synthesis of the three primary skin constituents: collagen, elastin, and HA. Specifically, collagen production has been demonstrated to be promoted by topical preparations of retinoids, vitamin C, and copper peptides, as well as oral vitamin C [93–95]. The synthesis of both HA and elastin has been shown, in animal models, to be stimulated by retinoids [96, 97], and HA levels are also thought to be enhanced through glucosamine supplementation [98]. Currently, no products have been shown to be effective, and no products have been approved, for spurring elastin synthesis. Inflammation reduction is also a significant target in wrinkle prevention, because inflammation is known to influence the degradation of collagen, elastin, and HA. Antioxidants, which protect the skin through several mechanisms, are used in this approach to mitigate ROS activity. This is important, because ROS act directly on growth factor and cytokine receptors in keratinocytes, and cutaneous inflammation can be initiated in these epidermal cells. In addition, it is known that growth factors and cytokines act synergistically in a complex
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process that involves several types of growth factors and cytokines [99]. Much more remains to be elucidated, however, regarding the direct impact of growth factors and cytokines on cutaneous aging. UV irradiation is theorized to initiate a cascade of events, acting on growth factor and cytokine receptors in keratinocytes and dermal cells, which result in downstream signal transduction by activating mitogen-activated protein (MAP) kinase pathways (extracellular signal-regulated kinase, c-jun N-terminal protein kinase, and p38). These then amass in cell nuclei, forming cFos/cJun complexes of transcription factor activator protein 1, and provoking the matrix metalloproteinases collagenase, 92 kDa gelatinase, and stromelysin to degrade collagen and other cutaneous connective tissue [100, 101]. The direct effects of ROS on cutaneous aging and the overall aging process are more clearly understood. Recently, Kang et al. have demonstrated that ROS activation of the MAP kinase pathways induces collagenase production, thus contributing to collagen degradation [101]. The use of antioxidants is believed to block these pathways, thus inhibiting the process of photoaging by preventing collagenase synthesis and its ensuing deleterious impact on collagen. Specifically, Kang et al. found that the pretreatment of human skin with the antioxidants genistein and N-acetyl cysteine hindered UV induction of the cJun-driven enzyme collagenase. The vast array of topical skin care products include several antioxidants as ingredients, including vitamins C and E as well as coenzyme Q10, and those originating from botanical sources, such as caffeine, coffeeberry, ferulic acid, feverfew, grape seed extract, green tea, idebenone, mushrooms, polypodium leucotomos, pomegranate, pycnogenol, reseveratrol, rosemary, and silymarin. The antioxidant capacity of these compounds is well established in the literature; however, their efficacy in topical formulations designed to reverse or diminish the cutaneous signs of aging is unclear. Nevertheless, several other practical measures can be employed to lessen or even prevent extrinsic skin aging, including avoiding/limiting solar exposure (especially from 10 am to 4 pm), using broad-spectrum sunscreen on a daily basis, avoiding cigarette smoke and pollution, eating a diet high in fruits and vegetables, taking oral antioxidant supplements (or using topical antioxidant formulations), and regularly using prescription retinoids. In the near future, technological innovations in tissue engineering and gene therapy may lead to breakthroughs in the therapeutic uses of growth factors, cytokines, and telomerase [102], including dermatologic applications.
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Skin Type Combinations and Alterations
Conclusion
Assessing the four skin type dichotomies together, as discussed above, provides insight into the simultaneous state or tendencies of an individual’s skin along four different spectra, yielding 16 different possible skin type permutations. Once individuals have identified their skin type and/or provided this information to their physician, the BSTS can be used by physicians as a valuable guide in treating specific skin problems, as well as identifying the most appropriate OTC products for their patient’s particular skin type. For example, formulations containing ingredients with the capacity to repair the skin barrier would be appropriate selections for a person with dry, sensitive, nonpigmented, tight skin (DSNT). The understanding of skin type, product ingredients, and basic science also dictates that an individual with oily, sensitive, nonpigmented, wrinkled skin (OSNW) would be best served by formulations containing retinoids and antioxidants. Practitioners, and particularly consumers, should be reminded that particular skin traits, propensities, or conditions are linked to certain skin types, and product selections should be made accordingly. For example, individuals characterized by pigmented, tight (PT) skin are more likely to have dark skin, whereas those with nonpigmented, wrinkled skin (NW) are more likely to have light skin. Further, a history of chronic sun exposure, resulting in wrinkles and solar lentigos, is typical for a person with pigmented, wrinkled (PW) skin. People with dry, sensitive (DS) skin are more likely than those with other combinations of the first two skin type parameters to present with eczema, and acne is correlated with oily, sensitive (OS) skin more than any other combination of the first two skin type spectra. Finally, people with the OSNW skin type are more likely to develop rosacea than individuals with other skin types. As noted briefly above, environmental conditions and stress can impact skin type. For this reason, it is recommended that individuals take a baseline BSTI questionnaire and re-take the test when stress, significant life changes, or cutaneous symptoms are present. Specifically, stress or discrete fluctuations in stress, pregnancy, menopause, exposure to variable climates or moving to a different climate, and various other significant exogenous or endogenous alterations can manifest in skin type changes. With baseline and updated BSTI scores, a physician is better equipped to arrive at a more holistic, integrated or informed skin-type assessment and treatment approach.
Significant growth and innovation have distinguished the skin care product market since the advent of the Helena Rubinstein and Elizabeth Arden cosmetics businesses. However, the breadth and depth of investigations into more accurately characterizing or classifying skin type have been disproportionately minimal. In fact, in the clinical setting, the four traditional labels used to describe skin type – dry, oily, sensitive, or combination – have been found to be inadequate in terms of accounting for the variations in observed skin types. In addition, these categories have seemed insufficient in the realm of matching skin care formulations with an individuals’ skin type, as actual skin types have defied such facile or limited labels associated with products. However, the author has devised and recently introduced a novel approach to more precisely characterize skin type, based on the perception of skin according to four basic parameters. The Baumann Skin Typing System (BSTS), based on the results of the Baumann Skin Type Indicator (BSTI), a self-administered questionnaire, evaluates skin according to four dichotomous spectra: dry or oily, sensitive or resistant, pigmented or non-pigmented, and wrinkled or tight (unwrinkled). The score on the questionnaire yields a four-letter designation indicating the individual’s Baumann Skin Type, with each letter of the code denoting an individual’s dominant proclivities along the spectrum of each parameter. To identify one’s skin type through the BSTS, skin qualities are rated simultaneously according to each dichotomous category, with the understanding that these skin type polarities are not mutually exclusive. Consequently, 16 skin type permutations are possible within the BSTS framework. A patient’s BSTI score provides a physician with considerable data, thereby contributing to or facilitating the practitioner’s approach to a treatment regimen for various cutaneous conditions, and increasing the probability of recommending the most appropriate OTC topical skin care products for that particular patient. This is especially important with regard to anti-aging skin care products, as a significant proportion of topical formulations are sought and intended to prevent or blunt the effects of cutaneous aging. Individual consumers can also benefit from knowing their BSTI score, as the information provided assists the consumer in choosing the most suitable topical treatments for their skin type. The vast array of skin care products can currently meet most basic cutaneous needs associated with all 16 BSTS skin types.
The Baumann Skin Typing System
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45. Mitsui T. (ed) New Cosmetic Science. New York: Elsevier, 1997, p. 134. 46. Del Rosso JQ. Cosmeceutical Moisturizers. In: Draelos ZD (ed) Procedures in Cosmetic Dermatology Series: Cosmeceuticals, 1st ed. Philadelphia: Elsevier, 2005, pp. 97–102. 47. Orth D, Appa Y, Contard E, et al. Effect of high glycerin therapeutic moisturizers on the ultrastructure of the stratum corneum. Poster presentation at the 53rd annual meeting of the American Academy of Dermatology, February 1995. 48. Kligman AM. Dermatologic uses of urea. Acta Derm Venereol. 1957;37(2):155–159. 49. Harding C, Bartolone J, Rawlings A. Effects of natural moisturizing factor and lactic acid isomers on skin function. In: Loden M, Maibach H (eds) Dry Skin and Moisturizers. Boca Raton: CRC Press, 2000, pp. 217–236. 50. Wohlrab W. The influence of urea on the penetration kinetics of topically applied corticosteroids. Acta Derm Venereol. 1984;64(3): 233–238. 51. Wohlrab W. Effect of urea on the penetration kinetics of vitamin A acid in human skin. Z Hautkr. 1990;65(9):803–805. 52. The Cosmetic Ingredient Review (CIR) Expert panel. Final report of the safety assessment of Urea. Int J Toxicol. 2005;24(Suppl 3):1–56. 53. Serup J. A double-blind comparison of two creams containing urea as the active ingredient. Assessment of efficacy and side-effects by non-invasive techniques and a clinical scoring scheme. Acta Derm Venereol Suppl (Stockh). 1992;177:34–43. 54. Van Scott EJ, Yu RJ. Control of keratinization with alpha hydroxy acids and related compounds. I. Topical treatment of ichthyotic disorders. Arch Dermatol. 1974;110(4):586–590. 55. Perricone NV, Dinardo JC. Photoprotective and antiinflammatory effects of topical glycolic acid. Dermatol Surg. 1996; 22(5):435–437. 56. Draelos ZD. Rediscovering the cutaneous benefits of salicylic acid. Cosm Derm Suppl. 1997; 4(10). 57. Van Scott EJ, Yu R. Hyperkeratinization, corneocyte cohesion, and alpha hydroxy acids. J Am Acad Dermatol. 1984;11(5)Pt 1: 867–879. 58. Berardesca E, Distante F, Vignoli GP, et al. Alpha hydroxyacids modulate stratum corneum barrier function. Br J Dermatol. 1997;137(6):934–938. 59. Stern E. Topical application of lactic acid in the treatment and prevention of certain disorders of the skin. Urol Cutan Rev. 1943;50:106. 60. Rawlings AV, Davies V, Carlomusto M, et al. Effect of lactic acid isomers on keratinocyte ceramide synthesis, stratum corneum lipid levels and stratum corneum barrier function. Arch Dermatol Res. 1996;288(7):383–390. 61. Stiller MJ, Bartolone J, Stern R, et al. Topical 8% glycolic acid and 8% L-lactic acid creams for the treatment of photodamaged skin. A double-blind vehicle-controlled clinical trial. Arch Dermatol. 1996;132(6):631–636. 62. Gonzalo MA, de Argila D, Garcia JM, et al. Allergic contact dermatitis to propylene glycol. Allergy. 1999;54(1):82–83. 63. Baumann LS, Spencer J. The effects of topical vitamin E on the cosmetic appearance of scars. Dermatol Surg. 1999;25(4):311–315. 64. Draelos ZD. Cosmetic selection in the sensitive-skin patient. Dermatol Ther. 2001;14:194. 65. http://www.wrongdiagnosis.com/a/acne/prevalence.htm#prevalence_intro. Accessed on October 5, 2008. 66. National Institute of Arthritis and Musculoskeletal and Skin Diseases. Available at http://www.niams.nih.gov/Health_Info/Acne/default.asp. Accessed on October 5, 2008.
67. National Rosacea Society. Available at: http://www.rosacea.org/ index.php. Accessed on October 5, 2008. 68. Yamasaki K, Di Nardo A, Bardan A, et al. Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nat Med. 2007;13(8):975–980. 69. Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol. 2006;24:147–174. 70. Shakoory B, Fitzgerald SM, Lee SA, Chi DS, Krishnaswamy G. The role of human mast cell-derived cytokines in eosinophil biology. J Interferon Cytokine Res. 2004;24(5):271–281. 71. Brown DJ, Dattner AM. Phytotherapeutic approaches to common dermatologic conditions. Arch Dermatol. 1998;134(11):1401–1404. 72. Basketter DA, Griffiths HA. A study of the relationship between susceptibility to skin stinging and skin irritation. Contact Dermatitis. 1993;29(4):185–188. 73. Lonne-Rahm SB, Fischer T, Berg M. Stinging and rosacea. Acta Derm Venereol. 1999;79(6):460–461. 74. Orton DI, Wilkinson JD. Cosmetic allergy: Incidence, diagnosis, and management. Am J Clin Dermatol. 2004;5(5):327–337. 75. Duarte I, Campos Lage AC. Frequency of dermatoses associated with cosmetics. Contact Dermatitis. 2007;56(4):211–213. 76. Mehta SS, Reddy BS. Cosmetic dermatitis – current perspectives. Int J Dermatol. 2003;42(7):533–542. 77. Jovanovic M, Poljacki M, Duran V, Vujanovic L, Sente R, Stojanovic S. Contact allergy to compositae plants in patients with atopic dermatitis. Med Pregl. 2004;57(5–6):209–218. 78. Freedberg IM, Eisen AZ, Wolff K, et al. Fitzpatrick’s Dermatology in General Medicine, 5th ed. New York: McGraw-Hill, 1999, p. 996. 79. Hermanns JF, Petit L, Martalo O, Pierard-Franchimont C, Cauwenbergh G, Pierard GE. Unraveling the patterns of subclinical pheomelanin-enriched facial hyperpigmentation: effect of depigmenting agents. Dermatology. 2000;201(2):118–122. 80. Wakamatsu K, Kavanagh R, Kadekaro AL, Terzieva S, Sturm RA, Leachman S, Abdel-Malek Z, Ito S. Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin. Pigment Cell Res. 2006;19(2):154–162. 81. Szabo G, Gerald AB, Pathak MA, Fitzpatrick TB. Racial differences in the fate of melanosomes in human epidermis. Nature. 1969; 222(198):1081–1082. 82. Jimbow K, Sugiyama S. Melanosomal translocation and transfer. In: Nordlund JJ, et al. The Pigmentary System. Physiology and Pathophysiology. New York: Oxford University Press, 1998. 83. Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, Shapiro SS. Inhibition of melanosome transfer results in skin lightening. J Invest Dermatol. 2000;115(2):162–167. 84. Paine C, Sharlow E, Liebel F, Eisinger M, Shapiro S, Seiberg M. An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway. J Invest Dermatol. 2001;116(4): 587–595. 85. Hakozaki T, Minwalla L, Zhuang J, Chhoa M, Matsubara A, Miyamoto K, Greatens A, Hillebrand GG, Bissett DL, Boissy RE. The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer. Br J Dermatol. 2002;147(1): 20–31. 86. Boukamp P. Ageing mechanisms: the role of telomere loss. Clin Exp Dermatol. 2001;26(7):562–565. 87. Boukamp P. Skin aging: a role for telomerase and telomere dynamics? Curr Mol Med. 2005;5(2):171–177. 88. Makrantonaki E, Zouboulis CC. Characteristics and pathomechanisms of endogenously aged skin. Dermatology. 2007;214(4):352–360.
The Baumann Skin Typing System 89. Kosmadaki MG, Gilchrest BA. The role of telomeres in skin aging/ photoaging. Micron. 2004;35(3):155–159. 90. Kappes UP, Luo D, Potter M, Schulmeister K, Runger TM. Shortand long-wave UV light (UVB and UVA) induce similar mutations in human skin cells. J Invest Dermatol. 2006;126(3):667–675. 91. Marrot L, Belaı¨di JP, Meunier JR. Importance of UVA photoprotection as shown by genotoxic related endpoints: DNA damage and p53 status. Mutat Res. 2005;571(1–2):175–184. 92. Baumann L. How to prevent photoaging? J Invest Dermatol. 2005;125(4):xii–xiii. 93. Varani J, Warner RL, Gharaee-Kermani M, Phan SH, Kang S, Chung JH, Wang ZQ, Datta SC, Fisher GJ, Voorhees JJ. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114 (3):480–486. 94. Nusgens BV, Humbert P, Rougier A, Colige AC, Haftek M, Lambert CA, Richard A, Creidi P, Lapiere CM. Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis. J Invest Dermatol. 2001;116(6):853–859. 95. Kockaert M, Neumann M. Systemic and topical drugs for aging skin. J Drugs Dermatol. 2003;2(4):435–441.
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96. Margelin D, Medaisko C, Lombard D, Picard J, Fourtanier A. Hyaluronic acid and dermatan sulfate are selectively stimulated by retinoic acid in irradiated and nonirradiated hairless mouse skin. J Invest Dermatol. 1996;106(3):505–509. 97. Tajima S, Hayashi A, Suzuki T. Elastin expression is up-regulated by retinoic acid but not by retinol in chick embryonic skin fibroblasts. J Dermatol Sci. 1997;15(3):166–172. 98. Matheson AJ, Perry CM. Glucosamine: a review of its use in the management of osteoarthritis. Drugs Aging. 2003;20(14):1041–1060. 99. Fitzpatrick RE. Endogenous growth factors as cosmeceuticals. Dermatol Surg. 2005;31(7)Pt 2: 827–831. 100. Fisher GJ, Voorhees JJ. Molecular mechanisms of photoaging and its prevention by retinoic acid: ultraviolet irradiation induces MAP kinase signal transduction cascades that induce Ap-1-regulated matrix metalloproteinases that degrade human skin in vivo. J Investig Dermatol Symp Proc. 1998;3(1):61–68. 101. Kang S, Chung JH, Lee JH, Fisher GJ, Wan YS, Duell EA, Voorhees JJ. Topical N-acetyl cysteine and genistein prevent ultraviolet-lightinduced signaling that leads to photoaging in human skin in vivo. J Invest Dermatol. 2003;120(5):835–841. 102. Ostler EL, Wallis CV, Aboalchamat B, Faragher RG. Telomerase and the cellular lifespan: Implications of the aging process. J Pediatr Endocrinol Metab. 2000;13 (Suppl 6):1467–1476.
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84 The Vaginal Microbiota in Menopause Miranda A. Farage . Kenneth W. Miller . Jack D. Sobel
Introduction The adult human is colonized by more than 100 trillion microbes [1], with an estimated 1012 bacteria on the skin, 1010 in the oral cavity, 1014 in the gastrointestinal tract, and 109 in the vaginal vault [2]. In any specific anatomical habitat, colonization is determined by pH, temperature, redox potential, endogenous secretions of lysozyme and immunoglobulins, and levels of nutrients including oxygen and water [3]. Colonization of an anatomical area by a specific microorganism also depends on the ability of the bacterium to adhere to the tissue [2]. Moist interior tissues, protected from the harsh external environment, are ideal habitats; the vaginal vault is colonized within 24 h of a female child’s birth and remains colonized until death [4]. Together, the human vagina and its resident microbiota comprise a dynamic yet fine-tuned ecosystem. Culture-based and biochemical methods have attempted to define the composition of ‘‘normal’’ vaginal microbiota, yielding guidelines which have been used to distinguish normal from pathogenic biota for nearly 2 decades [5]. Nugent criteria have defined the ‘‘normal’’ microbiota as that which is dominated by Lactobacillus species [5] and the diseased microbiota (bacterial vaginosis [BV]) as that lacking Lactobacillus dominance but displaying a preponderance of anaerobes and the presence of microorganisms believed to be pathogenic (e.g., Gardnerella and Bacteroides) [6]. The usefulness of the Nugent system, however, is limited by the facts that the onset of clinical symptoms does not correlate tightly with the types and numbers of bacteria identified and BV is often asymptomatic, regressing spontaneously in about 50% of cases [7]. In addition, a comprehensive description of the complex microbiota of the vagina has been frustrated by the available tools, which leave many organisms either uncultured or unidentified [8] and often provide only a snapshot of an environment characterized by continuous change [9]. Over a woman’s lifespan, the vagina microbiota will be in constant flux [10] – changing daily and even hourly [11] – as the ecosystem is buffeted by a variety of both internal and external insults that have the potential to
modulate the vaginal milieu [10]. Hormonal changes that mark the stages of a human female’s life – puberty, the menstrual cycle, pregnancy, and menopause – are in themselves a major influence [12]; ethnicity influences the microbiota as well [13]. External factors that may affect the composition of the microbiota include methods of contraception [10]; sexual behaviors (including the age of first sexual experience, the frequency of sex, number of sexual partners, and sexual practices); the presence of sexually transmitted infections; and even the introduction of semen [10, 14]. The use of personal hygiene products or medications may also play a role [15], as may cultural practices [16] and even diet [17]. The normal vaginal microbiota cannot be viewed as static or universal, but as a dynamic equilibrium dependent on both microbial and host characteristics. Recently developed culture-independent techniques have identified novel organisms yet uncultured (such organisms are believed to constitute 99% of the bacteria in the natural environment [18]), as well as a high level of diversity in the microbiota of healthy women; this has expanded the understanding of the dynamics of the vaginal microbiota. For example, molecular studies have identified several novel strains of Lactobacillus that inhabit the human vagina and revealed that the role of lactobacilli in producing lactic acid can be played by other previously unrecognized anaerobic bacteria [19]. Such techniques have also shown that organisms previously believed to signal a pathogenic state (Gardnerella vaginalis, Peptostreptococcus, Prevotella, and Streptococcus) are frequently found in healthy women [20]. Asymptomatic cases of BV (as identified by Nugent criteria) therefore may represent vaginal flora with functionally sufficient levels of lactobacillus, although these may be undetectable by gram stain. Moreover, other acidproducing bacteria, detectable only by molecular methods, may confer the necessary functionality to create an asymptomatic condition. These observations suggest that the normal vaginal microbiota cannot be rigidly and permanently defined for all women, but instead represent a spectrum of mutualistic and functional interrelationships. The spectrum of species in normal microbiota may vary among women (depending on host and
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_84, # Springer-Verlag Berlin Heidelberg 2010
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environmental variables) and over time even within an individual woman. For example, as will be discussed in this chapter, hormonal changes of a female lifecycle produce predictable variations in the vaginal microbiota.
. Table 84.1 Lactobacillus species recovered from the human vagina Most common L. crispatus L fermentum
Changes in the Vaginal Microbiota Over a Woman’s Lifespan
L. casei L. jensenii
Normal Microbiota During Childhood and Puberty During birth and shortly after that, maternal estrogens in the newborn’s circulatory system produce a high glycogen level in the vaginal epithelium, producing an environment in which lactic acid-producing microbes can thrive [4]. Because adrenal or gonadal hormones are inactive at this age, as maternal estrogen depletes, the prevalence of lactic acid-producing microbes present in neonates also decreases [12]. Vaginal pH during early childhood is neutral or slightly alkaline [12]. The morphology and physiology of the vulva and the vagina change at puberty [21]. With adrenal and gonadal maturation, cyclic hormonal patterns are established and menstruation begins. Mid-cycle estrogen levels produce peaks in the glycogen content of the vaginal epithelium, increasing the prevalence of lactic acid-producing microbes in the vaginal microbiota [12]. By adulthood, the morphology of the vulva is mature and the menstrual cycle becomes well established [12].
Normal Microbiota During the Reproductive Years Lactic acid-producing microbes are numerically dominant in most healthy adult women [12, 22] at levels of approximately 107 lactobacilli per gram of vaginal secretion [23]. Generally, only a single strain of Lactobacillus is cultured from any one individual [24]; in one report, 92% of individuals cultured had a single strain [25]. The most common Lactobacillus species identified by traditional culture techniques are L. crispatus and L. fermentum, although L. brevis, L. jensenii, L. casei, L. delbrueckii, and L. salivarius also are isolated [23]. Generally, levels of Lactobacillus spp. in the vagina rise and fall in parallel with circulating levels of estrogen [23] (> Table 84.1). Other bacteria commonly identified by traditional methods, but at lower population numbers, include Staphylococcus species, Ureaplasma, Corynebacterium, Streptococcus, Peptostreptococcus, Gardnerella, Bacteroides, Mycoplasma,
L. delbruckii Less common L. inersa L minutis L brevis L. leichmanni L. plantarum L salvarius L. gallinarum L. gasseri L. johnsonii L. paracasei L. rhamnosus L. reuteri L vaginalis a
Liners more often recovered via molecular techniques Table compiled from References [23, 28, 51, 64–66]
Enterococcus, Escherichia, Veillonella, Bifidobacterium, and Candida spp. [23] (> Table 84.2). The use of molecular techniques to evaluate the normal microbiota has confirmed that the vaginal ecosystems of most healthy fertile women are dominated by Lactobacillus species [10]. However, novel Lactobacillus species that do not grow readily on standard media also have been identified, as have other acid-producing species not formerly detected at numerically significant levels [10]. During the reproductive years, estrogen production stimulates vaginal epithelial cells to proliferate, producing a mid-cycle peak in intracellular glycogen levels in the vaginal mucosa [26] and a subsequent increase in lactic acid-producing microbes in the vaginal milieu [12]. By virtue of metabolizing glycogen, lactobacilli acidify the vaginal environment: pH in the vaginal vault of fertile women ranges generally from 3.8 to 4.5 [27]. The acidity of the vaginal environment appears to be the principal inhibitor of secondary bacterial infection [28]; H2O2 produced by some lactobacillus species also may assist in inhibiting colonization by other vaginal flora [29]. In
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. Table 84.2 Commensal and pathogenic organisms of the human vagina Organism Gram-positive cocci
Gram-positive bacilli
Facultative and aerobic
Prevalence
Anaerobic
Prevalence
Staphylococcus epidermis
+++
Peptostreptococcus +++
Staphylococcus aureus
+
Streptococcus*
+++
Streptococcus spp (a-hemolytic, nonhemolytic, groups B, D, S)*
+++
Micrococcus
++
Enterococcus faecalis*
+
Lactobacillus
+++
Lactobacillus
+++
Corynebacteria
+++
Propionibacteria
++
Clostridium Eubacteria
+++
Bifidobacteria Actinomyces Gram-negative cocci
Neisseria*
+
Veillonella
++
Gram-negative bacilli
Escherichia coli
+
Bacteroides*
+++
Klebsiella
Fusobacterium
Ureaplasma urealyticum*
+
Enterobacteria
+
Gram-variable bacillus
Gardnerella vaginalis*
+
Other organisms
Candida*
+
Mycoplasma hominis*
+
M. fermentans
+
+++ Very common, ++ common but only occur in low numbers, ++ occasional, + rare, *potentially pathogenic organism Table compiled from [2, 23, 67]
addition, some H2O2-producing lactobacilli appear to have viricidal activity [29]. It has been widely accepted that maintaining vaginal acidity through the production of lactic acid by Lactobacillus species is critical to vaginal health. However, up to 42% of women who do not exhibit lactobacilli-dominant microbiota are nonetheless able to maintain normal vaginal ecosystems [30–32]. Molecular techniques have detected other species capable of producing lactic acid in their microbiota, which indicates that although a community of lactic acid producers is maintained in healthy women, the composition of that community can vary [19]. Lactobacillus species and other lactic acid producers have the ability to inhibit growth of numerous other bacterial species in vitro [30]. Many factors contribute to this effect (> Table 84.3). Communities dominated by lactic acid producers can serve as a barrier to colonization by potentially pathogenic organisms and to overgrowth of
organisms that are otherwise commensal [19]. Such communities inhibit the transmission of sexually transmitted infections [15], including heterosexual transmission of human immunodeficiency virus (HIV) [33]. BV, a noninflammatory condition in which the vaginal microbiota exhibits a paucity of lactobacilli and a preponderance of anaerobes has long been an enigma. The chain of events, which tips the scales from a functional ecosystem to the development of BV, is still a mystery [9, 23]; the phenomenon seems to involve changes in the interplay between factors both endogenous and exogenous to the vagina that allow potentially pathogenic organisms to gain dominance [15, 23, 34]. However, healthy women with normal vaginal flora regularly experience transient yet significant changes dramatic enough to shift normal flora into a potentially pathogenic state [35], and substantial perturbation in the microbiota is often a prelude to vaginal disease [19].
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. Table 84.3 Role of Lactobacillus in stabilizing normal vaginal microbiota Reference Production of hydrogen Hillier SL et al. (1993) [24], peroxide Strus et al. (2006) [68] Production of bacteriocins
Reid et al. (2003) [69]
. Table 84.4 Changes in vaginal structure and function due to estrogen depletion after menopause Change Structural changes Vaginal epithelium thins
Mack et al. (1999) [71]
Penetration of E. coli biofilms
Reid et al. (2003) [69]
Dermis
Augment host immunity Kim et al. (2006) [72]
Normal Microbiota During Menopausal and Postmenopausal Years Functionally, menopause begins when the numbers of primordial follicles (which decline steadily from birth on) hit a critical predetermined low [36]. The age at which women enter menopause has a genetic component, as demonstrated by solid patterns with regard to time of onset in individual families [37]. The genetic contribution to age of onset is estimated by several studies to approach 50% [38]. Onset of menopause in women of European extraction has remained at about the current average of age 50 for at least the last 1,000 years [36]. However, both epidemiological and prospective studies have revealed a slightly but significantly earlier age of onset of menopause in black women both in the USA [39] and in Africa [40], and a slightly but significantly later onset in both ethnic Asian populations [41]. Moreover, the onset of the menopause is also influenced by the timing and number of pregnancies [36], nutrition [41], and smoking [41]. The plasma content of estrogen drops from about 129 ng/L in the reproductive years to about 18 ng/L after menopause [42], dramatically affecting the vagina [43] (> Table 84.4). The vaginal epithelium contains estrogen-alpha receptors: as estrogen levels drop, the vaginal epithelium atrophies and its glycogen content falls; this in turn depletes cell densities of lactobacilli [44], diminishes the production of lactic acid from glucose [45], and causes the vaginal pH to rise to about 6–8 [44]. This rise in vaginal pH, absent BV, or other vaginal pathology is a reliable indicator of menopausal status [46].
Loses rugae,
Farage et al. (2006) [12]
Pales, develops Pandit L et al. (1997) fine petechial [73] hemorrhages
Lactic acid depression of Ro¨nnqvist PDJ et al. (2006) [70] vaginal pH Inhibit Escherichia coli adherence
Reference
Vascular system
Atrophy
Maloney C et al. (2001) [74]
Collagen bundles fuse and undergo hyalinization
Pandit L et al. (1997) [73]
Elastin fibers fragment
Pandit L et al. (1997) [73]
Progressive Long et al. avascularization (2006) [75]
Functional changes Dyspareunia
Robinson D et al. (2003) [76]
Loss of elasticity
Long et al. (2006) [75]
Dryness
Robinson D et al. (2003) [76]
Decreased blood flow
Tsai et al. (1987) [77]
Vaginal pH appears to depend not only on constituents of the microbiota, but also on hydrogen ion secretion by ectocervical epithelial cells in the human vagina [47]. In postmenopausal women, these cells become atrophied and secretion of hydrogen ion (H+) is attenuated: postmenopausal estrogen treatment restores cell function. In premenopausal women with estrogen deprivation, estrogen treatment fully reactivates H+ secretion; this implies that treating perimenopausal women with estrogen early, before intracellular estrogen is depleted, would be advantageous [48]. Microbial constituents of the vaginal ecosystem adapt to changing pH and hormone levels [3]. After menopause, the prevalence and cell density of Lactobacillus drops [44, 46, 49], which is considered a normal maturational change: prevalence in postmenopausal women was zero in some studies [50]. Lactobacillus species confer considerable stability on the vaginal ecosystem [51], an assertion supported by the observation that Lactobacillusdominant vaginal microbiota retain a normal balance of
The Vaginal Microbiota in Menopause
84
. Table 84.5 Comparison of normal vaginal flora in healthy postmenopausal women with and without hormone replacement therapy Organism
Prevalence in non-HRT-treated women
Prevalence in HRT-treated women
Lactobacilli
41%
81%
Pabich et al. (2003) [56]
20%
95%
Gupta et al. (2006) [45]
49%
NA
Escherichia coli
Gardnerella
Reference
Hillier SL et al. (1997) [46] a
Devillard et al. (2004) [28]
NA
100%
40%
38%
Pabich et al. (2003) [56]
35%
10%
Gupta et al. (2006) [45]
40%
NA
10%
33.3%
Hillier SL et al. (1997) [46]
27%
NA
Gupta et al. (2006) [45] Hillier SL et al. (1997) [46]
Mobiluncus
30%
16.7%
Gupta et al. (2006) [45]
Bacteroides
40%
8.3%
Gupta et al. (2006) [45]
Group B Streptococcus
5%
16.7%
Gupta et al. (2006) [45]
Coryneforms Candida
Ureaplasma urealyticum
23%
NA
15%
16.7%
Hillier SL et al. (1997) [46]
58%
NA
0
23.3%
1%
NA
Gupta et al. (2006) [45] Hillier SL et al. (1997) [46] Gupta et al. (2006) [45] Hillier SL et al. (1997) [46]
a
NA
25%
Devillard et al. (2004) [28]
13%
NA
Hillier SL et al. (1997) [46]
a
Prevalence rates determined by molecular methodology HRT = hormone replacement therapy; NA = not available
flora for months, despite the presence of known pathogens [28]. In the perimenopause, depletion of the numbers of vaginal lactobacilli erodes the functional barrier to colonization by other bacteria. Consequently, after menopause, the vagina is likely to harbor more commensal species than during the reproductive years, which produces a more complex microbial community in the vaginal vault (> Table 84.5). The vaginal flora of postmenopausal women is altered, often being colonized by potentially pathogenic organisms [27]. The shift of the vaginal pH from a state of normal acidity (pH 3.8–4.5 in the estrogenized vagina [52]) to a state of alkalinity allows potentially pathogenic bacteria, particularly enteric bacteria [12], to invade or expand their colonization of the vagina. Culture-based techniques indicate that the alkaline vagina is often colonized primarily with fecal flora such as Enterobacteriaceae [53, 54], which rarely occurs when vaginal pH is less than 4.5 [55]. Other organisms isolated in substantial numbers from postmenopausal patients (by selective culture
techniques) are G. vaginalis (27%), Ureaplasma urealyticum (13%), Prevotella (33%), and coliforms (41%) [46]. Bacteroides has been isolated from 40% and Escherichia coli from 35% of postmenopausal subjects not treated with hormone replacement therapy (HRT) [45]. In postmenopausal women, vaginal colonization with E. coli was inversely associated with the presence of lactobacilli: relatively heavier growth of lactobacilli being associated with a lower frequency of E. coli colonization [56]. > Table 84.6 shows a summary of studies on the makeup of the postmenopausal vaginal microbiota. Interestingly, in a study of 48 postmenopausal women, molecular techniques demonstrated the presence of vaginal lactobacilli in every subject, yielding a technical (and actual) prevalence rate of BV of zero, contrast to the 10.5% prevalence rate ascertained by Nugent methodology at baseline [57]. It has been noted that the Nugent system of scoring was developed for pregnant women [58], and may not be suitable or appropriate for evaluating women in menopause. This is
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. Table 84.6 Studies on vaginal microbiota in postmenopausal women not using HRT
Study population
How menopause defined
25 Healthy women
NA
Culture
Increase in gram-negative bacteria
Tashjian JH et al. (1976) [78]
50 Postmenopausal women, aged 55–73 (mean age 63.7) 14 HRT users, 36 HRT nonusers
None described
Culture
No significant differences
Osborne NG et al. (1979) [54]
28 Healthy women (mean age 59)
Greater than Culture 2 years since last period
29% Cultures sterile, 19% of cultures contained anaerobesa
Blum M et al. (1981) [79]
46 Postmenopausal women
NA
Culture
65% Lactobacillus positive
Larsen et al. (1982) [80]
171 Healthy women in cancer NA prevention program
Culture
Increase in G. vaginalis
Ceddia et al. (1989) [81]
350 Menopausal UTI patients, Advanced aged 65–84 (mean age 72.3), age >3 months since any HRT therapy
Culture
20% Lactobacillus dominant, most subjects characterized by very sparse growth Fecal bacteria dominant Average KPI 3.0 0.9, only 16.3% greater than 10 pH 6.5 0.1
Milsom et al. (1993) [49]
Culture Gram stain/ Nugent Lactic acid production, H2O2 production
13% Lactobacillus dominant, 87% intermediate 49% Lactobacillus positive
Hillier et al. (1997) [46]
100 Healthy postmenopausal FSH women
Gram stain/modified Nugent score
44% Normal flora, 17% intermediate, 18% BV, 21% null flora
TaylorRobinson et al. (2002) [59]
20 Healthy postmenopausal women, aged 44–72 (mean age 58.9)
Culture, gram stain/Nugent score PCR/DGGE
95% Lactobacillus positive 30% Normal, 30% intermediate, 40% BV
Burton et al. (2002) [62]
73 Healthy postmenopausal women, no HRT use for 1 year
5 Years since last period, FSH level
None described
Experimental parameters
Results
Reference
a Anaerobes appeared only in the presence of aerobic bacteria BV = bacterial vaginosis; DGGE = denaturing gradient gel electrophoresis; FSH = follicle-stimulating hormone; H2O2 = hydrogen peroxide; HRT = hormone replacement therapy; KPI = karyopicnotic index; NA = not available PCR = polymerase chain reaction; UTI = urinary tract infection
underscored by the frequency of null flora – the absence of significant numbers of lactobacilli in postmenopausal women – yet without BV-associated microorganisms [59, 60]. Such a state is extremely rare in fertile women [44]. Null flora becomes more common with advancing age and increasing atrophy of the vaginal epithelium [59]. Null flora, representing a nearly sterile environment, meet the criteria for a Nugent score of 4, which is interpreted as an intermediate state between normal flora and BV. Yet, in postmenopausal women, this state is natural and nonpathogenic [58].
Vaginal Microbiota and Hormone Replacement Therapy in the Postmenopausal Woman In postmenopausal women, replacement estrogens can be successfully administered orally, intravaginally, and transdermally (> Table 84.7). Several decades ago, it was first demonstrated that estrogen therapy lowered vaginal pH and increased the presence of lactobacilli in the vagina [61]. Since then, studies have demonstrated convincingly that HRT normalizes vaginal pH and
The Vaginal Microbiota in Menopause
84
. Table 84.7 Effects of hormone replacement therapy on vaginal environment in menopausal women
Population
Definition of menopause
Experimental parameters
Results
Reference
15 Menopausal women (natural)
FSH levels Serum estrogen levels
Estadiol (dermal patch) Percentage of cultures in which anaerobes Ginkel et al. 0.1 mg/day were isolated dropped from 47% to 13% Post- (1993) [82] HRT, vaginal pH in subjects with Lactobacillusdominant flora was 4.4 compared to 5.2 in subjects with non-Lactobacillus-dominant flora
60 Postmenopausal women, aged 51–81, mean age 65 years
None described
36 Treated, 24 placebo, No Lactobacillus detected in any patient at Raz R, Stamm 8 months culture, pH baseline, after 1 month Lactobacillus species WE. (1993) [50] detected in 61% of treated subjects Enterobacteriaceae in treated group fell from 67% to 33.1% after 1 month
59 Postmenopausal women with vaginal dryness or discharge
NA
Oral estriol, 14 days Culture
Detection of lactobacilli in vaginal flora increased from 9% at baseline to 42% after treatment
Yoshimura T et al. (2001) [83]
258 Institutionalized menopausal women with UTIs
Advanced age (mean age 83 years)
Intravaginal estrogen, 0.5 g/day, three times per week
Vaginal pH Baseline: 7.4 0.71 At 6 weeks: 6.8 0.70 At 12 weeks: 6.7a
Maloney C et al. (2001) [74]
921 Healthy postmenopausal 590 HRT users, 331 HRT nonusers
Greater than 1 year since last menses
Oral and transdermal Users: 84.9% normal flora, 5.4% BV, 68.9% Cauci et al. estrogen Culture Gram with full Lactobacillus colonization Nonusers: (2002) [58] stain/Nugent score 46.3% normal flora, 6.3% BV, 34.1% with full Lactobacillus colonization
40 Healthy postmenopausal women, aged 41–82
None described
Gram stain/Nugent score PCR/DGGE History of HRT (Premarin1 – conjugated equine estrogen with progesterone) >2 years
HRT users: 100% Lactobacillus + 54% only one Heinemann C organism (90% of these Lactobacillus), et al. (2005) 87% three or less, 5.6% BV Nonusers: 91% [51] colonized by more than one organism, 31% BV Bacteria associated with BV and/or UTI 10¥ higher than in HRT users
89 Healthy postmenopausal women, 30 natural menopause with estrogen and progestin, 30 surgical menopause with estrogen replacement only, 20 natural menopause, untreated controls
Greater than 1 year since last period
HRT 2–24 months Culture/gram stain with Schroder’s score pH
Vaginal microbiota: HRT nonusers vs users Gupta et al. Lactobacillus 20% vs 90% Gardnerella (2006) [45] vaginalis 10% vs 33.3% Mobiluncus 30% vs 17% Bacteroides 40% vs 8% Gp B Strep 5% vs 16.7% Escherichia coli 35% vs 10% pH average 5.35 among nonusers, 4 among users
48 Menopausal women with vaginal complaints, 58–75 years of age
Greater than 5 years since last period
CEE at 0.625 for 90 days, gram stain with Nugent score pH, transvaginal sonography
Baseline 0% Nugent score type I (100% type Galhardo III) 30 Days 46% Type I 90 Days 74% Type I pH (2006) [43] dropped from 7.0 at baseline to 4.5 at 90 days
a pH still depressed at 6 weeks after estrogen therapy discontinued BV = bacterial vaginosis; CEE = conjugated equine estrogens; DGGE = denaturing gradient gel electrophoresis; FSH = follicle-stimulating hormone; HRT = hormone replacement therapy; NA = not available; PCR = polymerase chain reaction; UTI = urinary tract infection
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The Vaginal Microbiota in Menopause
vaginal microbiota in postmenopausal women [28, 45, 46, 50]. For example, in a group of postmenopausal women who were all devoid of vaginal Lactobacillus at baseline, intravaginal administration of estriol resulted in a 61% colonization rate by Lactobacilli after just 1 month, with a concurrent decline in vaginal pH from 5.5 to 3.8 [50]. In addition, the prevalence of colonization by Enterobacteriaceae, which was 67% at baseline, fell to 31% in the same time period [50]. In the postmenopausal stage of life, it is considered a paradox that despite the depletion of protective Lactobacillus in the vagina and the accompanying increase in colonization by fecal bacteria, the prevalence of clinically diagnosed BV remains low. Interestingly, although HRT reestablishes the Lactobacillus-dominated vaginal microbiota of the reproductive years, BV prevalence rates do not return to the levels seen in premenopausal women. Although the potential protective mechanisms in women undergoing hormone replacement therapy are not yet understood, HRT must induce changes beyond establishing a Lactobacillus-dominant flora that lower the risk of acquiring BV.
Normal Flora in Menopause and Urinary Tract Infection HRT provides other benefits to the ecosystem of the lower genital tract. Molecular profiles of the vaginal microbiota in untreated menopausal women sometimes reveals either colonization with a single type of pathogenic organism or the presence of an unstable vaginal microbial environment [62]. As noted earlier, the alkaline vaginal environment that exists after menopause may allow uropathogenic E. coli and other enterobacteria to displace vaginal lactobacilli. Molecular methods demonstrate that such E. coli infections can persist, creating a stable but abnormal vaginal microbiota that places these women at higher risk for urinary tract infection (UTI) [62]. Urinary tract infections contribute to urinary incontinence in older women [63]. In some but not all studies, orally or intravaginally administered HRT reduced the frequency of UTIs in postmenopausal women: the treatment restored acidic vaginal pH and improved vaginal health [50], reducing the risk of UTI (> Table 84.8). However, this indication remains controversial [89].
. Table 84.8 Effects of Hormone replacement therapy on urinary tract infections Population
Study design
Length of HRT use
Results
Reference
3,616 Patients with Case–control, first UTI, ages 50–69 observational
HRT use 1 year
Increased risk of UTI (odds ratio 1.9, Orlander 95% CI 1.5–2.2) et al. (1992) [84]
Patients with UTI, US, and Israel, aged 40–64
Case–control, observational
HRT use in recent past
Cases less likely than controls to Foxman report HRT use, but significant only et al. in the USA (2001) [85]
40 Healthy women, aged 66–91 years
Randomized, placebo-controlled clinical trial
Oral estriol vs placebo for Estriol significantly reduced UTI Kirkengen 8 weeks: 1 mg/day for 4 weeks frequency as compared to placebo et al. then 1 mg/day for 8 weeks over the last 8 weeks of the study (1992) [86] period
93 Postmenopausal Randomized, women with history double-blind of recurrent UTIs placebo-controlled clinical trial
Intravaginal estriol, 8-month study period
Incidence of UTI in estriol group significantly reduced (0.5 vs 5.9 episodes per patient-year, p < 0.001
53 Postmenopausal women
Multicenter randomized controlled open clinical trial
Intravaginal estrial (vaginal rings) for 36 weeks
45% of treated group remained UTI Eriksen B over study period compared to (1997) [87] 20% controls
72 Postmenopausal women, 60 years of age
Randomized, double-blind, placebo-controlled trial
Oral estriol (3 mg/day) vs placebo
No significant differences between Cardozo groups (authors concluded et al. problems with study design failed (1998) [88] to elicit clear differences)
HRT = hormone replacement therapy; US = United States; UTI = urinary tract infection
Raz R, Stamm WE. (1993) [50]
The Vaginal Microbiota in Menopause
Conclusion The vaginal ecosystem changes continually over a woman’s lifetime, as both intrinsic and extrinsic factors assail the fragile balance between competing organisms, sometimes on a daily basis. Lactobacillus and other lactic acid-producing microbes form the foundation of a healthy vaginal microbiota during the reproductive years. Vaginal lactobacilli depend on the presence of estrogen; after menopause, estrogen depletion elevates vaginal pH, reduces vaginal colonization by lactic acid-producing microbes and facilitates colonization by enteric organisms. This increases the risk of UTIs, a factor that contributes to incontinence in elderly women. HRT seems to restore vaginal pH and to reestablishe normal vaginal microbiota in postmenopausal women, thereby helping to promote a more healthful vaginal ecosystem. Because women in industrialized countries will spend one third of their lives in menopause, disorders of the aging vagina will continue to be a significant medical concern. A more comprehensive understanding is needed of the spectrum of microbes that constitute the normal vaginal microbiota and of the complex interplay of intrinsic and extrinsic factors that either maintain a healthy vaginal microbiota or promote disease. Molecular tools are shedding light on the array of microbiota that constitute healthy vaginal ecosystems and may eventually yield insights on changes in the microbiota that could signal the potential for pathogenesis. By better understanding the dynamics of vaginal environment in all stages of life, and by identifying those events that promote disease, there is hope that more effective therapies can be developed to promote vaginal health throughout a woman’s life.
Cross-references > Aging
Skin Microbiology
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47. Gorodeski GI. Effects of estrogen on proton secretion via the apical membrane in vaginal-ectocervical epithelial cells of postmenopausal women. Menopause. 2005;12:679–684. 48. Bachmann G. Quantifying estrogen treatment effect on vagina tissue: cellular age matters. Menopause. 2005;12:656–657. 49. Milsom I, Arvidsson L, Ekelund P, et al. Factors influencing vaginal cytology, pH and bacterial flora in elderly women. Acta Obstet Gynecol Scand. 1993;72:286–291. 50. Raz R, Stamm WE. A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infections. N Engl J Med. 1993;329:753–756. 51. Heinemann C, Reid G. Vaginal microbial diversity among postmenopausal women with and without hormone replacement therapy. Can J Microbiol. 2005;51:777–781. 52. Meltzer R. Vulvovaginitis. In Sciarra J (ed) Gynecology and Obstetrics. Philadelphia: JB Lippincott, 1987. 53. Bartlett JG, Onderdonk AB, Drude E, et al. Quantitative bacteriology of the vaginal flora. J Infect Dis. 1977;136:271–277. 54. Osborne NG, Wright RC, Grubin L. Genital bacteriology: a comparative study of premenopausal women with postmenopausal women. Am J Obstet Gynecol. 1979;135:195–198. 55. Stamey TA, Sexton CC. The role of vaginal colonization with enterobacteriaceae in recurrent urinary infections. J Urol. 1975;113: 214–217. 56. Pabich WL, Fihn SD, Stamm WE, et al. Prevalence and determinants of vaginal flora alterations in postmenopausal women. J Infect Dis. 2003;188:1054–1058. 57. Galhardo CL, Soares JMJ, Simo˜es RS, et al. Estrogen effects on the vaginal pH, flora and cytology in late postmenopause after a long period without hormone therapy. Clin Exp Obstet Gynecol. 2006;33:85–89. 58. Cauci S, Driussi S, De Santo D, et al. Prevalence of bacterial vaginosis and vaginal flora changes in peri- and postmenopausal women. J Clin Microbiol. 2002;40:2147–2152. 59. Taylor-Robinson D, McCaffrey M, Pitkin J, et al. Bacterial vaginosis in climacteric and menopausal women. Int J STD AIDS. 2002;13: 449–452. 60. Blum M, Elian I. The vaginal flora after natural or surgical menopause. J Am Geriatr Soc. 1979;27:395–397. 61. Molander U, Milsom I, Ekelund P, et al. Effect of oral oestriol on vaginal flora and cytology and urogenital symptoms in the postmenopause. Maturitas. 1990;12:113–120. 62. Burton JP, Reid G. Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (Nugent score) and molecular (polymerase chain reaction and denaturing gradient gel electrophoresis) techniques. J Infect Dis. 2002;186:1770–1780. 63. Fantl J, Newman K, Coiling J, et al. Urinary incontinence in adults: acute and chronic management. Rockville: US Department of Health and Human Services, Public Health Service, Agency for Health Care Policy and Research (AHCPR), 1996. 64. Burton JP, Cadieux PA, Reid G. Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Appl Environ Microbiol. 2003;69:97–101. 65. Ison CA. Factors affecting the microflora of the lower genital tract of healthy women. In: Hill MJ, Marsh PD (eds) Human Microbial Ecology. Boca Raton: CRC Press, 1990. 66. Pascual LM, Daniele MB, Pa´jaro C, et al. Lactobacillus species isolated from the vagina: identification, hydrogen peroxide production and nonoxynol-9 resistance. Contraception. 2006;73:78–81. 67. Onderdonk A, Wisseman K. Normal vaginal microflora. In Elsner P, Martius J (eds) New York: Vulvovaginitis Marcel Dekker, 1993.
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Cosmetic Surgeries
104 A New Paradigm for the Aging Face Samuel M. Lam
Introduction This textbook is dedicated to a thorough understanding of the aging skin that encompasses both basic science and clinical topics. However, the effects of skin aging extend well beyond the inherent microarchitecture of the cutis itself. Concomitant aging processes have a remarkable impact on the appearance of the skin as well, which will be covered in this chapter. An outdated perspective of the aging process for the human face is that the only two components of facial aging include manifest skin changes, e.g., rhytids, textural worsening, solar elastosis, and dyschromias, and gravitational effects, e.g., brow, periorbital, midface, jawline, and neck descent. However, the concept of volume deflation via loss of both soft-tissue and bone has increasingly been recognized not only as an ancillary part of the aging process, but also as a core facet of aging [1]. Although various components of the aging process will be discussed in this chapter, the central role of volume loss will be elucidated in great detail and the effect that volume loss and volume repletion have on the appearance of the skin will be explored. Although some fundamental scientific ideas will be evaluated, this chapter is almost entirely of a clinical nature. It discusses how the facial aging process is seen as well as how surgical and nonsurgical interventions are made to ameliorate this condition. Autologous facial fat transfer is the mainstay of therapy for facial rejuvenation followed by other adjunctive measures including rhytidectomy, hair restoration, and skin therapies. Regarding facial fat transfer, some new theories about the observed stem-cell changes to the skin in which scarring, dyschromias, and rhytids diminish over a period of 1–2 years following a fat transfer will also be discussed. From a surgeon’s perspective, this textbook is principally focused on educating the students and researchers on aging skin, and in this case, the overall aging process related to cutaneous aging. Accordingly, very little will be addressed as far as elaborate technical execution of surgical methods is concerned. Instead, the primary focus will be in alignment with the objectives of this textbook, which are to provide current and comprehensive understandings of the aging process, i.e., to teach the reader to see more than to do.
Understanding the Facial Aging Process Using a Volume-Centric Model As alluded to earlier, the effect of gravity on the facial aging process has been greatly overestimated. What was once thought to be distinctly gravitational has been reconsidered as representing volume deflation. Lambros studied the aging process by superimposing the face of a mature individual over the exact pose of that same individual at 20 years of age [2]. He then evaluated the effects that accounted for periorbital, midface, and jawline aging. His results are nothing short of remarkable: the brow was seen on occasion to descend approximately 1 mm but no further; the lower eyelid–cheek interface did not fall at all, the cheek and nasolabial groove did not descend at all, and the jawline actually went up with aging. This latter observation can be quite confusing without further explanation. The jowl that appears with aging has been thought in the past to represent descent of that part of the jawline. What Lambros discovered was that the entire jawline that was once volumetrically full (by virtue of replete fat, softtissue, and bone) was actually, at one point, situated below the jowl along its entire length. In other words, the entire jawline recedes superiorly to expose the jowl. In addition, by examining the relative position of nevi to the surrounding anatomic landmarks he found that nevi do not fall downward, but either do not change position at all or migrate horizontally along muscle pull directions, indicating a volumetric deflation rather than a descent. With this almost unassailable empiric study, it is hard to argue that gravity is the principal mechanism by which facial aging occurs. Further, simply having patients bring in their own photographs during their youth will help educate both the patient and the surgeon regarding what techniques would be ideal to rejuvenate that patient so that the result will approximate their own youthful mien and avoid the unnatural alteration of identity that can occur through overzealous, traditional, excisional techniques. Lambros states: ‘‘The brows do not fall as much as we pick them up’’ [3]. Accordingly, using old photographs can be an instructive guide to plan surgical intervention. Interestingly, traditional textbooks on plastic surgery are perhaps the worst source of information to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_104, # Springer-Verlag Berlin Heidelberg 2010
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understand the aging process and what is required to rejuvenate the face. Brows are arbitrarily lifted upward and upper eyelid skin aggressively removed along with upper eyelid fat. Lower eyelids have fat extracted and skin tightened leaving the area hollower and the canthal position unnaturally changed. Instead, the ‘‘textbooks’’ that have the most accurate information on what defines youth are entitled Glamour, Allure, and Vogue and can be purchased at any newsstand. The reader is encouraged to review these exemplars of youthful beauty to understand facial shape, proportion and what exactly defines youth. In many instances, the very low brows that are robustly full will perhaps shock a
complacent surgeon/physician into rethinking the aging process entirely.
Framing the Eye Traditional reductive surgery, especially in the periorbital region, oftentimes leads to an acceleration of the aging process in which the hollower eyelid and brow are manifestly more so after the procedure (> Fig. 104.1). In short, traditional eyelid and brow surgery can actually render an individual even older in appearance than the desired rejuvenation. A model to understand what in fact happens
. Figure 104.1 This 45-year-old woman is shown (a) before fat transfer but after eyelid and browlift surgery (done elsewhere) that have in combination left her looking different and not more youthful. She is shown (b) 1 week following full facial fat transfer, (c) 3 months after the procedure with a slight dip in appearance, and (d) 1 year after a single session of fat transfer
A New Paradigm for the Aging Face
with the brow is to think of a balloon that deflates, in which the goal is not to excise the hanging and deflated skin but to refill the lost volume (> Fig. 104.2). Now, in selected individuals, a very small degree of skin (1–3 mm) is removed but almost always in conjunction with fat transfer to the brow and upper eyelid for the aforementioned reasons. Performing browlifts was stopped several years ago realizing today that a browlift is rarely, if ever, indicated and can be counterproductive in the ultimate goal of framing the eye with fat. The lower eyelid ‘‘eyebag’’ that appears most oftentimes does not need to be removed but additional fat added along the exposed
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periorbital bony rim. This again represents the opposite strategy of traditional surgery in which the eyebag is removed and the skin is tightened. A model for the lower eyelid may be informative to understand this concept better: the rocks (eyebag) are covered by high tide (fat along the orbital rim) but when the shoreline recedes (panfacial fat loss) the rocks that were once covered become evident. In a lecture, William Little explained it this way, and his analogy makes quite a bit of sense. In other words, the exposed orbital fat may be thought of as being camouflaged in youth with the presence of fat surrounding it along the orbital rim.
. Figure 104.2 This 46-year-old woman appears to require traditional eyelid surgery. However, with closer inspection she exhibits a hollow eye deformity due to volume loss that also involves the entire face. She is shown (a) before fat transfer, (b) 1 week after fat transfer, (c) 1 month after, (d) 3 months after, (e) 1 year after, and (f) 1.5 years following a single session of fat grafting
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A transconjunctival blepharoplasty is performed to remove excessive orbital fat only in about 5% of operative cases, but almost always in conjunction with adding fat along the bony orbital rim. Skin resurfacing and botulinum toxin are used to address the cutaneous changes in the lower periorbital region and no skin is almost ever removed along the lower half of the periorbital frame for fear of changing the lower eyelid and/or canthal position in an unfavorable way. Another finding that Lambros discovered in his seminal study was that the eyelid shape is more almond configured in youth no matter the race and that in turn becomes increasingly smaller and rounder in shape with age. This narrower eyelid shape of aging is more accentuated via traditional browlifting techniques and aggressive reductive eyelid blepharoplasty, which has been facetiously termed blepherectomy (> Fig. 104.1). Although there is no known method to safely re-enlarge the lateral canthus to resemble the almond shape of youth without risking unpredictable scleral show, fat transfer to the lateral brow can help draw one’s attention to the illusory widening of the eye shape rather than a further narrowing of it. These concepts are very difficult to explain with superlative clarity, and the reader is advised to study young and old (unoperated) eyes in the quest to understand what takes place in nature rather than what happens in the preconceived brain or biased surgical training history that can taint perceptions of the aging process.
Recalibrating Perceptions and Redefining Aging Most individuals, specifically women, seek microchanges to their face, namely, improvement in the appearance of small folds and creases around their mouth, reduction of the ‘‘crepiness’’ of their upper eyelid, etc. The reason for this desire in change is that women in particular apply makeup at close range so that the small facial flaws take center stage and appear to be the focus for aesthetic improvement. The first goal of the aesthetic surgeon/ physician is to recalibrate what may be more important for the patient when evaluating the face for aging and related cosmetic enhancement. Gladwell’s [4] brilliant thesis, Blink, argues that we judge another individual in a blink of an eye. When evaluating the face for aging, it should be understood what programs us to make an almost immediate, visceral response to another person’s attractiveness and aging. It is certainly not the micro skin effects in the perioral region in most cases, but a larger gestalt that is quickly apparent upon first glance.
Before wrinkles and small flaws can be appreciated, the gut response of a bystander judging another individual regarding age and attractiveness at even a relatively far social distance of 10–20 feet should be the measure of how aging is understood. What is the fundamental element, then, if it is not the prescribed traditional vocabulary of wrinkles, folds, and other minor flaws? In a word – geometry. The facial shape of another as being older or younger is recognized almost instantaneously. This concept is further refined in this chapter. The shape of the face of a baby, a young child, a teenager, and an individual in his/her early 20s is round owing to the abundance of so-called ‘‘baby fat’’ despite overall body weight or habitus. The ongoing volume loss of the face is a continuous process that begins from infancy forward. An individual in his/her early 20s has proportionately less fat in the face than a child or a baby. Accordingly, this process continues for the remainder of one’s life. Most women in fact who are dreadfully afraid of looking fat oftentimes when they pass 35 years of age look retrospectively at their youth and in the majority of cases prefer the look of their face in their early 30s than in their 20s due to the slimming effect that further soft-tissue loss affords them. However, passing through the early 30s into the mid to late 30s, a slight fatigue and aging become more apparent as they mature passing through the narrow window of full-framed youth to thinner 30-something youth to now slight aging with further volume loss. These volume changes can be redefined more precisely with geometric terminology in broad strokes. From infancy to early 20s, the predominant facial shape is round. With a slight slimming effect that occurs in the early 30s and loss of fat in the buccal area among other areas, the face transforms into a triangle with the apices in the anterior cheek and chin. In the intervening mid to late 20s a hybrid shape is observed somewhere between a circle and a triangle, i.e., a less circular circle or a slightly widened triangle. Keep in mind that these geometric assumptions are not meant to describe each and every individual person, as variances occur owing to gender, race, genetics, weight, and environmental insults like sun exposure, smoking, etc. As volume loss progresses from mid 30s into the early 40s, the face assumes a more masculine appearance whether the individual is a man or a woman. The eyes and the cheeks flatten, and the padding of the anterior chin starts to dissipate exposing the underlying malar and chin bone protuberances. With volume loss across the expanse of the jawline minus the jowl region, the apices of a new geometry shift toward the appearance of a square: the malar bone and the jowl become the new apices of this square. The flattening effect of the face
A New Paradigm for the Aging Face
further accentuates the masculine contour along with the exposed bone, which is a masculinizing attribute. Many male models are chosen for their flatter anterior cheek profile, as they look more chiseled and attractive for these masculine hallmarks. It has been noticed that even very young male models are chosen for this attribute of greater bone exposure than their female counterparts. As metabolism slows in the late 30s and beyond, weight gain is oftentimes more prevalent at this juncture. The mid to late 40s and thereafter exposes the curious mixture of weight gain and soft-tissue volume loss further unbalancing the face. The soft tissue of the periorbital region, upper anterior cheek, and anterior chin continue to dissipate in the face of weight gain that becomes more pronounced in the lower anterior cheek and the jowl region along with neck adiposity. The dominance of the lower face and ongoing recession of the upper face with marked depressions running supero-medially down infero-laterally in the anterior cheek transform the face into the shape of an upside triangle. These progressive changes become even more apparent with further aging of the 50s and beyond as the lower face dominates with concurrent volume loss of the periorbital region and midface.
Beyond Geometry: Understanding Transitions and Highlights Is gross geometry then the only perception of aging? Obviously not, as the neck does suffer from gravitational forces with the exposure of loosening platysma. There are also readily apparent signs of cutaneous damage with the onset of rhytids, dyschromias, etc. However, what can be more important than both neck descent and skin changes is what could be termed microgeometry. The aforementioned geometry in the previous section can be smoothly presented with minimal transitions or with multiple abrupt demarcations. Take for example an overweight child or young adult versus an overweight 50 years something. If they are both replete with fat, how does the brain determine their aging even before a wrinkle or a hanging neck is perceived? The answer lies in the fact that an overweight youth is uniformly convex and uniformly round. An overweight person past 35 years of age or so will exhibit areas of marked hollowness that become even more pronounced alongside pockets of excessive weight gain in the lower cheek, jowl, and neck area. These abruptions in gross geometry further accentuate perception of aging. That is how it is possible to tell if someone is young and simply full with their natural baby fat, someone overweight and young, and someone overweight and older.
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For example, someone who is relatively thin or too thin in youth as compared with aging. The young person even when very thin will still maintain a soft-tissue padding that is relatively uniform unless they are so thin that they look emaciated. An older individual will show abrupt transition points as the underlying bone is exposed and retaining ligaments exacerbate transition points. In short, a young face exhibits relative uniformity, whereas an older face displays signs of obvious transitions between various facial regions despite weight, neck descent, and signs of skin aging. With that respect, another word to introduce in perception is convexity. The reader is reminded that in daily life most situations involve the play of overhead lighting. Indoors, top-down lighting is the norm, and even outdoors, the sun shines from a relatively high vantage point. Flash photography, on the other hand, tends to wash out facial features that can improve one’s appearance. Daily life is not so kind. With overhead lighting, everyone can look a bit worse. The more pointed the light source from above, the worse that facial features can appear. The wellknown ‘‘mug shot’’ look of celebrities caught after a bad night of partying reflects as much their torrid state as the harsh overcast lighting. With all of that in mind, two attributes of the aging process become sharply defined in relief with standard overhead lighting: the appearance of unwelcome facial transition zones (previously discussed) and the presence/absence of light convexity. The flatter the face with aging, the less is the light bounce that the face transmits back to the viewer. Relative convexity of the lateral brow, upper cheek, and chin with reflected light bounce back to the viewer are hallmarks of a youthful face. Softening abrupt transitions and creating facial convexity are two major objectives of facial fat transfer. Interestingly, when it comes to the appearance of skin, more light on the skin will make it look brighter and thereby more youthful. Other effects of fat transfer on the skin will be discussed next.
Stem-Cell Changes and Other Cutaneous Manifestations Following Fat Transfer Although skin resurfacing techniques and botulinum toxin remain the gold standards for addressing the signs of aging skin, there has been a consensus among fattransfer surgeons that favorable skin changes can occur down the road following fat transfer [5]. Wrinkles, scars, pores, texture, and other pathologies have been noted to diminish in areas overlying transplanted fat. These cutaneous effects, if they manifest, require months if not a year
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or more to start to show up. Personal findings noted the reduction of acne scarring after a year or more as well as scar reductions in areas that failed to be corrected with conventional scar revision surgery. Reports have claimed improvement in conditions such as radiation damage, chronic ulceration, breast capsular contracture, and damaged vocal cords. Thoughts include that transplanted fat cells may contain adipocyte-derived stem cells or preadipocytes that can repair surrounding tissues and perhaps even transform into bone, cartilage, muscle, blood vessels, nerves, and skin [6]. Thinking and research on these purported claims is still in its nascent phase, but the clinical evidence is difficult to deny.
Long-Term Outcomes Using a Hair-Transplant Model A major drawback that has been expressed regarding transplanted fat is its equivocal longevity. Simply put, this problem was not observed. With proper hand harvesting using gentle negative pressure, centrifugation to purify the fat cells, and atraumatic cannula injection techniques using microdroplet technique, transplanted fat holds remarkably well. It is with rare exception that an additional session is required to attain the optimal results. However, to understand the nature of this longevity, the evolution of fat grafting results over the first 2 years and beyond must be understood. A critical study that is in the process of publication by a major journal [7], documents the evolution of a fat grafting result over the first 18 months using threedimensional computer VECTRA modeling. Using this method, the author quantified the volume a fat-transfer result would have preoperatively and at points measured at 3 months and every 3 months thereafter for the first year and a half. What they found was that the fat-transfer results at 3 months in many cases had the same volume as preoperatively, i.e., there was no appreciable volumetric gain. However, at 6 months the result started to increase in volume and steadily did so in each recorded interval, i.e., every 3 months. The obvious question then is why should this happen? Why would the result apparently evaporate at 3 months and then steadily increase thereafter. An attempt to reframe the thinking of this concept has been made using a hair-transplant model in the author’s forthcoming book, Aging Face: The New Paradigm [8]. Hair transplant surgery using follicular-based grafts is very similar to a fat transfer for the following reasons. First, they both rely on free grafts, i.e., no direct microvascular attachment, just freely transplanted with
surrounding blood supply creeping in over time. Second, both types of grafts are relatively small and numerous (tiny parcels of fat the size of 1/50th of a cubic centimeter compared with tiny hair grafts containing 1–4 hairs). Third, they are both transplanted into the same general body region, i.e., the head. Finally, albeit least importantly, they are both performed for cosmetic purposes. Walter Unger’s book, Hair Transplantation [9], explains how transplanted hair grafts attain their blood supply over time. During the first few days, nutrients from the surrounding tissue enter the graft through a process known as plasmatic imbibition. Thereafter, a tenuous blood supply maintains graft viability through a process known as primary and secondary inosculation. It is not until 6 months following a hair transplant that formal neovascularization is fully attained. This time period also correlates with clinical onset of substantive hair growth. From experience, except for occasional examples of significant hair growth at 3–4 months, in most cases pronounced clinical growth is generally evident starting approximately 6 months following hair transplant surgery. Hair grafts then continue to grow at variable rates for the first 18 months. Not surprisingly, a similar progress in the evolution of a fat grafting result was clinically observed. The only difference would be that hair grafts typically fall out after the first few weeks, whereas a fat grafting result can persist for the first 6 weeks or so owing to the presence of edema since fat grafting is contingent upon soft-tissue volume, whereas a hair transplant result obviously is not. Like a hair transplant, a process of vascular inosculation maintains the fat graft alive, which does not become clinically apparent until typically 6 months following the procedure at which time neovascularization has been established with ongoing growth of a result in the majority of cases for the first 18 months or so just like a hair transplant. The longevity of a hair transplant and fat grafting result is also correlative. An individual who undergoes a hair transplant will retain the transplanted grafts but suffer ongoing hair loss in susceptible hairs that have not been transplanted. Similarly, once the fat grafting attains a mature blood supply, the grafts survive but ongoing volume loss of nontransplanted fat occurs with ineluctable aging. Generally, a fat transfer may require a minor touch-up procedure 3–4 years later in someone with a genetic predisposition toward more accelerated aging but will not require anything further for 5–10 years in most individuals. It has been speculated that fat grafting has been so roundly condemned in the past with regard to longevity due to several reasons. First, poor technique can compromise longevity with speculated errors including traumatic donor harvesting, inadequate or excessive processing, and
A New Paradigm for the Aging Face
improper infiltration techniques. Second, physicians may not sufficiently follow clinical results over time or understand the nature of a transplant result. More specifically, the 3-month interval that often presents a clinical situation that is quite unimpressive and may discourage surgeons from continuing, since the result appears to have dissipated. Conversely, filling a patient repeatedly during these time intervals of volume descent may lead to an uncorrectable overfilling when the fat grafting result attains maturity 2 years later. Some of the stem-cell changes that have been proposed for fat transfer have also been observed clinically when hair transplanted into regions of scarring alopecia can actually heal the damaged and cicatricial skin. Obviously, the effect that a transplanted hair graft has on surrounding tissues may arise through a recognized stem-cell process. The pilosebaceous unit is considered the source for skin regeneration with stem cells understood to reside in the bulge region of the hair shaft. In addition, the longstanding premise of modern hair transplant surgery that transplanted hairs fully retain the native characteristics of their donor region is being recently challenged [10]. For example, hairs grafted from the occiput into the eyebrow region have shown a retardation of hair growth rate to match that of native eyebrow hairs. In addition, hairs transplanted from the body to the scalp in individuals who have depleted their occipital donor hair have been shown to start growing more rapidly and become finer in caliber over time. These profound clinical observations reveal how little is known about the nature of a transplanted graft and to skin and hair changes in general.
Conclusion The landscape of understanding facial aging is constantly a shifting terrain. Most of the traditional thinking that dominated perception of aging (i.e., gravity and wrinkles) this past century has been recently upended by distilling the concept of aging through the primary mechanism of
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soft-tissue (and bony) volume depletion. The juncture between dermatologic and surgical worlds to restore the aging face has become more apparent than ever. The novel concepts of stem-cell changes that can manifest following fat transfer push this idea to an even greater measure. Ongoing clinical and basic-science research will ensure that more natural results are attained for facial rejuvenation that also more closely parallel the true nature of facial aging with possible derivative benefits to the understanding and treatment of the aging skin.
Cross-references > Cosmetic > Facial
Surgery in the Elderly Rejuvenation: A Chronology of Procedures
References 1. Lam S, Glasgold M, Glasgold R. Complementary Fat Grafting. Philadelphia: Lippincott, 2007. 2. Lambros V. Observations on periorbital and midface aging. Plast Reconstr Surg. 2007;120(5):1367–1376. 3. Lambros V. Lecture at Cedars-Sinai Medical Center. Los Angeles, October 26, 2008. 4. Gladwell M. Blink: The Power of Thinking Without Thinking. Boston: Little, Brown, & Company, 2005. 5. Having spoken to well-respected and experienced fat transfer surgeons over the years, the consensus is that these surgeons have clinically observed cutaneous benefits that have been thought to be related to fat transplant beyond what would have been observed through any other more direct skin therapy. 6. Coleman S. Structural fat grafting: More than a permanent filler. Plast Reconstr Surg. 2006;118:108S–120S. 7. Meier JD, Glasgold RA, Glasgold MJ. Autologous fat grafting: Longterm evidence of its efficacy in midfacial rejuvenation. Arch Facial Plast Surg. 2009;11(1):24–28. 8. Lam S, Karam A, Goldman M. Aging Face: The New Paradigm. London: Elsevier, 2009. 9. Unger W, Shapiro R. (eds) Hair Transplantation, 4th ed. London: Informa Healthcare, 2004. 10. Lee S, Kim D, Jun J, et al. The changes in hair growth pattern after autologous hair transplantation. Dermatol Surg. 1999;25:605–609.
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Ingredients and Products for Aging Skin
97 Aging and Anti-aging Strategies Giuseppina Candore . Giovanni Scapagnini . Calogero Caruso
Introduction Nowadays, people are living much longer than they used to do, however they are not free of disabilities and diseases, which still represent the dark side of aging and longevity. Aging is a relentless process that affects all cells, tissues, organs, and organisms, diminishing homeostasis and increasing organism vulnerability [1]. Aging progression causes a reduction of the response to environmental stimuli. It may be defined as a systemic loss of molecular fidelity that, after reproduction, reaches levels that exceed repair, turnover, or maintenance capacity. Loss of molecular fidelity occurs because energetics determines maintenance of the vital structure and functional integrity of the biomolecules. Hence, molecular fidelity is maintained for time periods. Thus, aging at molecular level results from increasing entropy that exceeds repair and turnover capacity. This progressive loss of molecular fidelity, in which the involvement of the reactive oxygen species (ROS) and free radicals has been implicated, increases predisposition to illness and death. Current biological thinking emphasizes that organisms are encoded for early survival and reproduction to prevent the species from extinction. These considerations suggest that the biological determinants of human aging lie in the fact that human cell maintenance and repair systems evolved when human life expectancy was only half what it is today. The host response mechanisms are limited because they occur at cost of investments in early survival and reproduction [2–6]. In the Western countries, comparing the mortality rate in people over 65 years, versus individuals in the age range between 25 and 44 years, it increases to 100-fold for stroke, and chronic lung disease, 92-fold for heart disease, 89-fold for pneumonia and influenza, and 43-fold by cancer [2], pointing out the importance of immuneinflammatory responses in aging. In fact, both innate and instructive immunity are implicated in almost age-related diseases. The modifications of the immune system in elderly are evaluated as a deterioration of the immune system, the so called immunosenescence [4]. On the other hand, aging in good condition seems directly correlated with a good functioning of the immune system,
suggesting that genes regulating the immune inflammatory responses are involved in longevity [7, 8]. On the other hand, the social–economical environment, that deeply affects the dietary and sanitary conditions, also has a remarkable role on the life expectancy, which was very short in the past. At the beginning of the twentieth century, the improved hygienic conditions determined, in the industrialized countries, a raise in life expectancy up to about 50–60 years. At the end of 1900, the improved hygienic conditions, the proper diet, the better health condition, and the decreased infant mortality, elevated life expectation up to 80 years [2]. In the last century, both human life expectancy and maximum lifespan potential increased, and the analysis of north-European mortality curves, suggests that a relevant role was played by the reduction of lifetime pathogen burden [9, 10]. In fact, increasing longevity and declining mortality in the elderly, occurred among the same birth cohorts that experienced a reduction in mortality at younger ages; so, the decline in old age mortality, by reducing deaths from cardiovascular diseases (CVD), was promoted by the reduced burden of infections and inflammation [11]. Thus, a low grade systemic inflammation characterizes aging and inflammatory markers are significant predictors of mortality in old humans [12].
Cellular Senescence Cellular senescence was first described by Hayflick and Moorfield in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrested [13–15]. The majority of senescent cells assume a characteristic flattened and enlarged morphology, and over the years a large number of molecular phenotypes have been described, such as changes in gene expression, protein processing, and chromatin organization [16–20]. The growth arrest occurs mostly in G1 phase [21]. Although individual cells arrest rapidly, probably within the duration of a single cell cycle, cultures are typically quite asynchronous with increasing proportions of cells withdrawing into senescence over a period of several weeks [22]. Senescent cells
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_97, # Springer-Verlag Berlin Heidelberg 2010
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maintain metabolic activity, and can remain viable essentially indefinitely. An important component of this stability in culture may be the capacity of senescent cells to resist apoptosis [23, 24]. Cellular senescence can be activated by various types of stressful stimuli, including telomere shortening, oncogenic, or tumor suppressor signals, and DNA damage. Progressive telomere shortening in successive cell divisions induces senescence due to the loss of terminal sequences during DNA replication. Maintenance of the telomere sequences at human chromosome ends is essential for immortalized cells to escape from the normal limitations of the proliferation capacity [14]. Conceptually, there are two broad categories of replicative cellular senescence. The first is initiated by dysfunctional telomeres or other forms of genotoxic stress eliciting a DNA damage response mediated primarily by the p53 tumor suppressor pathway [22, 25]. The second, much less understood response does not involve telomeres or DNA damage, and is characterized by the upregulation of the cyclin-dependent kinase inhibitor p16INK4a gene. These basic distinctions are however complicated by the fact that p16 can be upregulated by a wide variety of stresses, including some forms of genotoxic damage. Although inflammation is primarily triggered by the engagement of the immune system, it has been known since 1999 that the establishment of replicative cellular senescence in human skin fibroblasts, leads to a strong shared inflammatory response involving the transcriptional upregulation of cytokines, such as interleukins (IL-1, IL-15), their receptors (TLR4), and chemotactic secreted factors (Gro-a and MCP-1 among others) [17]. These results were initially interpreted as idiosyncratic of dermal fibroblasts and to recapitulate in vitro the inflammatory process associated with wound healing, a suspicion strengthened by the observation that such a response was augmented by high concentrations (10%) of serum. It is now known that senescence leads to a coordinated secretion of a large number of soluble factors [26, 27] – the socalled secretory phenotype – and this occurs in different cell types, following different stresses. Thus, skin aging is accompanied by a local pro-inflammatory status.
Skin Aging The aging process is noticeable within all organs of the body, and manifests itself visibly in the skin. So, skin aging is particularly important because of its social impact, and also represents an ideal model organ for investigating the aging process. Human stem cells have recently attracted so much general interest in many fields of biology and
clinical medicine including gerontology. In fact, in healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Despite accumulation of senescence markers in aged skin, epidermal stem cells are maintained at normal levels throughout life. Therefore, skin aging is induced by impaired stem cell mobilization or reduced number of stem cells able to respond to proliferative signals. In the skin, existence of several distinct stem cell population has been reported. The self-renewal and multi-lineage differentiation of skin stem cells make these cells attractive for aging process studies, but also for regenerative medicine, tissue repair, gene therapy, and cell-based therapy with autologous adult stem cells not only in dermatology. In addition, they provide in vitro models to study epidermal lineage selection and its role in the aging process [28]. However, cutaneous aging consists of distinct processes due to either intrinsic or extrinsic factors [29]. A stochastic process that implies random cell damage as a result of mutations during metabolic processes due to the production of free radicals is also implicated [30]. Intrinsic aging depends on time and reflects the genetic background. Hormones are decisively involved in intrinsic aging. Over time important circulating hormones decline due to a reduced secretion of the pituitary, adrenal glands, and the gonads or due to an intercurrent disease. Among them, growth factors (i.e., growth hormone and insulin-like growth factor-I) and sex steroids (i.e., androgens and estrogens) show significant changes in their blood levels, and play a distinct role in the generation of the aging phenotype [31]. The extrinsic aging, also know as photoaging, is clinically, biologically, and molecularly distinct from intrinsic aging. Photoaging is typically characterized by prominent alterations of the cellular components and the extracellular matrix of the connective tissue. Photoaging depends primarily on the degree of sun exposure and skin pigment. Individuals who have outdoor lifestyles, live in sunny climates, and are lightly pigmented will experience the greatest degree of photoaging [32]. Solar UV radiations hurt epidermal and connective tissues, activating complex molecular cascades able to accelerate physiological aging [33, 34]. Photoaging is characterized by specific and peculiar clinical and histopathologic features. The former include deep wrinkles, roughness and dryness, laxity, atrophy, yellowish complexion, hyperchromic areas (solar lentigo, flat seborrheic keratoses, freckles) and hypochromic areas, telangectasie, purpura, cutaneous fragility and pseudostellate scars, finally resulting in preneoplastic and neoplastic lesion development on
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chronically photoexposed areas. UV damages can be linked mostly to the photochemical overproduction of ROS and reactive nitrogen species (RNS). ROS and RNS UV-generated can directly alter cellular components (DNA, proteins, lipids), and also affect regulation of gene expression of signalling molecules/cascades such as mitogen activated protein kinases (MAPKs) and interrelated inflammatory cytokines as well as NF-kB and activator protein-1 (AP-1) [35]. It is also well documented that photoexposure induces the activation of the enzymatic systems, e.g., lipoxygenase (LOX) and cyclooxygenase (COX), which are responsible for the production of inflammatory mediators. Of particular interest is gene regulation and oxidative activation of matrix metalloproteinase (MPP), a family of Zn-dependent endopeptidases which are produced by different cell types and taken together are capable of degrading all the components of the intercellular matrix of the connective tissue. MPP takes part in the development of the alterations, typical of photoaging. Even limited exposure to solar light may induce MMP synthesis beyond the control of specific inhibitors [36, 37]. The role played by ROS in controlling MPP activity has been largely documented, and a critical role is due to the activation of the transcription factor AP-1 [38]. The adverse acute and long-term effects of solar exposure are well established, and in general, are related to skin type. It is widely assumed that sensitivity to UV is directly related to pigmentation or tanning ability, and this assumption is primarily based on epidemiological evidence that shows that skin cancer and photoaging are much less common in people who tan well or who have high levels of constitutive pigmentation [39, 40]. Furthermore, studies comparing dark-skinned peoples with related albinos [41] show the latter to have a higher incidence of photoaging. Pigmentation, whether constitutive (i. e., base skin color) or facoltative (induced by UV), depends on the balance between two classes of melanins: the eumelanins that are insoluble black or brown nitrogenous pigments and phaeomelanins that are alkali-soluble yellow to reddish-brown pigments that usually contain sulphur as well as nitrogen [42, 43]. Unlike eumelanins, which are mainly protective, phaeomelanins are considerably photolabile, and may produce highly cytotoxic and mutagenic free radical species on photoexcitation, which would account for the greater proclivity of red-haired Celtic-type population to photoaging, skin cancer, and sunburn [44, 45]. Apart phototype, individual ability to counteract noxious molecular events induced by UV, depends by the activation of a complex defence system against oxidative
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stress. Among the numerous defensive genes expressed during cellular stress response to UV exposure, a critical role seems to be played by an heterogeneous family of proteins, the heat shock proteins (HSPs) [46, 47]. This family includes the HSP70, an inducible protein able to refold damaged proteins, and Heme Oxygenase 1 (HO-1), a redox sensitive enzyme with strong cytoprotective effects in several tissue, including skin [48, 49]. Cellular ability to maintain adequate expression levels of protective genes such as HSP70 and HO-1, in response to a stressful insult, such as UV exposure, seems to be essential to preserve cellular homeostasis, and to delay aging related degenerative processes [50, 51]. Individual variability in the efficacy to activate these defensive genes is due to mechanisms not completely understood that include genetic makeup, responsible also for different phototypes and age. At molecular level post trascriptional regulation might represent a putative mechanism to modulate individual efficiency in the activation of cellular stress response. Post transcriptional regulation is fundamental to modify the half-life of some messenger RNAs [52]. Both HSP70 and HO-1 have been shown to be post transcriptionally regulated in various cell lines [53, 54], and this process is thought to be altered during aging. This evidence has been proposed as a possible cause for the impaired efficacy of defensive genes such as HSPs [55]. In recent years, natural derived polyphenols have attracted considerable attention because of their skin photoprotection effects [56]. Many of these substances have been shown to activate specifically the expression of some HSPs and in general genes involved in cellular stress response [57, 58]. Having a better understanding of the protective role played by HSPs in photoaging processes and mechanisms, regulating their activation, will allow identifying the novel pharmaceutical strategies to prevent photoaging. The immune system may either have a protective role against sunburn and skin cancer, or conversely, promote solar damage. The skin is poised to react to infections and injury, such as sunburn, with rapidly acting mechanisms (innate immunity) that precede the development of acquired immunity and serve as an immediate defense system. Some of these mechanisms, including activation of defensins and complement, modify subsequent acquired immunity. An array of induced immune-regulatory and pro inflammatory mediators is evident, at the gene expression level, from the microarray analysis of both intrinsically aged and photoaged skin. Thus, inflammatory mechanisms may accentuate the effect of UV radiation to amplify direct damaging effects on molecules and cells, including DNA, proteins, and lipids, which cause
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immunosuppression, cancer, and photoaging. A greater understanding of the cutaneous immune system’s response to photo-skin interactions is essential to comprehensively protect the skin from adverse solar effects. Sunscreen product protection, measured only as reduction in redness (current ‘‘sun’’ protection factor) may no longer be sufficient, as it is becoming clear that protection against UV-induced immune changes is of equal if not of greater importance [59]. However, both intrinsic and extrinsic mechanisms, in a vicious circle, through ROS production and telomere shortening, are responsible for a pro inflammatory status of skin, hence worsening skin aging.
Anti-aging Strategies A constant dream of humankind is to stop, postpone, and/or reverse the aging process. During the last years, an increasing number of scientific meetings, articles, and books have been devoted to anti-aging strategies and therapies. This topic is very popular among the general public, whose imagery has been fascinated by all possible tools and tricks to retard aging, thus, approaching immortality, but it is also full of misleading, simplistic, or wrong ideas. It is timely to clarify the rationale and the biological basis of possible scientific strategies, and remember the constraints which have to be taken into account within such a perspective [4, 60]. In almost all instances, claims of drugs, health supplements, and other types of intervention are not based on any evidence supported by sound scientific knowledge. Based on a report by the United States General Accounting Office on the questionable and even harmful effects of anti-aging health products for seniors, some years ago, George Wick concluded that many of the benefits claimed by such health products have no scientifically-proven effect on the aging process, and in many instances, have the inherent potential for both physical and economic damage to the consumer. Of course, other approaches, including intervention in telomere shortening, cell cycle control, interfering with the oncogene/anti-oncogene homeostatic balance, etc. are being studied as possible ‘‘anti-aging’’ manipulations. Although solid data suggesting increased survival time in vitro or life span expansion in experimental animals in vivo has been reported in some of these instances, these interventions are still far from being applicable in the complex human situation, and must therefore await further scrutiny, particularly with regard to still unknown side effects [61]. Thus, in the present chapter, anti-aging strategies aimed not to rejuvenate, but to slow aging and to delay
or avoid the onset of age-related diseases are discussed, allowing to substantially slow down the aging process, extending people’s productive, youthful lives. In particular, the chapter will briefly focus on the control of inflammation that is involved in the pathophysiology of most age-related diseases and on dietary intake. Finally, it will discuss specific skin anti-aging treatment. As previously stated, aging is accompanied by a lowgrade inflammation held responsible for many age-related diseases, so a decrease in the rate of inflammation should prevent the activation of the immune system. The ageassociated increase in pro inflammatory cytokines in the elderly, raises the possibility that some pro inflammatory cytokine-blocking antibodies or soluble receptors could also be beneficial for the elderly. Moreover, there are other less potent, well-known inflammation-modulating drugs, such as statins and non-steroidal anti-inflammatory drugs that have few side effects and can be used with safety even in very old subjects. On the basis of this, it is reasonable to assume that anti-inflammatory treatments could be useful to counteract and reduce the age-dependent inflammatory status [60, 62, 63]. Another crucial intervention is to provide elderly subjects with a correct dietary intake. It is well accepted that nutrition can influence or even play a leading role in the development of various diseases such as infections, cancer, and CVD. Approximately, 40 micronutrients (vitamins, essential minerals, and other compounds required in small amount for normal metabolism) have been reported as essential components in the diet [64]. The dietary intake of essential macro and micronutrients is usually inadequate in the elderly [65]. Several causes contribute to this gap. First of all, the poor socio-economic conditions present in a large part of old people may lead to consumption of inexpensive food items deficient in micronutrients, such as carbohydrates [66]. The gap is worsened by loss of appetite, lack of teeth, and intestinal malabsorption that lead to the final result of frailty, disability, and mortality [67]. So, a crucial topic is to provide elderly subjects with a correct dietary intake. To the extent that after conscious control of one’s own behavior, environment, life style, and diet, a degree of control of both the aging and diseases of aging are potentially possible. Macronutrients such as anti-oxidant, dietary fibre, omega-3, as well as micronutrients such as vitamins, zinc, iron and copper, and selenium are of particular interest. Thus, nutritional interventions could be beneficial for the prevention, retardation, or even reversal of established immunosenescence and aging. In particular, Zinc plays a relevant role in the control of immune response in aging [68, 69]. There is a strong interrelationship
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between Zinc with Niacin and Selenium. Zinc is important for immune efficiency, energy utilization and hormones’ turnover, and antioxidant activity. Accordingly, improved immune performance, metabolic harmony, and antioxidant defence occur in elderly after physiological zinc supplementation, which also induces prolonged survival in old, nude, and neonatal thymectomized mice. Interestingly selenium provokes zinc release by Metallothioneins, and the association ‘‘zinc plus selenium’’ improves humoral immunity in old subjects after influenza vaccination [70]. Red wine represents a source of polyphenols which exhibit a number of biological effects on various systems; in this respect, there is evidence that red wine polyphenols constitute one of the ingredients of the Mediterranean diet which is associated with reduced all cause and cause-specific mortality as CHD [71]. Recently a series of papers [72–74] focused on some aspects of the effects of polyphenols. The authors have investigated the ability of red wine polyphenols to promote the in vitro release of both pro inflammatory and anti-inflammatory cytokines from human healthy peripheral blood mononuclear cells (PBMC), as well as of immunoglobulins from B cells. Following red wine cell pretreatment, results show a production of regulatory interleukin (IL)-12, proinflammatory (IL-1beta and IL-6), and anti-inflammatory (IL-10) cytokines, as well as of IgA and IgG. They discussed the fine balance between inflammation and anti-inflammation, as well as the role of humoral immune response either systemic or mucosal as a consequence of red wine intake. Turning on the molecular mechanisms elicited by polyphenols from red wine on PBMC, they investigated their involvement in the activation of p38 and ERK1/2 molecules belonging to the MAPK kinase family involved in release of interferongamma, and therefore, in nitric oxide (NO) production. Results demonstrated that in cells both expression of p38 and ERK1/2 augments in presence of red wine polyphenols, but their expression drops in presence of polyphenols plus LPS. This indicates that in Gram-negative infections, polyphenols may attenuate triggering of inflammatory mediators as a response to LPS stimulation. Concerning the wellknown idea that red wine might favor anti-atherogenic mechanisms of red wine in the course of cardiovascular disease, an important aspect pointed out is the release of NO from PBMC stimulated by red wine polyphenols. Release of NO from mononuclear cells may play an important role in cardiovascular disease, because it is known that this molecule acts as an inhibitor of platelet aggregation [75–77]. Ultimately, as previously discussed, aging is characterized by immunosenescence; hence these results suggest that moderate use of red wine may be beneficial in
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age-related disorders where the host immune response is very often not effective against a variety of antigens. Finally, in a survey recently published, it has been found that higher adherence to a Mediterranean diet was associated with a statistically significant reduction in total mortality. The dominant components of the Mediterranean diet score as a predictor of lower mortality are moderate consumption of ethanol, low consumption of meat and meat products, and high consumption of vegetables, fruits and nuts, olive oil, and legumes. Minimal contributions were found for cereals and dairy products, possibly because they are heterogeneous categories of foods with differential health effects, and for fish and seafood, the intake of which is low in this population [78]. UV induced skin damage by ROS is a rapid process, and antioxidants can prevent the damage when applied at the beginning or during the development of oxidative stress. Antioxidant supplementation is an integral part of a multi-faceted approach in photoprotection. Topical application of vitamin E has shown to induce smoothening of fine lines and wrinkles whereas when given as a diet supplement, a limited cutaneous bioavailability was indicated which is insufficient to scavenge ROS generated in photoaged human skin. Topical delivery of these agents is an attractive alternative, so that they can be used as cosmetic ingredients against skin aging, especially as curative/therapeutic in addition to their prophylactic action [79].
Conclusion A long life in a healthy, vigorous, youthful body has always been one of humanity’s greatest dreams. According to De Grey [80], recent progress in genetic manipulations and calorie-restricted diets in laboratory animals hold forth the promise that someday science should enable to exert total control over own biological aging. To conclude, at present, aging must be considered an unavoidable end point of the life history of each individual. Nevertheless increasing knowledge about the mechanisms regulating aging, allows to envision many different strategies to delay the onset of age-related diseases, in order to endow everybody with a long and good final time in life.
Cross-references > Cosmetics
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26. Krtolica A, et al. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA. 2001;98:12072–12077. 27. Coppe JP, et al. Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J Biol Chem. 2006;281:29568–29574. 28. Zouboulis CC, et al. Human skin stem cells and the ageing process. Exp Gerontol. 2008;43:986–997. 29. Uitto J, et al. Molecular aspects of photoaging. Eur J Dermatol. 1997;7:210–214. 30. Puizina-Ivic´ N. Skin aging. Acta Dermatovenerol Alp Panonica Adriat. 2008;17:47–54. 31. Farage MA, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 32. Fischer GJ, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol. 2002;138:1462–1470. 33. Cole J, et al. Comparison of normal human skin gene expression using cDNA microarray. Wound Repair Regen. 2001;9:77–85. 34. Curto EV, et al. Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods. Biochem Biophys Res Commun. 2002;291:1052–1064. 35. Angel P, et al. Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene. 2001;20:2413–2423. 36. Brenneisen P, et al. UVB wavelength dependence for the regulation of two major matrix metalloproteinases and their inhibitor TIMP1 in human dermal fibroblasts. Photochem Photobiol. 1996;64: 649–657. 37. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med. 2004;37:768–784. 38. Callaghan TM, Wilhelm KP. A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part I: cellular and molecular perspectives of skin ageing. Int J Cosmet Sci. 2008;30:313–322. 39. Weinstock MA. Ultraviolet radiation and skin cancer: epidemiological data from the United States and Canada. In: Young AR, Bjorn LO, Moan J, Nultsch W (eds) Environmental UV Photobiology. New York: Plenum Press, 1993, pp. 295–344. 40. Kollias N, et al. Photoprotection by melanin. J Photochem Photobiol B Biol. 1991;9:1135–1160. 41. McFadden AW. Skin disease in the Cunan Indians. Arch Dermatol. 1961;84:1013–1023. 42. Hearing VJ. Regulation of melanin formation. In: Norlundss J, Boissym RE, Hearing VJ, King RA, Ortonne JP (eds) The Pigmentary System. Oxford: Oxford University Press, 1998, pp. 423–438. 43. Land EJ, Riley PA. Spontaneous redox reactions of dopaquinone and the balance between the eumelanic and phaeomelanic pathways. Pigment Cell Res. 2000;13:273–277. 44. Ito S, Jimbow K. Quantitative analysis of eumelanin and pheomelanin in hair and melanomas. J Invest Dermatol. 1983;80: 268–272. 45. Novellino L, et al. Isolation and characterization of mammalian eumelanins from hair and irides. Biochim Biophys Acta. 2000;1475: 295–306. 46. Morris SD. Heat shock proteins and the skin. Clin Exp Dermatol. 2002;27:220–224. 47. Trautinger F. Heat shock proteins in the photobiology of human skin. J Photochem Photobiol B. 2001;63:70–77. 48. Holzer AM, et al. Heat-shock proteins as drugs: potential applications in cancer, infections, and autoimmune and atopic diseases. J Drugs Dermatol. 2007;6:393–399.
Aging and Anti-aging Strategies 49. Rattan SI, Ali RE. Hormetic prevention of molecular damage during cellular aging of human skin fibroblasts and keratinocytes. Ann N Y Acad Sci. 2007;1100:424–430. 50. Tyrrell RM. Solar ultraviolet A radiation: an oxidizing skin carcinogen that activates heme oxygenase-1. Antioxid Redox Signal. 2004;6:835–840. 51. Abraham NG, et al. Human heme oxygenase: cell cycle-dependent expression and DNA microarray identification of multiple gene responses after transduction of endothelial cells. J Cell Biochem. 2003;90:1098–1111. 52. Brennan CM, et al. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol. 2000;151:1–14. 53. Kaarniranta K, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci USA. 1998;95:2319–2324. 54. Gallouzi IE, et al. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci USA. 2000;97:3073–3078. 55. Wang W, et al. Loss of HuR is linked to reduced expression of proliferative genes during replicative senescence. Mol Cell Biol. 2001;21:5889–5898. 56. Baumann L. How to prevent photoaging? J Invest Dermatol. 2005;125:xii–xiii. 57. Scapagnini G, et al. Curcumin activates defensive genes and protects neurons against oxidative stress. Antioxid Redox Signal. 2006;8: 395–403. 58. Scapagnini G, et al. Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers. Mol Pharmacol. 2002;61:554–561. 59. Bennett MF, et al. Skin immune systems and inflammation: protector of the skin or promoter of aging? J Investig Dermatol Symp Proc. 2008;13:15–19. 60. Capri M, et al. Complexity of anti-immunosenescence strategies in humans. Artif Organs. 2006;30:730–742. 61. Wick G. ‘‘Anti-aging’’ medicine: does it exist? A critical discussion of ‘‘anti-aging health products.’’ Exp Gerontol. 2002;37:1137–1140. 62. Franceschi C, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci. 2000;908:244–254. 63. Vasto S, et al. Inflammatory networks in ageing, age-related diseases and longevity. Mech Ageing Dev. 2007;128:83–91. 64. Alvarez-Leon EE, et al. Dairy products and health: a review of the epidemiological evidence. Br J Nutr. 2006;96:S94–S99. 65. Ames BN. Low micronutrient intake may accelerate the degenerative diseases of aging through allocation of scarce micronutrients by triage. Proc Natl Acad Sci USA. 2006;103:17589–17594.
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66. Kant AK. Consumption of energy-dense, nutrient-poor foods by adult Americans: nutritional and health implications. The third National Health and Nutrition Examination Survey, 1988–1994. Am J Clin Nutr. 2000;72:929–936. 67. Semba RD, et al. Low serum micronutrient concentrations predict frailty among older women living in the community. J Gerontol A Biol Sci Med Sci. 2006;61:594–599. 68. Zhao W, et al. Migration and metalloproteinases determine the invasive potential of mouse melanoma cells, but not melanin and telomerase. Cancer Lett. 2001;162:S49–S55. 69. Giacconi R, et al. Pro-inflammatory genetic background and zinc status in old atherosclerotic subjects. Ageing Res Rev. 2008;7: 306–318. 70. Mocchegiani E, et al. Zinc, Metallothioneins and longevity: interrelationship with niacin and selenium. Curr Pharm Des. 2008;14: 2719–2732. 71. Trichopoulou A, Dilis V. Olive oil and longevity. Mol Nutr Food Res. 2007;51:1275–1278. 72. Magrone T, et al. Polyphenols from red wine modulate immune responsiveness: biological and clinical significance. Curr Pharm Des. 2008;14:2733–2748. 73. Magrone T, et al. Elicitation of immune responsiveness against antigenic challenge in age-related diseases: effects of red wine polyphenols. Curr Pharm Des. 2008;14:2749–2757. 74. Magrone T, et al. Molecular effects elicited in vitro by red wine on human healthy peripheral blood mononuclear cells. Potential therapeutical application of polyphenols to diet-related chronic diseases. Curr Pharm Des. 2008;14:2758–2766. 75. Bradamante S, et al. Resveratrol provides late-phase cardioprotetion by means of a nitric oxide and adenosine-mediated mechanism. Eur J Pharmacol. 2003;465:115–123. 76. Das S, et al. Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NF-kB. Free Radic Res. 2006;40:1066–1075. 77. Shen MY, et al. Inhibitory mechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK and NO/cyclic GMP. Br J Haematol. 2007;139:475–485. 78. Trichopoulou A, et al. Anatomy of health effects of Mediterranean diet: Greek EPIC prospective cohort study. BMJ. 2009, Jun 23;338: b2337. doi: 10.1136/bmj.b2337. 79. Kaur IP, et al. Role of novel delivery systems in developing topical antioxidants as therapeutics to combat photoageing. Ageing Res Rev. 2007;6:271–288. 80. De Grey A. Ending Aging. New York: St. Martin’s press, 2007.
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96 Aging and Skin Sensitivity Michael K. Robinson
Introduction Any attempt to assess and understand skin sensitivity or reactivity differences within and among human subpopulations is complicated by a variety of factors. Perhaps, the most important of these is a simple statistical reality of the inherent difficulty in making population-level assertions on the basis of studies conducted with relatively small numbers of subjects. > Figure 96.1 illustrates the dilemma by providing two different scenarios: one in which two sample populations with very different reactivity profiles are drawn from two relatively nonoverlapping parent populations (A) and a second in which the sample populations (again with different reactivity) are drawn from virtually indistinguishable parent populations (B). In the first instance, measured (and statistically significant) differences between the two sample populations would be representative of the actual populations from which the samples were drawn, whereas in the second case, the sampling would be done only within the overall reactivity ranges of the parent populations. In the case of population differences in skin reactivity, the exact nature of the parent populations or the dynamics of those populations over time is uncertain. Only a limited number of subjects of the representative groups can be tested, and based on the results obtained, presumptions are made about the global nature of populations from which those subjects were drawn. Observations of differences in reactivity among subsets of any population must be tempered by this uncertainty. This explains why the literature on population differences in fundamental skin biology or skin responsiveness is so often conflicting [1]. Skin aging is a dynamic process involving both intrinsic biological changes and the chronic strain imposed by environmental stressors such as UV irradiation [2]. Within the past decade, the term ‘‘inflammaging’’ has been used to describe the cascade of proinflammatory responses that accumulate with age and that lead to a variety of chronic inflammation-related disease states [3]. Inflammaging of the skin is itself one such state. It is central to the dichotomy of aging skin function that the inflammatory and immune responses act in both protection (from harmful agents such as bacteria, viruses, and cancer cells) and
restoration of the skin (wound healing), while also promoting the skin aging process [4]. Inflammation has long been known to accompany skin aging; however, it has been difficult to determine whether it is a driving force in the aging process or an epiphenomenon [5]. Recent genome-wide assessment of changes in gene expression associated with chronological aging and photoaging has clearly demonstrated that inflammation is a central theme in both processes, but exacerbated in photoaging [6, 7]. It is on top of this ever changing proinflammatory milieu that an attempt is being made to ascribe true differences in skin reactivity and their underlying mechanistic bases.
Aging and Basic Skin Biology/Physiology Although beyond the scope of this chapter, it is pertinent to at least briefly consider differences in some of the basic characteristics of skin that may be influenced by age. Characteristics such as skin thickness, barrier function, elasticity, wound repair potential, etc. can certainly influence overall skin reactivity profiles. A selection of published studies and reviews has indicated the following: 1. Reduced forearm skin elasticity with age (comparing pre and postmenopausal women) [8] 2. Lack of differences in epidermal thickness with age [9] 3. Likely reduction in barrier function and delayed recovery after barrier insult in photoaged skin [10, 11] 4. Qualitatively similar wound-healing response in elderly versus young subjects, but delayed response in the elderly [12] 5. Other literature disparity on effects of aging on wound-healing rates [13] 6. Defective removal of UV-induced pyrimidine dimers from epidermis of older (70–78 years) versus younger (22–26 years) subjects [14] 7. Reduced noradrenalin-induced vasoconstriction during skin cooling in older (62–76 years) versus younger (18–30 years) subjects [15] In general, there is a tendency toward a gradual diminution in skin structure and function with age, although the
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_96, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 96.1 Schematic representation of two small study populations with different reactivity profiles, but drawn from (a) two parent populations with distinctly different profiles or (b) two parent populations with virtually identical reactivity profiles (From Robinson SK. [41]. With permission)
variability in results across these studies makes it difficult to make very definitive conclusions around specific endpoints. In fact, there is a fairly extensive literature on the above topics and individual research studies commonly report alternative findings; particularly when different assessment methods are used. Again, this points to the difficulty in extrapolating from small-scale studies to the population as a whole.
Effect of Age on Objective Skin Irritation Response If aging skin is subjected to even low-level chronic inflammation, one might expect that the response to acute irritant exposure would be heightened due to
the known priming effect of underlying skin irritation on other types of skin reactivity, like allergic contact dermatitis [16]. In contrast, the literature on age-related susceptibility to skin irritation generally shows a reduced sensitivity in older (i.e., >60 years) versus younger adults. A comparison of two widely different age groups (18–25 and 65–84) for reactivity to a strong irritant stimulus (24-h patch exposure to 5% sodium lauryl sulfate [SLS]) showed greater mean reactivity in the young versus old subjects (4.57 vs. 2.62 on a 0–5 visual grading scale) [17]. A similar study using a 20-fold lower SLS concentration gave comparable results [18]. The mean response and percent positive responders were greater in young (average age 25.9) versus older (average age 74.6) test subjects. In some areas of the body (e.g., thighs), the response difference was quite dramatic. The lower visual grades in the older subjects were matched by a decrease in the magnitude of SLS-induced changes in barrier function. Grove et al. [19] studied different types of chemicalinduced skin irritation in young and old subjects. Using ammonium-hydroxide-induced blistering responses, they saw a more rapid initiation of blistering in their older (65–75) versus younger (18–30) subjects, but a much slower development of the full blister response. They also examined the response to a variety of irritants (e.g., histamine, DMSO, 48/80, chloroform–methanol, lactic acid, and ethyl nicotinate). In all cases, the visual grades were greater in the younger subjects. The decline in histamine reactivity with advancing age was, in fact, a confirmation of a much earlier study, which showed a trend of decreasing skin reactivity to intracutaneous injection of various concentrations of histamine across 10-year-age cohorts from 0–10 to >70 years of age [20]. Elderly subjects also showed reduced reactivity to ultraviolet irradiation [21]. Very few studies have shown the opposite effect (increased irritation in the elderly), although some exist [22]. Barrier function can also be more susceptible to the compromising effects of UV treatment among older subjects, even though visible skin reactivity is diminished [21]. In a recent survey of acute patch-test reactions to common irritants [23], results from multiple studies conducted over a period of 4 years were combined. In comparing response profiles across different age clusters, the oldest cohort of study subjects showed significantly reduced reactivity to two strong irritants (> Fig. 96.2). Responses to weaker irritants were directionally, but not significantly, reduced in these subjects (data not shown). Therefore, this larger population analysis supported the conclusions from smaller base size studies that elderly subjects are somewhat less susceptible to common skin
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. Figure 96.2 (a, b) The mean (+SE) acute irritation response grades for the indicated age group clusters to 20% SDS and 100% OAc, respectively. For SDS, the numbers of test subjects in each age cluster were 18–25 years (44), 26–35 years (77), 36–45 years (118), 46–55 years (80), 56–74 years (64), and 62–74 years (22) (one subject did not have his/her age recorded and was omitted from the analysis). For OAc, the numbers of test subjects in each age cluster were 18–25 (21), 26–35 (21), 36–45 (29), 46–55 (30), and 56–74 (34). The mean grades for each age cluster were cross-compared by statistical analysis. Abbreviations: SDS, sodium dodecyl sulfate; OAc, octanoic acid (From Green BG. [24]. With permission)
irritants. The results of all of these acute irritation studies would suggest that the underlying proinflammatory state of photoaged skin does not appear to prime the skin for increased reactivity to acute irritants. Thus, while promoting the structural breakdown commonly associated with aging skin, the inflammatory response is apparently less effective in its fundamental role of skin protection.
Effect of Age on the Subjective (Sensory) Skin Irritation Response In addition to objective skin irritation responses, there are many parameters of skin reactivity that are purely subjective or symptomatic in nature. These include the self-assessed quality of ‘‘sensitive’’ versus ‘‘normal’’ skin.
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They also include the various severities (e.g., mild, moderate, severe) and qualities (e.g., sting, burn, itch) of skin sensations to physical, chemical, or thermal insult. There are ways to quantify these symptomatic responses, but such measurements must be carefully controlled to avoid artifacts [24]. Also, the type of scales used to measure such responses can greatly influence the utility of the data for comparative analyses [24] like population response patterns or differences. Direct testing of sensory irritation involves application of chemical (e.g., lactic acid, capsaicin, histamine), thermal (heat, cold), or mechanical (tactile) stimuli to various parts of the body and capture of the resulting sensation by symptom grading. Examples include the lactic acid, chloroform/methanol, and capsaicin stinging/burning tests [25–28]. Although studies of this type are plentiful, few have directly addressed age-related differences in response profiles. One study of noxious and thermal stimulation of different areas of the face indicated disparate findings on age-related effects, although a slight reduction in response thresholds was observed with increased age (age range 20–89) [29], consistent with results on objective skin irritation described above. A recent histological study has shown reduced expression of nerve growth factor in the epidermis of older (age 51–81) versus younger (age 6–18 and age 19–50) subjects [30]. There has also been a reported decrease, with aging, of the epidermal nerve density (at least in the face) [31]. These observations may provide a mechanistic explanation for a reduced sensory response threshold in older adults.
Effect of Age on the Skin Sensitization Response Skin sensitization differs from skin irritation in its requirement for initial immune recognition of cutaneously encountered low-molecular-weight allergens. However, the dermatitis that is elicited after a secondary exposure to the same allergen can be very similar in visual appearance to primary irritant dermatitis. The requirement for immune recognition and response introduces an additional factor into the consideration of age-related differences in skin reactivity; i.e., immune competence. This is particularly noteworthy given the commonly accepted notion of reduced immune competence with advancing age [32]. Age-related studies of skin sensitization are mainly limited to retrospective analysis of elicitation patterns and incidences across patch-testing cohorts. Here the data is mixed and, because of the nature of the testing
approach, it is difficult to separate age-related inherent susceptibility from age-related patterns of exposure. An early study [33] showed no age-related differences in the incidence of allergic patch-test reactions across four common contact allergens (nickel, neomycin, ethylenediamine, and benzocaine). However, other investigators have seen different trends. Goh [34] studied the response incidence to common patch-test tray allergens and saw a general increase in incidence in subjects >40 versus those 59 versus those 70 years of age) showed similar incidence of response across 0–7, 8–14, and 20–50 age groups, but a reduced incidence in the oldest (>70) age group [36]. Kwangsukstith and Maibach have suggested that the incidence of allergic contact dermatitis gradually increases from birth to 14 years of age, then holds steady in overall incidence but greatly varies by allergen based on exposure patterns. The incidence declines with advancing age in terms of both the severity of response (possibly related to similar reduction in irritation responses discussed above) and a waning of the allergic response in previously sensitized individuals [37]. The only way to truly assess allergic sensitivity is to evaluate the induction of allergic contact sensitization in previously naive subjects. Studies of this type are not generally done today, but studies from the earlier literature do shed some light on age-related susceptibility. Back in the early 1950s, Schwartz [38] studied the induction of sensitization to dinitrochlorobenzene (DNCB) in previously naive subjects of different ages. Approximately half of 174 subjects became sensitized. Among three age cohorts (21–59, 60–79, >80) there was no significant
Aging and Skin Sensitivity
difference in the incidence of sensitization. Thus, for very potent sensitizers, there appears to be little age-related difference in allergic sensitivity. Although focused on the very young, a different result was obtained more recently using DNCB induction and challenge in infants from birth to 9 months of age [39]. Using a single DNCB dose for induction and one tenth of that dose for challenge, sensitization rates increased from approximately 7% at birth (up to 15 days of age), to 26% at the end of the first month, to 63% by the third month, and to 91% by the ninth month. These results are consistent with the understanding of the fact that the immune system is still maturing in the first months of life. Looking at a different type of immune response, immediate skin hypersensitivity (an IgE-mediated response), there is also ‘‘historical’’ evidence of an age-related decline in skin immune reactivity [20]. In this study, the investigators simply analyzed intracutaneous skin testing records for reactivity to both common and uncommon food allergens from among 100 subjects from their allergy practice in each 10-year-age cluster by decade (0–10, 11–20, 21–30, etc.) to >70 years of age. For the common allergens there was a dramatic age-related decline in the number of positive skin reactions (both slight positive and moderate to marked positive reactions). They showed a fairly progressive linear decline in percentage responses with each advancing decade of patient age. The percentage positive reactions to uncommon allergens were decidedly fewer, but a similar age-related decline was observed. Hence, the prevailing evidence on skin reactivity (for both immediate and delayed skin hypersensitivity responses) supports the notion of some degree of senescence in skin immune competence with advancing age.
Conclusion As noted above, there is a general caution that needs to be applied to studies that report differences in skin biology, reactivity, or symptoms. The caution simply relates to the fact that known intra- and interindividual differences in skin reactivity [23, 40] and the potential breadth of reactivity across large population clusters makes it difficult to draw definitive conclusions from studies on limited numbers of subjects. Age-related differences in skin reactivity tend to be more consistent in the response patterns that have emerged from individual studies, than studies of other population comparisons (e.g., gender or ethnicity) [23]. The trend toward reduced skin irritation responsiveness in elderly subjects is a fairly common observation. Self-perception of skin sensitivity and sensory skin
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responses, as a function of age, have not been studied well enough to draw firm conclusions, though the limited data available also support the age-related reduction in responsiveness. The situation is a bit more complicated with allergic responses. Here, except for the very young developing immune system, sensitivity may be as much related to exposure history than as to inherent differences in susceptibility per se, although the prevalence and severity of elicitation responses can show a decline with age. The interest in evaluating and understanding inherent patterns of skin reactivity and significant differences in reactivity across populations continues to grow. Much of this is due to the current trend in the development of products with improved tolerance profiles for all consumers, especially those with heightened skin sensitivity. It may also reflect the interest in marketing to specifically targeted segments of the population, for example, the development of skin anti-aging products. Regardless of the driving forces, clinical research on this topic is likely to continue and improve the understanding in the future.
Cross-references > Cutaneous
Effects and Sensitive Skin with Incontinence in the Aged > Perceptions of Sensitive Skin with Age
References 1. Robinson MK. Population differences in skin structure and physiology and the susceptibility to irritant and allergic contact dermatitis: implications for skin safety testing and risk assessment. Contact Dermatitis. 1999;41:65–79. 2. Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol. 2008;7(S2): s12–s16. 3. Franceschi C, Bonafe´ M, Valensin S, Olivieri F, De Luca M, Ottaviani E, De Benedictis G. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci. 2000;908:244–254. 4. Bennett MF, Robinson MK, Baron ED, Cooper KD. Skin immune systems and inflammation: protector of the skin or promoter of aging? J Invest Dermatol Symp Proc. 2008;13:15–19. 5. Krutmann J, Gilchrest BA. Photoaging of Skin. In: Krutmann J, Gilchrest BA (eds) Skin Aging. Berlin: Springer-Verlag, 2006, pp. 33–44. 6. Robinson MK, Tiesman JP, Binder RL, Juhlin KD. Immune and inflammatory gene expression profiles of chronological skin aging and photoaging. J Am Acad Dermatol. 2008;58(S2):408. 7. Robinson MK, Binder RL, Griffiths CEM. Genomic-driven insights into changes in aging skin. J Drugs Dermatol. 2009;8(7s):s8-s11. 8. Sumino H, Ichikawa S, Abe M, Endo Y, Ishikawa O, Kurabayashi M. Effects of aging, menopause, and hormone replacement therapy
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on forearm skin elasticity in women. J Am Geriatr Soc. 2004; 52:945–949. Sandby-Moller J, Poulsen T, Wulf HC. Epidermal thickness at different body sites: Relationship to age, gender, pigmentation, blood content, skin type and smoking habits. Acta Derm Venereol. 2003; 83:410–413. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model. J Clin Invest. 1995;95:2281–2290. Reed JT, Elias PM, Ghadially R. Integrity and permeability barrier function of photoaged human epidermis. Arch Dermatol. 1997;133:395–396. Gosain A, Luisa MD, DiPietro A. Aging and wound healing. World J Surg. 2004;28:321–326. Norman D. The effects of age-related skin changes on wound healing rates. J Wound Care. 2004;13:199–201. Yamada M, Udono MU, Hori M, Hirose R, Sato S, Mori T, Nikaido O. Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo. Arch Dermatol Res. 2006;297:294–302. Thompson CS, Holowatz LA, Kenney WL. Attenuated noradrenergic sensitivity during local cooling in aged human skin. J Physiol. 2005;564:313–319. Agner T, Johansen JD, Overgaard L, Volund A, Basketter D, Menne T. Combined effects of irritants and allergens. Contact Dermatitis. 2002;47:21–26. Lejman E, Stoudemayer T, Grove G, Kligman AM. Age differences in poison ivy dermatitis. Contact Dermatitis. 1984;11:163–167. Cua AB, Wilhelm KP, Maibach HI. Cutaneous sodium lauryl sulphate irritation potential: age and regional variability. Br J Dermatol. 1990;123:607–613. Grove GL, Duncan S, Kligman AM. Effect of ageing on the blistering of human skin with ammonium hydroxide. Br J Dermatol. 1982; 107:393–400. Tuft L, Heck VM, Gregory DC. Studies in sensitization as applied to skin test reactions. III. Influence of age upon skin reactivity. J Allergy. 1955;26:359–366. Gilchrest BA, Stoff JS, Soter NA. Chronologic Aging Alters the Response to Ultraviolet-Induced Inflammation in Human-Skin. J Invest Dermatol. 1982;79:11–15. Nilzen A, Voss Lagerlund K. Epicutaneous tests with detergents and a number of other common allergens. Dermatologica. 1962; 124:42–52. Robinson MK. Population differences in acute skin irritation responses – race, sex, age, sensitive skin and repeat subject comparisons. Contact Dermatitis. 2002;46:86–93. Green BG. Measurement of sensory irritation of the skin. Am J Contact Dermatitis. 2000;11:170–180.
25. Frosch PJ, Kligman AM. A method for appraising the stinging capacity of topically applied substances. J Soc Cosmet Chem. 1977;28:197–209. 26. Soschin D, Kligman AM. Adverse subjective responses. In: Kligman AM, Leyden JJ (eds) Safety and Efficacy of Topical Drugs and Cosmetics. New York: Grune & Stratton, 1982, pp. 377–388. 27. Christensen M, Kligman AM. An improved procedure for conducting lactic acid stinging tests on facial skin. J Cosmet Sci. 1996;47:1–11. 28. Green BG, Bluth J. Measuring the chemosensory irritability of human skin. J Toxicol Cutan Ocul Toxicol. 1995;14:23–48. 29. Heft MW, Cooper BY, Obrien KK, Hemp E, Obrien R. Aging effects on the perception of noxious and non-noxious thermal stimuli applied to the face. Aging Clin Exp Res. 1996;8:35–41. 30. Adly MA, Assaf H, Hussein MR. Age-associated decrease of the nerve growth factor protein expression in the human skin: preliminary findings. J Dermatol Sci. 2006;42:268–271. 31. Besne´ I, Descombes C, Breton L. Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. 2002;138:1445–1450. 32. Oyeyinka GO. Age and sex differences in immunocompetence. Gerontology. 1984;30:188–195. 33. Prystowsky SD, Allen AM, Smith RW, Nonomura JH, Odom RB, Akers WA. Allergic contact hypersensitivity to nickel, neomycin, ethylenediamine, and benzocaine. Relationships between age, sex, history of exposure, and reactivity to standard patch tests and use tests in a general population. Arch Dermatol. 1979;115:959–962. 34. Goh CL. Prevalence of contact allergy by sex, race and age. Contact Dermatitis. 1986;14:237–240. 35. Young E, van Weelden H, van Osch L. Age and sex distribution of the incidence of contact sensitivity to standard allergens. Contact Dermatitis. 1988;19:307–308. 36. Wantke F, Hemmer W, Jarisch R, Gotz M. Patch test reactions in children, adults and the elderly – a comparative study in patients with suspected allergic contact dermatitis. Contact Dermatitis. 1996;34:316–319. 37. Kwangsukstith C, Maibach HI. Effect of age and sex on the induction and elicitation of allergic contact dermatitis. Contact Dermatitis. 1995;33:289–298. 38. Schwartz M. Eczematous sensitization in various age groups. J Allergy. 1952;24:143–148. 39. Cassimos C, Kanakoudi-Tsakalidis F, Spyroglou K, Ladianos M, Tzaphi R. Skin sensitization to 2, 4 dinitrochlorobenzene (DNCB) in the first months of life. J Clin Lab Immunol. 1980;3:111–113. 40. Robinson MK. Intra-individual variations in acute and cumulative skin irritation responses. Contact Dermatitis. 2001;45:75–83. 41. Robinson MK. Age and gender as influencing factors in skin sensitivity. In: Berardesca E, Fluhr JW, Maibach HI (eds) Sensitive Skin Syndrome. New York: Taylor & Francis, 2006, pp. 169–180.
94 Aging in Asian Skin Low Chai Ling
Introduction
Melanin
Asian skin differs from Caucasian skin in both structure and physiology (> Fig. 94.1). As a result of these distinctions, Asian skins with their darker pigmentation respond differently when exposed to ultraviolet light, lasers and other light devices. This needs to be recognized by cosmetic surgeons and laser practitioners. Asian skins also exhibit alternate clinical manifestations of photoaging. As a result, Asian skins may benefit from skin treatments targeting different aging issues as compared to those affecting Caucasian skins. This chapter will review the biology of Asian skin and discuss a clinical approach to the aesthetic management of Asian skin.
There are no clear differences in the overall number of melanocytes between the races [3]. Differences in skin colour between the races are mainly attributed to variations in melanosomes. Melanosomes are tissue-specific lysosome-related organelles of pigment cells in which melanins are synthesized and stored. The main determinants of skin colour have been attributed to (1) the quantity and type of melanin within the epidermis, and (2) the number, size, aggregation pattern, and distribution of melanosomes [4]. In fact, it has been well documented that AfricanAmerican and Caucasians exhibit different distribution patterns of melanosomes, thereby accounting for the differences in their skin colour. The skin’s response to UV irradiation is dependent on the epidermal content of melanin as well as the distribution of melanosomes. Melanin confers some degree of photoprotection on the skin. This inherent photoprotection influences the rate of the skin aging changes between the different racial groups. As a result of the higher melanin content in Asian skin compared with white skin, Asian skins generally manifests the classic signs of photoaging later in life, typically beyond the fifth decade [5]. Despite a degree of photoprotection conferred by melanin, pigmented Asian skins can still experience significant photodamage if inadequate sun protection is used [6]. In fact, because Asian skin is less susceptible to the immediate deleterious effects from UV light exposure (sunburn), patients may not be aware that their skin is reactive to the long-term, cumulative effects of unprotected exposure. Photodamage is present histologically as epidermal atypia and atrophy, dermal collagen and elastin damage, and marked hyperpigmentation [6]. Apart from pigmentation from photodamage, Asian patients are most susceptible to post-inflammatory hyperpigmentation as a direct consequence of cutaneous inflammation or injury due to the increased melanin content.
Structural Differences between Asian and Caucasian Skin Skin Structure There have been no firm conclusions to suggest clear racial differences in the structure of skin. Some studies do suggest that heavily pigmented skin is more compact, and may have more cell layers compared with lightly pigmented skin. Changes in skin biophysical properties with age demonstrate that the darker skin types retain younger skin properties compared with the fair skin types. There seems to be ethnic variability in the structure of dermal collagen and the abundance of surface lipids. Darker skin types found in certain Asian groups seems to have larger fibroblasts and varying structure of collagen bundles. Stratum corneum lipid content is higher in Asian patients compared with other ethnicities [1]. Asian skins are reported to have an overall weaker skin barrier function. Several studies indicate that Asian skin maybe more sensitive to exogenous chemicals probably due to a thinner stratum corneum and higher eccrine gland density [2]. Clearly, this is an area which needs to be studied in depth as there is more to uncover in the differences in skin structure between Asian skins and the other skin types.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_94, # Springer-Verlag Berlin Heidelberg 2010
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Skin Phototype The Fitzpatrick skin phototype system is used to categorize skin types in people of all skin colour [7]. The classification depends on the amount of melanin pigment in the skin. Melanin, along with water and hemoglobin, is the target chromophore in skin for lasers and light devices. This is an important consideration because laser energy intended for deeper targets can be absorbed by melanin in the basal layer of the epidermis and can cause unintended epidermal damage, risking blistering, permanent dyspigmentation, textural changes, focal atrophy, and scarring in darker skin types. Patients are categorized from very fair skin types (Fitzpatrick skin type I) to very dark skin types (VI) based on two parameters: constitutive skin colour and response to sunlight and UV radiation (> Table 94.1). The Fitzpatrick classification describes the propensity for sun reactivity. In general, all skin types are susceptible to photoaging but of different degrees. Pale or white skin burns easily and tans slowly and poorly: it needs more protection against sun exposure. Higher Fitzpatrick phototypes are less susceptible to sun burns, likely due to the protective role of melanin. However, higher skin
. Figure 94.1 Asian skins exhibit different manifestations of aging from their Caucasians counterparts
phototypes are more prone to develop post-inflammatory pigmentation after cutaneous injury (brown marks). Asians generally fall into categories IV–VI, with Chinese subjects showing the predominant skin type to be type III, followed by type II and then type IV [8]. It is worth noting that in Asian patients; constitutive skin colour does not always correlate with skin response to UV radiation [9]. It is important to assess specifically for Fitzpatrick skin phototype when evaluating a patient before laser or light therapies, because their constitutive skin colour alone may not predict response to such melanintargeted therapies. Patients with higher Fitzpatrick skin types are extremely prone to post-inflammatory hyperpigmentation. When treating such patients with light and laser therapies, practitioners are advised to proceed with caution and a conservative approach may be appropriate. For example, an ablative laser treatment may also be replaced by a fractionated resurfacing laser or a nonablative laser treatment to minimize the risk of postinflammatory hyperpigmentation in such individuals. Such patients may also benefit from pre-treatment and post-treatment topical skin lightening regimens to minimize this undesired result. Common skin lightening regimens include hydroquinone 2–4%, kojic acid 1–4%, azelaic acid 20%, arbutin 1%. Hydroquinone is banned in the European Union, Australia and Japan due to its carcinoma inducing concerns. With topical hydroquinone, there is still a small risk of contact dermatitis though this usually responds promptly to topical steroids. An uncommon, yet important, adverse effect of Hydroquinone is exogenous ochronosis.
. Table 94.1 Fitzpatrick skin phototype Skin Phototype
Skin Colour
Features
I
Ivory white
Always burns, never tans
II
Fair white
Usually burns, tans poorly
III
White
Sometimes burns, average tan
IV
Beige to olive, lightly tanned
Rarely burns, tans easily
V
Moderate brown or tanned
Very rarely burns, profuse tan
VI
Dark brown to black
Never burns, always tans darkly
Aging in Asian Skin
Ochronosis is characterized by progressive bluish black darkening of the skin area exposed to hydroquinone. Histologically, degeneration of collagen and elastic fibers occurs, followed by the appearance of characteristic ochronotic deposits consisting of crescent-shaped, ochre-coloured fibers in the dermis. Carbon dioxide lasers and dermabrasion have been reported to be helpful in the treatment of exogenous ochronosis. Reports have also described effective therapy with the Q-switched alexandrite 755-nm laser.
Aging Processes Anatomy of Aging Skin Aging skin exhibits progressive changes such as thinning, skin laxity, fragility, and wrinkles. Sun-exposed areas demonstrate additional skin changes, including dyschromia, premature wrinkling, actinic elastosis and telangiectasias [10]. When a cell stops replicating, it enters into a period of decline known as ‘‘cell senescence’’. Aging at the cellular level is thought to be related to cellular senescence. This refers specifically to the shortening of telomeres (the terminal portions of chromosomes) with each cell cycle. Telomere shortening ultimately results in apoptosis once a critical length is reached. Histopathologically, photoaging is characterized by disorganized collagen fibrils, decreased normal collagen and increased abnormal elastic fibers termed solar elastosis at the upper dermis, as well as flattening of the dermal–epidermal junction with elongated and collapsed fibroblasts [11].
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and hormones. Intrinsic aging is characterized by dryness, laxity, and skin atrophy, affecting both sun-exposed and non–sun-exposed skin. In Asian patients, intrinsic aging predominantly results in a decrease in skin elasticity.
Extrinsic Aging Extrinsic aging is environmentally induced. It has been established that chronic inflammation at the cellular level is the cause of extrinsic aging [12]. The two most important factors in Asian patients that contribute to the extrinsic aging process are a history of smoking and chronic UV light exposure. As in white skinned patients, the effects of cigarette smoking and excessive chronic sun exposure seem multiplicative [13]. In Asian patients, extrinsic aging manifests as pigmentary changes, rhytides, laxity, and coarseness of skin texture with pigmentary changes predominating as the initial signs of the extrinsic aging process. Ultraviolet irradiation leads to dermal damage with histologic evidence of disorganized collagen fibrils and abnormal solar elastotic material. Elevated matrix metalloproteinases and collagen degeneration leads to dermal breakdown [14]. This manifests clinically as progressive skin laxity and the formation of rhytids.
Aging Skin
Cutaneous aging is an interplay of intrinsic and extrinsic aging processes. Intrinsic aging occurs naturally and can be exacerbated by extrinsic aging. Most of the efforts in facial rejuvenation are targeted toward counteracting the effects of extrinsic aging.
There are numerous factors that contribute to aging skin. Even among patients of Asian heritage, these factors appear in varying degrees in each individual resulting in great discrepancies in the aging process even among individuals of the same ethnicity. The clinical signs of aging include dyschromia, loss of smooth surface skin texture, loss of translucency, skin volume loss, and functional loss [15]. By accurately identifying the relative contributing factors to an aged appearance, individual-specific skin treatment can be selected, and the risk-benefit ratios of the possible therapies weighed. This is an essential part of a comprehensive cosmetic consultation and ultimately, a successful and safe skin rejuvenation treatment process.
Intrinsic Aging
Photoaging
Intrinsic or chronologic aging is a genetically determined process of aging which occurs in skin; this type of aging also occurs in photo-protected skin. Additional contributing factors include the effects of gravity, expression
Ethnic and genetic differences in skin structure and function mean that the clinical manifestations of photoaging in Asian skin differ from those of white skin. Asian skins have different natural defense mechanisms against chronic
Intrinsic Versus Extrinsic Skin Aging
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Aging in Asian Skin
UV exposure. In addition, different cultural habits related to UV exposure should also be taken into consideration. While photoaging in white individuals manifests as premature and progressive development of fine lines and rhytides, the primary manifestation of photoaging in Asian skin is pigmentary changes, with rhytides being less conspicuous and usually not noticeable until after the fifth decade [16, 17].
Photoaging Scale A photoaging scale was devised by Glogau (> Table 94.2) to aid in assessing severity of skin photodamage [18]. This can be helpful in discussing potential results of facial cosmetic procedures with patients. Although initially used to describe white skin, the Glogau photoaging scale, with slight alteration, is also very useful in analysis of Asian skin and has the advantage of practical clinical application. In type I or early photoaging, no wrinkles are present. There is little or no visible pigmentary changes present. Female patients in this category usually require no makeup. Asian patients may not be aware that their skin is reactive to the long-term, cumulative effects of unprotected exposure because Asian skin is less susceptible to the immediate deleterious effects from UV light exposure (sunburn). Daily sun protection should nonetheless be advocated. In type II photoaging, pigmentary changes become more prevalent, with development of mottled skin tone and solar lentigo. Superficial brown pigmentation and freckling can be accentuated with Wood’s lamp illumination. These mild and flat pigmentary changes respond readily to topical agents, such as retinoids, ascorbic acid (vitamin c), hydroquinone, and alpha-hydroxy acids [19]. . Table 94.2 Photoaging scale (Glocau RG [18]) Skin type
Age in years
I (mild)
20–30
Clinical findings Little or no photoaging, little or no wrinkling, no keratoses
II (moderate) 30–50
Early to moderate photoaging, wrinkles present with motion
III (advanced) 50–60
Advanced photoaging, wrinkles with rest, visible keratoses, noticeable discolorations
IV (severe)
60 and Severe photoaging, deep wrinkles – over both dynamic and gravitational wrinkling, actinic keratoses
Superficial chemical peels, microdermabrasion, and intense pulsed light therapies also yield results at this early stage [20]. Lasers such as the Q-switched ND:Yag laser can also be considered for more resistant pigmented lesions. Although no obvious wrinkles appear when facial musculature is at rest, dynamic wrinkles appear when the face is in motion. These rhytides often first appear on the upper third of the face along the lateral orbits as ‘‘crow’s feet’’, the glabella as ‘‘frown lines’’, and forehead as ‘‘horizontal lines’’ [21]. Chemical muscle denervation with botulinum toxin can also be considered to relax dynamic wrinkles and to improve the appearance of the face in motion. In patients exhibiting type III photoaging, pigmentary changes are advanced. Deeper pigmentary disorders, such as melasma and Hori’s nevus, may manifest. These lesions are more difficult to treat. Raised brown lesions, such as seborrheic keratoses, further disrupt the surface texture of the skin. Laser treatment of pigmentary disorders of this type is challenging. Patients in this stage also display obvious static wrinkles. Skin no longer has a smooth texture and exhibits reduced reflectance. In addition to chemical denervation, soft tissue augmentation is indicated to replace volume loss and to plump up static wrinkles such as the nasolabial lines. Fractional ablative lasers in addition to the above light therapies listed for type II photoaging can be considered to improve skin texture and skin dyschromia. However because of increased risk for post-inflammatory hyperpigmentation, these techniques must be used with caution and with adequate counseling and preparation of patient expectations. Finally, in patients with type IV photoaging, wrinkles predominate while numerous severe pigmentary lesions are also present. Hardly any normal skin remains. Topical therapy does not effect a significant benefit and noninvasive aesthetic treatments may only give limited improvements. Surgical correction in addition to skin rejuvenation measures may be considered.
Pigmentary Changes In Asian patients, photodamage predominantly presents as pigmentary changes rather than wrinkling. This difference is partially due to the higher epidermal melanin content, which can predispose these patients to a higher risk for hyperpigmentation from light source treatment. Long-term UV light damage to the skin is the most common trigger of dyschromia. Visible colour changes include early appearance of uneven skin tone, followed by various pigmented growths. The most common pigmentary disorders seen in chronically UV-exposed skin include
Aging in Asian Skin
ephelides (freckling), solar lentigo, melasma, and seborrheic keratosis [16]. The development of seborrheic keratosis seems to be more prevalent in Asian men, whereas hyperpigmented maculae are more prominent in women [13]. Each of these pigmentary disorders is caused by pathology in varying levels of the epidermis and dermis. To differentiate epidermal from dermal lesions, one can use a Wood’s lamp in the examination of a patient with dyspigmentation. Superficial (epidermal) melanin is accentuated with Wood’s lamp illumination, whereas deeper (dermal) melanin does not appear enhanced. Accurate diagnosis of the etiology of pigment alteration allows for targeted, more effective treatment. For epidermal pigmented lesions, IPL can be effective [20]. Q-switched laser and fractional resurfacing laser are options used to remove dermal pigment.
Textural Changes Textural changes begin with a loss of smoothness and reflectance, gradually progressing to the development of raised growths, such as seborrheic keratoses. Yellow thickened bumps (elastosis or heliosis) appear and are due to tangled masses of damaged elastin protein in the dermis. There is also increased dermal collagen due to scarring from repeated inflammation. This thick dermis loses elasticity and is weaker than normal, putting aging skin at higher risk for injury. Rubbing or pulling on the skin can cause skin tears. Skin appears sallow and dull. Visible coarseness of the skin may also develop because of sebaceous hyperplasia, enlarged pore size, and thickening of unwanted facial hair [22]. As the skin and underlying supporting structures lose elasticity over time, it becomes less resistant to the cumulative effects of gravity and underlying musculature. With early aging, wrinkles appear only when the face is in motion, usually at the expression lines around the eyes and mouth. Over time, as elasticity of skin further diminished, wrinkles are visible even when the face is at rest.
Volume Loss Facial aging is associated with loss of soft tissue fullness in certain areas, and descent or hypertrophy in others [23]. An overall loss of volume occurs in the periorbital, forehead, malar, temples, mandibular, mental, glabellar, and perioral sites, while hypertrophy of fat occurs in the submental, lateral nasolabial folds, and labiomental crease,
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jowls, infraorbital pouches, and malar fat pad. Gradual relaxation of the ligamentous support leads to descent and separation of the fat pads. Remodeling of the craniofacial skeleton leads to a reduction in facial height [23]. This decreases the space available to support the overlying soft tissues causing the face to collapse in on itself. The orbits increase in diameter as the orbital shelf descends, resulting in the malposition of the lower eyelid which presents as lateral bowing and scleral show. Maxillary resorption causes loss of support in the upper lip, and this contributes to perioral wrinkling, thinning and inversion of the lips. Assessment of the aging face should include an analysis of the quality and position of the underlying fat.
Functional Loss The main functional losses experienced by aging skin are a decrease in cell replacement, barrier function, chemical clearance, mechanical protection, immune responsiveness, wound healing, thermoregulation and vitamin D production. With aging, the barrier function is compromised. There are some differences in barrier function between Caucasians and Asian skins. Barrier function relates to the total architecture of the stratum corneum as well as its lipid levels. Asian skin is reported to possess a similar basal transepidermal water loss (TEWL) to Caucasian skin and similar ceramide levels but reduced stratum corneum natural moisturizing factors [2]. Upon mechanical challenge it has weaker barrier function compared to Caucasian skins. Both skin types also exhibit differences in intercellular cohesion. The frequency of skin sensitivity is quite similar across different racial groups but the stimuli for its induction shows subtle differences. Nevertheless, several studies indicate that Asian skin may be more sensitive to exogenous chemicals probably due to a thinner stratum corneum and higher eccrine gland density [2]. Due to decreases especially in melanocytes and the dermis, there is a disruption in the mechanical protection served by the skin. This results in increased UV penetration and damage as well as decreased nutrient transfer. With aging, wound healing is impaired with increased breakdown in tissue. This explains why the elderly has a predisposition to chronic wounds. Immune responsiveness is impaired in aging skin as Langerhans cells exhibit a reduction in number as well as an impairment of their antigen presenting capacity with age. Acute inflammatory reactions are less noticeable.
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Aging in Asian Skin
Aging skin shows a decrease in sweat glands, vasculature, nerves, sweat glands, and sebaceous secretions. These changes results in an overall reduction in thermoregulation and a lower threshold for pain perception and reaction. Vitamin D production decreases with age. This may in turn decrease calcium levels and predispose the elderly to osteomalacia. The melanin in darker skin types acts as a sun screen and may slow down production of Vitamin D3. However, race had only a marginal effect on the production of active vitamin D metabolites. While racial pigmentation has a photoprotective effect, it does not prevent the generation of normal levels of active vitamin D metabolites [24].
Conclusion Recognizing the unique characteristics of Asian skin allows physicians to optimize cosmetic results and ultimately to a successful facial rejuvenation for an aging Asian skin. Differences in the structure and physiology of Asian skin, particularly in melanin content, accounts for variations in response to UV light exposure and alternate clinical manifestations of photoaging. Cosmetic surgeons and laser practitioners should be aware of the distinct presentations of photodamage in Asians and understand the increased risk of post-inflammatory hyperpigmentation associated with various therapeutic modalities.
Cross-references > Determinants
in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
References 1. Richards GM, Oresajo CO, Halder RM, Structure and function of ethnic skin and hair. Dermatol Clin. 2003;21(4):595–600. 2. Rawlings AV, Ethnic skin types: are there differences in skin structure and function? Int J Cosmet Sci. 2006 Apr;28(2):79–93. 3. Staricco RJ, Pinkus H, Quantitative and qualitative data on the pigment cells of adult human epidermis. J Invest Dermatol. 1957;28(1):33–45. 4. Goldschmidt H, Raymond JZ, Quantitative analysis of skin color from melanin content of superficial skin cells. J Forensic Sci. 1972;17(1):124–131.
5. Siegrid SY, Roy C. Grekin, MD, Aesthetic Analysis of Asian Skin. Facial plast surg clin North Am. 2007 Aug;15(3):361–365. 6. Kotrajaras R, Kligman AM, The effect of topical tretinoin on photodamaged facial skin: the Thai experience. Br J Dermatol. 1993; 129(3):302–309. 7. Fitzpatrick TB, The validity and practicality of sun reactive skin type I through VI. Arch Dermatol. 1988;124:869–871. 8. Liu W, Lai W, Wang XM, et al. Skin phototyping in a Chinese female population: analysis of four hundred and four cases from four major cities of China. Photodermatol Photoimmunol Photomed. 2006; 22(4):184–188. 9. Choe YB, Jang SJ, Jo SJ, et al. The difference between the constitutive and facultative skin color does not reflect skin phototype in Asian skin. Skin Res Technol. 2006;12(1):68–72. 10. Gilchrest BA, Yaar M. Ageing and photoageing of the skin: observations at the cellular and molecular level. Br J Dermatol. 1992;127 (Suppl 41):25–30. 11. Bernstein EF, Chen YQ, Kopp JB, et al. Long-term sun exposure alters the collagen of the papillary dermis: comparison of sunprotected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy. J Am Acad Dermatol. 1996;34(2 Pt 1):209–218. 12. Thornfeldt CR, Chronic Inflammation is the Etiology of Extrinsic Aging. J Cosmet Dermatol. 2008; 7: 78–82. 13. Chung JH, Lee SH, Youn CS, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol. 2001;137(8):1043–1051. 14. Fisher GJ, Wang ZQ, Datta SC, et al. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997;337: 1419–1428. 15. Calderone DC, Fenske NA. The clinical spectrum of actinic elastosis. J Am Acad Dermatol. 1995;32(6):1016–1024. 16. Chung JH, Photoaging in Asians. Photodermatol Photoimmunol Photomed. 2003;19(3):109–121. 17. Nouveau-Richard S, Yang Z, Mac-Mary S, et al. Skin ageing: a comparison between Chinese and European populations. A pilot study. J Dermatol Sci. 2005;40(3):187–193. 18. Glocau RG. Aesthetic and anatomic analysis of the aging skin. Semin Cutan Med Surg. 1996;15(3):134–138. 19. Griffiths CE, Goldfarb MT, Finkel LJ, et al. Topical tretinoin (retinoic acid) treatment of hyperpigmented lesions associated with photoaging in Chinese and Japanese patients: a vehicle-controlled trial. J Am Acad Dermatol. 1994;30(1):76–84. 20. Feng Y, Zhao J, Gold MH, Skin rejuvenation in Asian skin: the analysis of clinical effects and basic mechanisms of intense pulsed light. J Drugs Dermatol. 2008 Mar;7(3):273–9. 21. Glogau RG, Physiologic and structural changes associated with aging skin. Dermatol Clin. 1997;15(4):555–559. 22. Bolognia JL, Aging skin. Am J Med. 1995;98(1A):99S–103S. 23. Sydney R Coleman, Rajiv Grover. The anatomy of the aging face: volume loss and changes in 3-dimensional topography. Aesthetic Surg J. 2006 Jan;26(1):S4–S9. 24. Matsuoka LY, Wortsman J, Haddad JG, Kolm P, Hollis BW, Racial pigmentation and the cutaneous synthesis of vitamin D. Arch Dermatol. 1991 Apr;127(4):536–8.
90 Aging Skin: Some Psychosomatic Aspects Madhulika A. Gupta
Introduction The skin, especially the facial skin, is a powerful organ of communication and one of the most easily visible indicators of age, health, and disease, and of various socially important attributes such as social status, wealth, and sexual attractiveness [1]. The face is the part of the body invested with the greatest interpersonal meaning and is the focus of attention during communication. The aging of the facial skin secondary to both intrinsic and extrinsic factors (e.g., photodamage and smoking) and the development of hyperfunctional facial lines due to repeated expression of emotion over time can lead to aging of the appearance. Over the last several decades, the cultural and social meanings of growing old have changed and old age has started to acquire increasingly negative connotations. Often normal intrinsic aging is viewed as a medical and social problem that needs to be addressed by health-care professionals. The idea that chronological age itself does not signal the beginning of old age, and that one can get older without the signs of aging, has become increasingly prevalent, with a high value placed by the society on the maintenance of a youthful appearance [1]. Facial appearance and expressions, for example, as a result of the corrugator muscle activity of the forehead (resulting in a frown), play a substantial role in the expression of emotions in addition to signaling attributes such as age [2]. From a Darwinian evolutionary perspective [3], the interpretation of facial expression is an integral component of interpersonal communication and tends to be universal and constant across time and cultures [2]. The face is the focus of human communication and facial expressions have evolved as a means of nonverbal communication and as a way of enhancing verbal communication [2]. The repeated expression of emotion over time produces hyperfunctional facial lines. The presence of these lines when the face is at repose may give the person an aged appearance or give an erroneous impression of emotions or personality characteristics. As the skin ages and the support of the underlying cutaneous structures is lost, more wrinkles and folds develop, and
gradually the dynamic lines that communicate emotion change to static lines ingrained on the face at rest. The orientation and depth of these folds is greatly influenced by the underlying activity of the facial muscles. These hyperfunctional lines are common in the forehead, between the brows, around the eyes, and in the area of the mouth. For example, hyperfunctional forehead lines may give an impression of aging, and frown lines or deep vertical creases in the glabellar region give the impression of anger or dissatisfaction. It has been observed that these hyperfunctional lines can result in a ‘‘malfunction of the facial organ of communication’’ [2, 4]. With aging, the corner of the mouth will often droop creating an appearance that may be misinterpreted as displeasure or sadness, or the drooping of the brow or sagging of the upper eyelid may result in the appearance of drowsiness and exhaustion [2]. Therefore, the internal emotion may be quite different from the message received by others, and the disparity between the internal mood and the external appearance can be a significant source of anxiety and may culminate in a feeling of disconnect between the inner self and the face that the individual sees in the mirror. This incongruity may be confirmed in social interactions, resulting in a sense of alienation. These miscues may further affect reciprocal behavior; for example, a frown is more likely to elicit a frown rather than a smile from another person, and negative responses usually reinforce negative behavior, resulting in greater social alienation. The increasing presence of hyperfunctional facial lines with age therefore has implications far beyond considerations of attractiveness, as they affect the perception of emotions and perceived personality traits of the individual, and treatments to smooth the hyperfunctional facial lines may be warranted because of their positive social ramifications [2]. Data from the American Society of Plastic Surgeons [1] report 10.9 million cosmetic procedures in 2006; just under two million of these treatments were traditional cosmetic surgical procedures such as liposuction, rhinoplasty, and breast augmentation, and the vast majority representing over 9.1 million procedures involved
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_90, # Springer-Verlag Berlin Heidelberg 2010
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minimally invasive procedures such as botulinum toxin A injections and chemical peels, which are largely used to rejuvenate the appearance. It is further reported [5] that the use of these procedures has increased eightfold since 1992, and these numbers are likely a gross underestimate of the total number of procedures to rejuvenate the appearance, since a large number of the minimally invasive procedures are being performed by non-plastic surgeons. Another aging-related phenomenon is that a greater number of individuals are becoming concerned about aging-related changes at a much younger age. A study of nonclinical subjects reports that over 50% of women under the age of 30 reported dissatisfaction with the appearance of their skin, and some of the attributes they were dissatisfied with such as wrinkles and ‘‘bags’’ and ‘‘darkness’’ under the eyes are the signs of aging of the skin [6]. This emerging trend, where younger individuals may be seeking treatments for cutaneous rejuvenation, further emphasizes the importance of the psychosocial dimension in the overall management of these patients. This chapter reviews some of the psychosomatic aspects of aging skin that may be of importance in the clinical management of patients seeking treatment for rejuvenation of their appearance using surgical, minimally invasive procedures and topical therapies.
Review of Literature There is an extensive literature [7] on the psychosocial factors in cosmetic surgery; however, few studies have examined the psychosocial factors in the treatment of aging skin or aging of the face.
Surgical Treatments An earlier (1964) comprehensive study [8, 9] of 106 consecutive patients seen at the Johns Hopkins Hospital over a period of 12 years for the surgical correction of facial evidences of aging evaluated the preoperative and postoperative psychiatric state of 46 of the 64 patients (mean age 48.5 years, seven males) who qualified for the surgery. Forty-two of the 106 patients that were excluded had a higher incidence of previous psychiatric treatment and suffered from a much higher incidence of family disruption during childhood. Among the patients that received surgery ‘‘two patterns of interpersonal relationships’’ were observed; 43% were ‘‘emotionally distant or mistrustful’’ and were ‘‘diagnosed psychiatrically as manifesting hysterical tendencies,’’ and ‘‘two thirds of them
described an unhappy marriage’’; the remaining 57% were described as having ‘‘passive dependent’’ personalities. Edgerton et al. [9] further observe that ‘‘over 74% (of the 46 patients who were psychiatrically evaluated) were diagnosed as having some associated but not primary psychiatric disorder’’ and ‘‘only four patients had been previously hospitalized for mental illness’’ and ‘‘only one of these had been found psychotic.’’ The psychiatric diagnoses among the 46 patients who were evaluated psychiatrically were as follows: ‘‘Neurotic depressive reaction (often after husband’s death)’’ in 15 of 46 or 32.6%, ‘‘Personality trait disturbance’’ in 12 out of 46 or 26.1%, ‘‘Schizoid personality’’ in six patients, three of whom were men, and ‘‘Anxiety reaction’’ in one patient; the remaining 12 patients had ‘‘No psychiatric disorder.’’ The motivations for patients coming for face-lifts differed by age groups: the ‘‘emotionally dependent group’’ (age 29–39 years) represented 22% of patients who ‘‘tended to be insecure and dependent on their spouses,’’ reported significantly more family disruption during childhood than the older patients, and demonstrated ‘‘problems of adjustment to adult responsibilities’’; the ‘‘worker group’’ (age 40–49 years) constituted 37% of patients whose ‘‘major motivation for surgery was to meet vocational requirements for a youthful attractive appearance’’; and the ‘‘grief group’’ (age 50 years and older) who comprised 40% of the sample, and ‘‘two thirds of these were suffering grief over the death of a spouse or separation from children’’ and sought surgery to give them ‘‘self-confidence,’’ ‘‘self-esteem,’’ and ‘‘a new chance to make friends’’ but ‘‘underlying depression was very common in this group.’’ Examination of the gender differences revealed that in contrast to the female patients all the seven male patients had a history of emotional illness and all received a psychiatric diagnosis. As for their reason for seeking surgery, none of the men cited the loss of a loved one as principal motivation. All male patients were reported as facing ‘‘a critical life decision at the time of their first visit’’ and wished to look ‘‘less stern,’’ ‘‘not so old and tired,’’ and ‘‘to adjust to American living’’ (in the case of an immigrant). The authors caution that ‘‘plastic surgeons should seek to uncover the nature of the decision and determine whether rhytidectomy will realistically aid the outcome.’’ The early postoperative course [8, 9] was ‘‘generally mild and without serious emotional disturbance’’: nine patients showed ‘‘mild depression or transient tears’’ usually on the third or fourth postoperative day, and ‘‘some reexperienced the grief previously suffered at the loss of a loved one’’; paresthesia and numbness of the facial skin after operation was common and ‘‘sometimes augmented the patient’s feeling of unreality.’’ If blepharoplasty was
Aging Skin: Some Psychosomatic Aspects
performed, the blindfolding resulting from pressure bandages over the eyes during the first 48 h postoperatively was associated with heightened anxiety in some patients. This was in contrast with other procedures such as rhinoplasty and augmentation mammaplasty where up to 40% of patients have been reported to experience significant short-term emotional disturbances. Patients were followed up psychiatrically between 6 months and 12 years postoperatively and ‘‘over 85% of patients reported significant improvement’’ in each of the following areas: ‘‘personal comfort,’’ ‘‘less self-critical,’’ ‘‘better satisfied with their lives,’’ ‘‘less self-conscious,’’ ‘‘more social ease,’’ ‘‘more self-esteem,’’ and ‘‘happier.’’ Furthermore, 55% had obtained one or more of the following: ‘‘a new job,’’ ‘‘marriage,’’ ‘‘a promotion or raise,’’ ‘‘a merit award,’’ ‘‘formation of other new, close relationships,’’ or ‘‘termination of an old, detrimental relationship without emotional upset.’’ No patients reported ‘‘guilt feelings’’ or ‘‘having any feelings of deception about her age.’’ The authors conclude that ‘‘satisfactory psychologic result of face-lifting depends on several variables’’ such as ‘‘how the patient approaches surgery, with confidence or mistrustful attitudes,’’ ‘‘a genuine and personal interest on part of the surgeon and whether the surgery constitutes a therapeutic or ‘rebirth’ experience,’’ ‘‘the potential for realistic improvement in the patient’s personal environment as a result of the surgical experience,’’ ‘‘how much positive feedback the patient receives from their friends and associates regarding an improvement in their appearance,’’ and finally ‘‘the actual anatomic improvement that the facial skin and subcutaneous tissue permit’’; contraindications to surgery where ‘‘basically good procedures may produce poor results’’ included ‘‘unresolved emotional conflicts’’; and ‘‘the effectiveness of a psychiatrist in helping the plastic surgery patient is directly proportional to his interest and experience with the problems of deformity.’’ Goin et al. [10] evaluated 50 female face-lift patients preoperatively and postoperatively for up to 6 months with semistructured psychiatric interviews and psychological tests. The 50 patients were chosen from 117 consecutive face-lift consultations; 20% of patients were rejected by the surgeon for psychological reasons, which were as follows: patients were ‘‘unable or unwilling to listen, were excessively fearful, idealized the surgeon (believing he could accomplish what others had failed to do), or had a history of severe psychological disturbance following other operations.’’ Preoperatively, psychological testing, e.g., with the Minnesota Multiphasic Personality Inventory (MMPI) revealed ‘‘a relatively normal group’’ and ‘‘there were no clear diagnostic groupings.’’ Only one patient showed neurotic pathology with high scores on
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several MMPI scales and none were psychotic. Four patients were ‘‘in the midst of grieving over dead loved ones’’ at the time of surgery. Clinical evaluation revealed ‘‘some evidence of clinical depression,’’ rated as mild to moderate, in seven or 14% of patients. Postoperatively, 27 or 54% of patients ‘‘displayed clinical evidence of psychological disturbance’’ and 30% of these patients experienced depression 6 months postoperatively. The patients with depression reactions were divided into four categories [10]: six patients or 12% described ‘‘feelings of depression or anxiety occurring sometime within the first 5 days’’ and these symptoms were gone by the end of the first week; and, another 12% had ‘‘transient episodes of depression occurring around the second or third week, which lasted 3–5 days’’ and ‘‘the depression was related to some new stress in the patient’s life’’ such as divorce and illness in the family. Thirty percent of patients reported more prolonged depression, which was present upto 6 months postoperatively; among these 16% ‘‘were depressed within the first 5 days and continued to be depressed for several weeks’’ and 14% ‘‘developed a clinical depression in the second or third postoperative week, which lasted for several weeks.’’ This group with prolonged clinical depression was reported preoperatively, to have ‘‘either a preexisting and clinically detectable depression or a high depression score on the MMPI’’ and the authors conclude that the surgery either ‘‘intensified’’ or ‘‘unmasked’’ their depression. No other preoperative factors were associated with postoperative depression. The subgroup that became depressed within the first 5 days were more ‘‘independent, self-reliant, and wanted to control their lives,’’ ‘‘did not anticipate any changes in their self-esteem’’ and ‘‘had hoped that the face-lift would slow down the aging process.’’ In contrast, the subgroup with later onset depression comprised ‘‘passive-dependent women who wished to be cared for and did not want to be in charge of their lives.’’ This group also had less favorable surgical results. Therefore, overall decrease in the support from the immediate postoperative period and disappointment with results of surgery contributed to the depressive reaction in this last group of women. Postoperatively, improvement was noted in other areas including ‘‘increased self-esteem’’ (28%), ‘‘better able to cope with life’’ (8%), ‘‘more assertive and comfortable at work’’ (8%), and ‘‘diminished grief reactions’’ (8%). Some of the factors associated with a postoperative improvement in psychological state were as follows: preoperatively, the desire for an improved self-image; a higher-than-average score on the Paranoid subscale of the MMPI pre-operatively and greater reinvolvement of these patients with friends and colleagues after surgery, which reduced the intensity of their previous distorted perceptions about people; and the
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patient’s desire preoperatively to improve the chances of retaining a job, advancing her career, or if she had previous cosmetic operations. A French study [11, 12] measured psychosocial factors in 103 facial cosmetic surgery patients using standardized rating scales and semistructured interviews, both presurgery and 9 months after facial cosmetic surgery. Presurgery, 50% of patients reported that they had used a psychotropic therapy of which 27% were antidepressants, 20% were seeking employment, and 59% were ‘‘motivated by a search for well-being.’’ The patients had high depression scores presurgery and this did not change significantly after the surgery. Presurgery patients had high scores on measures of social anxiety, especially fear of speaking in public rather than a fear of social interaction and this decreased significantly postsurgery. Of several psychological motives studied, a lack of self-confidence associated with a desire to create and enhance interpersonal relationships predicted the greatest improvement on postsurgical scores. In a Brazilian study [13] 32 female Caucasian patients, aged 46–68 years, undergoing rhytidoplasty, were examined preoperatively and 2 and 6 months postoperatively. Measures of health perception, energy, and social function were significantly improved at 6 months postoperatively, while measures of mental health which were related to anxiety and depression, and self-esteem showed improvement both at 2 and 6 months postoperatively. The authors discuss the improvement in the patients’ overall sense of well-being and not just their psychological health, after the surgery for facial rejuvenation. A recent Canadian study [14] examined 93 patients (82 females and 11 males) who had undergone rhinoplasty (49%) and surgery for the aging face (51%). All patients were administered the 59-item Derriford Appearance Scale (DAS59), a validated instrument that measures body image-related distress and dysfunction, preoperatively and 3 months after surgery. Patients were routinely screened for psychiatric disorders and patients with body dysmorphic disorder (BDD) or related psychiatric disorders were excluded from participation. Facial aging patients and patients in the highest age category (51 years) had the lowest baseline DAS59 scores indicating the least amount of appearance-related emotional concern. Postoperatively, there was a significant reduction in all dimensions of the DAS59, with the greatest mean reduction in the factor measuring ‘General Self-consciousness of Appearance’, whereas the least improvement was noted for ‘Self-consciousness of Sexual and Bodily Appearance’. Men had higher preoperative levels of distress in contrast to women, especially the males undergoing rhinoplasty;
men also exhibited a greater overall percentage decrease in scores postsurgery. The greatest mean percentage improvement in presurgery or postsurgery DAS59 scores was noted in the >50 years age group. Therefore, while the 51 years age group showed a decline in appearancerelated concerns presurgery according to their DAS59 scores, the greatest relative benefits postsurgery were derived for the oldest subgroup of patients.
Nonsurgical or Minimally Invasive Treatments A study of 20 patients with mild to moderately photodamaged skin [15] who had entered a study to evaluate the efficacy of topical tretinoin for the treatment of photodamaged skin reported that at baseline the subjects had high scores on the Interpersonal Sensitivity and Phobic Anxiety subscales of the Brief Symptom Inventory (BSI). The Interpersonal Sensitivity (BSI) subscale measures a lack of ease during interpersonal interactions, and the Phobic Anxiety (BSI) subscale provides an index of a persistent fear response to certain situations including social situations that lead to avoidance of the situations that provoke anxiety. High scores on these BSI subscales therefore suggest that the subjects with photodamage, who were concerned enough about the photodamage-related skin changes to seek treatment, were experiencing uneasiness during their interpersonal interactions [15]. After 24 weeks of therapy, both the Interpersonal Sensitivity (BSI) and Phobic Anxiety (BSI) scores decreased significantly (p < 0.05) in the topical tretinoin, but not in the control group that was receiving the inactive vehicle [15]. These findings were confirmed in another study [16] involving 40 additional subjects with moderate to severe photodamage. In this study [16] a significant decrease in Phobic Anxiety (BSI) (p < 0.05) was observed after 24 weeks of therapy with topical tretinoin, while an increase in Phobic Anxiety (BSI) (p < 0.05) was noted in the group receiving the inactive vehicle. General body-image concerns related to body weight and shape were measured with the Eating Disorder Inventory (EDI) pretreatment and posttreatment with topical tretinoin. The patients receiving the active treatment with topical tretinoin and not the control group reported a significant decline (p < 0.01) in Drive for Thinness (EDI) and Body Dissatisfaction (EDI), which measure an excessive concern about thinness, body shape, and body weight. These findings indicate that aging-related changes affecting the skin caused increased social anxiety and concerns about general aspects of body image related to body weight and
Aging Skin: Some Psychosomatic Aspects
shape, and this anxiety and general dissatisfaction with body image decreased with the treatment of some of the cutaneous changes of photodamage [16]. Treatment of glabellar frown lines with Botulinum Toxin A has been associated with a favorable psychosocial outcome. In one study [17] 20 women between 35 and 60 years of age, assessed as having moderate to severe glabellar rhytids, received Botulinum Toxin A treatment to the forehead and crow’s feet area and had standardized frontal and lateral view photographs taken, which were rated for ‘‘first impressions’’ on the following domains: social skills, academic performance, dating success, occupational success, attractiveness, financial success, relationship success, and athletic success. Botulinum Toxin A improved first impressions scores for dating success, attractiveness, and athletic success ratings; the first impressions on academic performance and occupational success demonstrated a significantly lower (i.e., lower degree of agreement with the descriptive statement associated with the domains) rating after treatment with Botulinum Toxin A and this effect was no longer observed when a ‘‘smile/relax’’ variable was added to the model. Another preliminary study [18] used Botulinum Toxin A to treat glabellar frown lines in ten female patients, ranging in age between 36 and 63 years, diagnosed with DSMIV [19] criteria for major depressive disorder (MDD) despite treatment with psychotropic drugs and psychotherapy. The time period for which the patients had been depressed ranged from 2 to 17 years and seven out of ten patients had been tried on two or more antidepressant medications. The patients were evaluated 2 months later, and nine out of ten patients were no longer depressed both by clinical criteria and scores on standardized rating scales, and the remaining patient who had an improvement in her mood had bipolar disorder. The authors [18] discuss the Darwinian notion that ‘‘the free expression, by outward signs, of an emotion intensifies it. On the other hand, repression, as far as this is possible, of all outward signs softens our emotions.’’ Increased frown muscle activity has been associated with depression and patients that have their frown lines treated with Botulinum Toxin A appear to be happier and enhancement of the facial expression of happiness may also make the treated individuals feel happier [18].
Psychosomatic Assessment of the Patient The request for cosmetic procedures is typically emotionally or psychosocially motivated. Cosmetic procedures
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are supposed to be life enhancing, not life saving. The primary responsibility of the clinician who is performing an aesthetic procedure is to ensure that (1) he or she can accomplish what the patient desires; and (2) the patient is satisfied with the outcome of the procedure. An acceptable indication for an aesthetic procedure is that the procedure will improve the patient’s quality of life. The most common psychiatric comorbidities that can be associated with an unfavorable treatment outcome (> Table 90.1) are discussed below in detail; the presence of body dysmorphic disorder is generally considered to be a contraindication for cosmetic procedures. Edgerton et al. [20] have reported the course of 87 severely psychologically disturbed patients ‘‘ranging from moderate degrees of neurosis to frank psychosis’’ who underwent aesthetic plastic surgery and were followed up for an average of 6.2 years. As many as 82.8% of patients had a ‘‘positive psychological outcome,’’ 13.8% experienced ‘‘minimal improvement’’ from surgery, and three patients or 3.4% were ‘‘negatively affected’’; among the ‘‘negatively affected’’ the first patient who had rhinoplasty said that ‘‘she had expected to erase the emotional scars from an early childhood trauma,’’ the second patient with blepharoplasty had a poor surgical outcome, and the third patient who had rhinoplasty was identified as having untreated bodyimage issues. The authors [20] report that there were no suicides, psychotic decompensations, or lawsuits, and further observe that patients with severe psychological disturbances benefited from a ‘‘combined surgical–psychiatric treatment designed to address the patient’s profound sense of deformity.’’ Some general demographic considerations include the fact that male patients seeking cosmetic procedures tend to have more severe psychopathology than their female counterparts; and individuals in their late 40s tend to have the maximum concern about aging of their appearance as at this life stage for the first time ‘‘losses are uncompensated by new gains’’ [8]. Other considerations during assessment include other life stresses especially bereavement. Patients should be asked regarding their motivation for seeking the procedure; those seeking to enhance selfesteem have been consistently shown to have a favorable outcome. During the clinical evaluation the clinician should assess gender identity concerns which may be covert, and a history of abuse. When abuse is suspected it may be prudent to refer the patient to a mental health specialist, as direct enquiry about the abuse can lead to psychiatric decompensation in patients who are excessively somatically focused. The clinician should be aware of the two to three times higher suicide rate among
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. Table 90.1 Clinical features (Diagnostic Statistical Manual for Mental Disorders (DSM IV-TR) [19]) of psychiatric disorders commonly encountered in patients seeking cosmetic procedures Body image pathologies 1. Body dysmorphic disorder (BDD) (also referred to as ‘‘Dysmorphophobia’’): (a) The patient is preoccupied with an imagined or a minimal defect in appearance and the patient’s concern is markedly excessive. (b) There is marked distress or impairment in social, occupational, or other areas of functioning resulting from the preoccupation about the appearance and the preoccupation is not attributable to another psychiatric disorder. (c) Common complaints in BDD include imagined or slight flaws affecting the face or head such as wrinkles, vascular markings, paleness or redness of the complexion, hair thinning, scars, excessive facial hair, acne, swelling, and facial asymmetry, or disproportion. Other preoccupations involve the shape, size, or some other attributes affecting the nose, eyelids, eyes, eyebrows, ears, mouth, lips, teeth, jaw, chin, cheeks, or head. Other body parts such as the genitals, breasts, buttocks, abdomen, upper and lower extremities, overall body size, body build, and muscularity can also be the focus of concern and several attributes may be the focus of concern simultaneously. (d) The complaint may be specific, e.g., ‘‘bump’’ on nose or vague, e.g., ‘‘falling face’’ or ‘‘inadequately firm eyes.’’ (e) Insight about the perceived defect is typically poor, and some patients may be frankly delusional, i.e., they are totally convinced that their view of the ‘‘defect’’ is accurate and undistorted and they cannot be convinced otherwise. (f) Patients may frequently check their ‘‘defect’’ either directly or in a reflecting surface such as a mirror or store window, or there may be excessive grooming behavior such as excessive hair combing, elaborate make-up application, skin picking, and hair removal. Sometimes excessive checking and grooming intensifies the preoccupation about the appearance instead of reassuring the patient, and in such cases patients may avoid mirrors. (g) BDD has been associated with variable degrees of psychiatric morbidity, and may be associated with repeated psychiatric hospitalizations, suicidal ideation, suicide attempts, and completed suicide. Other emergencies may be associated with the BDD patients attempts to correct their perceived flaws by, for example, self-surgery and other self-administered remedies. 2. Eating disorders (anorexia nervosa and bulimia nervosa) (a) Anorexia nervosa (AN) is characterized by a refusal to maintain a minimally normal body weight ( Table 90.1.
Body Dysmorphic Disorder The reported rates of body dysmorphic disorder (BDD) (> Table 90.1) in cosmetic surgery and dermatology setting range between 6% and 15% [19]; various studies have reported a higher prevalence of BDD among patients seeking cosmetic procedures [22]. The studies on the psychosocial correlates of the treatment of aging skin do not specifically refer to increased body dysmorphic disorder in this patient population; this is in part because severely psychiatrically ill patients were typically excluded from most of these studies, and secondly, body-image concerns of patients seeking cosmetic procedure have been evaluated only since the 1990s [22]; BDD first appeared in the psychiatric nosology in 1980, and the term ‘‘body dysmorphic disorder’’ was not used till 1987. Some of the major body image-related concerns of BDD patients involve cutaneous changes associated with aging such as wrinkles and sagging of the skin, and it is likely that BDD is more prevalent among patients requesting cosmetic procedures for aging skin. Various studies have
suggested that patients with BDD who undergo cosmetic procedures experience no change or worsening of their symptom, or develop a preoccupation with another imagined flaw [22]; and BDD patients have been known to become violent toward their cosmetic surgeons when they are dissatisfied with the outcome of surgery. BDD is a contraindication for cosmetic procedures and these patients require psychiatric management of their disorder. The patient seeking treatment for aging skin who also has BDD is not likely to have a typical presentation. Preoccupation with an imagined or slight defect in appearance (> Table 90.1), the first diagnostic criterion of BDD describes the presentation of the majority of cosmetic surgery patients [22], and it may be difficult to assess, for example, whether the concerns of a 35-year-old woman about some wrinkles or sagging of facial muscles is out of proportion to the clinical severity of the problem. BDD usually begins during adolescence; however, the disorder may not be diagnosed for many years often because the patient is reluctant to reveal their symptoms [19]. The disorder usually has a fairly continuous course, although the intensity of symptoms may vary over time, and the part of the body which is the focus of concern may remain the same or change [19]. Therefore, the clinician should obtain a history of body-image concerns starting in adolescence and enquire about a history of other cosmetic procedures which initially may appear to be unrelated to the presenting concern. Secondly, the clinician should enquire about the degree of distress and impairment in functioning caused by the current aging-related problem or other body-image problem with a question like ‘‘What does your concern (i.e., the body-image problem) stop you from doing?’’ For example, if the patient reports that their appearancerelated concern has prevented them from maintaining a job or significantly impaired their social functioning, diagnosis of BDD should be considered.
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Eating Disorders Anorexia nervosa (AN) and bulimia nervosa (BN) (> Table 90.1) usually start during adolescence, and among some patients can be relapsing with exacerbations and remissions, and persist into late life. Patient often do not disclose the fact that they have an eating disorder and are ashamed of their chaotic eating patterns, which can range from severe dietary restriction to bingeing and purging, which is often carried out in secrecy. Some patients can experience significant fluctuations in body weight, which can in turn lead to redundant skin folds, and premature aging of the appearance. Some women with chronic eating disorders develop osteoporosis, and may be of shorter stature as a result of malnutrition during adolescence. In some eating-disordered patients the concern about cutaneous body image may be grossly inconsistent with the norms for their age [6]. In a crosssectional study [6] examining concerns about various aspects of skin appearance among under 30-year-old eating-disordered patients (n ¼ 32) and nonclinical controls (n ¼ 34), it was observed that 81% of the eating-disordered patients versus 56% of controls reported dissatisfaction with the appearance of their skin (p ¼ 0.03). The cutaneous attributes that were of the greatest concern to the eating-disordered patients were those that are also associated with aging and photodamage, e.g., ‘‘darkness’’ under the eyes, freckles, fine wrinkles, and patchy hyperpigmentation (in addition to dryness and roughness of the skin, which are often secondary to the eating disorder). One of the central psychopathological factors underlying eating disorders, which have a peak incidence during the teenage years, is difficulties in dealing with the developmental tasks of adolescence and young adulthood. It is possible that the greater concerns about aging skin in the eating-disordered sample is an index of the overall difficulties experienced by these patients in dealing with ‘‘growing up and growing old,’’ which may lead this group of patients to seek treatments for their aging face. It is also interesting to note that the largest study of psychosocial factors among face-lift patients [8, 9] identified that 22% of their patients between the age of 29 and 39 years were ‘‘emotionally dependent’’ and demonstrated ‘‘many problems of adjustment to adult reponsibilities.’’ A youthful look is typically associated with a slim and welltoned body, and some individuals may become excessively preoccupied with diet and exercise as their appearance ages. In a small group of individuals who have other risk factors for the development of an eating disorder, the fear
of aging precipitated by the cutaneous changes of aging, can culminate in anorexia nervosa [23]. The association between concerns about aging of the appearance and drive for thinness has been studied in nonclinical samples. In a survey of 71 men and 102 women who were all nonclinical subjects attending a shopping mall [24], it was observed that concerns about the effect of aging on the appearance correlated directly (r ¼ 0.4; p < 0.05) with the Drive for Thinness subscale of the Eating Disorder Inventory (EDI) even after the possible confounding effect of body mass index and chronological age were partialled out statistically. This correlation was significant among both men and women. The Drive for Thinness (EDI) subscale measures an excessive preoccupation with dieting and exercise and an ardent desire to lose weight. Furthermore, among the women the belief that having younger-looking skin is a prerequisite to good looks correlated with Drive for Thinness (EDI) (r ¼ 0.3; p < 0.01) and Body Dissatisfaction (EDI) (r ¼ 0.4; p < 0.01) after the effects of age and body mass index were partialled out statistically. The Body Dissatisfaction (EDI) subscale measures dissatisfaction with body shape and weight and the concern that certain body regions such as the abdomen, hips, and thighs are too fat. This finding has been replicated among another randomly selected sample of nonclinical subjects [25]. These findings highlight the impact of aging skin on satisfaction with overall body image that is not necessarily related to aging, and this relation was observed independent of chronological age.
Mood Disorders Most of the psychosocial studies on the treatment of aging skin observed the importance of depressive symptoms of some type; around 14% [10] to 33% [8, 9] of patients described having depressive symptoms prior to their facelift surgery; during the early postoperative course, upto 24% of patients [10] were observed to experience a transient flare-up of depressive symptoms, which were partly related to emergence of feeling about the recent death of a loved one and other psychosocial stressors; 30% [10] of patients experienced a more prolonged course of depression and this group also had more depressive symptoms preoperatively. In one study [11, 12], the depression scores did not change significantly presurgery to postsurgery. In nonsurgical studies, patients seeking treatment for photodamaged skin with topical tretinoin [15, 16] did not have high depression scores at baseline; and a
Aging Skin: Some Psychosomatic Aspects
preliminary study indicates treatment of glabellar frown lines with Botulinum Toxin A, in patients with major depressive disorder was associated with remission of depression, which was previously treatment resistant in all patients except one who turned out to have bipolar disorder [18]. These findings from a wide range of studies suggest that patients seeking cosmetic procedures should be screened for depressive disease with a special focus on recent bereavement or other significant losses (e.g., children leaving home); management of depressive illness prior to therapy is likely to be associated with a more favorable postoperative course. The clinician should specifically assess for bipolar disorder (> Table 90.1) because a patient who presents with depressive symptoms may in fact be bipolar. The bipolar patients who are most likely to be overlooked are those with more subtle symptoms, i.e., patients with Bipolar II disorder. The patient with Bipolar II disorder who is hypomanic may present as a social, extroverted individual who is highly motivated to improve her appearance, or she may have a grandiose and unrealistic view of how the cosmetic procedure can further improve her appearance. Hypomanic patients can be very pleasant and complimentary, or they may be irritable, in which case they are more likely to get psychiatric attention. The patient’s motivation for surgery or desire to have a cosmetic procedure may totally change once the patient is no longer hypomanic. A hypomanic patient may be on a spending spree and not be able to afford procedures that they have signed up for. Some bipolar patients may ‘‘overcompensate’’ psychologically and have a hypomanic reaction after major bereavement, e.g., death of a spouse. It is important to identify such situations, especially as the literature [8–10] suggests that a significant number of patients seeking treatments for aging skin have recently suffered the loss of their spouse.
Anxiety Disorders The literature suggests that patients seeking treatment for aging skin suffer from a range of anxiety-related symptoms, but generally do not meet all the criteria for an anxiety disorder [19]. Prior to treatment, some patients reported anxiety during interpersonal interactions and increased self-consciousness [8–10, 15, 16]. These represent some features of social phobia or social anxiety disorder [19], which is characterized by clinically significant anxiety provoked by exposure to certain types of social or performance situations, often leading to avoidance
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behavior. Overall, there was an improvement in social anxiety-related symptoms post treatment. Some patients with obsessive–compulsive disorder may experience obsessive thoughts about some aspect of their appearance that they do not perceive as ‘‘perfect’’ and may seek cosmetic treatments to correct the problem; patients with body dysmorphic disorder can have obsessive thoughts about their imagined deformity. The clinician should be aware that some patients seeking body-image surgery are survivors of childhood sexual abuse [26] and may be suffering from posttraumatic stress disorder (PTSD) [19]. It has been observed that ‘‘plastic surgeons treat child sexual abuse survivors without being aware of it.’’ Such patients often appear well adjusted and may become symptomatic under the specific stresses of surgery [27], for example, they may start having flashbacks of their trauma. For some patients, the decision to have cosmetic procedures is their attempt, albeit unconscious, to ‘‘fix’’ a body that is tainted by abuse. Summit [27] notes that the abused child ‘‘will tend to blame his or her body for causing the abuse and will tend to search for the idealized authority figures who might both redeem the body and undo the abuse.’’ Some patients may have a history of multiple cosmetic procedures [27] which they may not have found to be satisfactory; many patients with histories of childhood sexual abuse may not have conscious recollection of the their traumatic experiences. Summit [27] cautions against being too intrusive as a direct enquiry regarding a history of abuse can seriously psychiatrically destabilize some patients with chronic PTSD; he observes that ‘‘walking the fine line between support and intrusion requires experience and deserves consultation with specialist colleagues.’’ Several epidemiologic studies have reported that the suicide rate among women with cosmetic breast implants is two to three times the expected rate [21]; it is not difficult to speculate that this may be related to the fact that patients with childhood sexual abuse and PTSD who are at a much greater risk for suicide are also more likely to seek body-image surgeries.
Dissociative Disorders Patients with dissociative disorders especially dissociative identity disorder (DID) (or multiple personality disorder) may experience psychiatric decompensation after bodyimage surgery [28]. A DID patient with a history of severe sexual abuse in a cult, who was previously morbidly obese, underwent panniculectomy after losing over 100 lb. The patient had one personality that felt ‘‘safe’’ in her obese
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Aging Skin: Some Psychosomatic Aspects
body and warded off men while ‘‘protecting’’ the younger personalities (that were formed during the course of severe abuse in the cult). The patient was relatively stable after the weight loss, but decompensated psychiatrically when some men in her apartment complex commented on how good she was looking after the panniculectomy [28]. While the panniculectomy was not a strictly cosmetic procedure, it is conceivable that the patient would have a similar reaction after a cosmetic procedure to rejuvenate her appearance.
Psychotic Disorders Cosmetic procedures for facial rejuvenation have generally not been associated with psychotic decompensation [20]; this is in contrast to aesthetic rhinoplasty patients who, for example, have been shown to develop psychotic disorders such as schizophrenia years after the surgery. Schweitzer et al. [29] have described the case of a woman who underwent routine rhytidectomy with satisfactory aesthetic results. The patient had no past psychiatric history. Twenty-four hours post surgery she became delusional; however, her sensorium was clear. Her symptoms cleared within 2 weeks after antipsychotic drug therapy was started. The patient had a history of severe abuse and neglect during her childhood; appearance was of overriding importance for the female family members and the patient’s mother used to be very critical of the patient’s appearance. It is possible that the patient’s decision to seek surgery was related to some unresolved issues from her childhood which surfaced after surgery and resulted in a psychotic decompensation. In such cases, it is helpful to ascertain the symbolic significance of the procedure for the patient.
Personality Disorders Patients seeking procedures for rejuvenation of their appearance have been identified as having some ‘‘Cluster B’’ (Narcissistic and Histrionic) and ‘‘Cluster C’’ (Obsessive– compulsive and Dependent) personality traits [19]. Individuals with severely narcissistic personalities may develop a major adjustment disorder in reaction to the cutaneous signs of aging. Among such narcissistic individuals who typically have pervasive pattern of grandiosity and need for admiration, having a youthful appearance is often a precondition for self-acceptance and trusting that they will be accepted by others; and an aging appearance can result in a significant emotional crisis, including a severe depressive reaction. A patient with severe narcissistic personality traits is therefore more likely to have
unreasonable expectations of cosmetic procedures for facial rejuvenation. The patient with histrionic personality traits tends to be excessively emotional and attention seeking, and may use her physical appearance to draw attention to herself, and will tend to react negatively to decreased attention from others as a result of aging-related changes; such patients are also likely to have unrealistic expectations of what treatment has to offer. Several studies have shown that patients seeking cosmetic procedures have a greater need to be in control of their lives and have obsessive–compulsive personality traits [10]; and the younger group of patients seeking face-lifts [8, 9] tended to have dependent personality traits. Some of these patients experienced short-term depressive reactions post surgery; however, generally they were satisfied with the outcome of the cosmetic procedures. Goin et al. [10] have further observed that a patient with paranoid personality traits improved after surgery for a face-lift even though ‘‘ordinarily psychiatrists are quite wary about recommending elective operations for patients known to be paranoid.’’ They observe that the paranoid patient had a favorable adjustment as the alterations produced by a face-lift are not drastic body alterations and the changes do not necessitate ‘‘extreme personality organization.’’ The paranoid patient became more socially interactive following her face-lift and this reduced her paranoid thinking.
Conclusion Some of psychosomatic aspects of aging skin should be considered as they may be importance in the clinical management of patients seeking treatment for rejurenation of their appearance using either surgical, minimally invasive procedures or topical therapies.
Cross-references > Assessing
Quality of Life in Order Adult Patients with Skin Disorders > Psychological and Social Implications of Aging Skin: Normal Aging and the Effects of Cutaneous Disease
References 1. Gupta MA, Gilchrest BA. Psychosocial aspects of aging skin. Dermatologic Clinics. October 2005;23(4):643–648. 2. Finn JC, Cox SE, Earl ML. Social implications of hyperfunctional facial lines. Dermatol Surg. 2003;29:450–455.
Aging Skin: Some Psychosomatic Aspects 3. Heckmann M, Ceballos-Baumann A. Botulinum Toxin overrides depression: not surprising yet sensational. Dermatol Surg. 2007; 33(6):765. 4. Khan JA. Aesthetic surgery: diagnosing and healing the miscues of human facial expression. Opthal Plast Reconstr Surg. 2001;17:4–6. 5. Sarwer DB, Crerand CE. Body dysmorphic disorder and appearance enhancing medical treatments. Body Image. 2008;5:50–58. 6. Gupta MA, Gupta AK. Dissatisfaction with skin appearance among patients with eating disorders and non-clinical controls. Br J Dermatol. 2001;145:110–113. 7. Honigman RJ, Phillips KA, Castle DJ. A review of psychosocial outcomes for patient seeking cosmetic surgery. Plast Reconstr Surg. 2004;113:(4): 1229–1237. 8. Webb LW, Slaughter R, Meyer E, Edgerton M. Mechanisms of psychosocial adjustment in patients seeking ‘‘face-lift’’ operation. Psychosom Med. 1965;27(2):183–192. 9. Edgerton MT, Webb WL, Slaughter R, Meyer E. Surgical results and psychosocial changes following rhytidectomy, an evaluation of facelifting. Plastic Reconstr Surg. June 1964;33(6):503–521. 10. Goin MK, Burgoyne RW, Goin JM, Staples FR. A prospective psychological study of 50 female face-lift patients. Plastic Reconstr Surg. April 1980;65(4):436–442. 11. Meningaud JP, Bernadiba L, Servant JM, Herve C, Bertrand JC, Pelicie Y. Depression, anxiety and quality of life among scheduled cosmetic surgery patients: multicentre prospective study. J Craniomaxillofac Surg. 2001;29(3):177–180. 12. Meningaud JP, Benadiba L, Servant JM, Herve C, Bertrand JC, Pelicier Y. Depression, anxiety and quality of life: outcome 9 months after facial cosmetic surgery. J Cranio Maxillofac Surg. 2003;31: 46–50. 13. Alves MC, Alba LEF, Santos R. de Arruda, Ferreira LM. Quality of life and self-esteem outcomes following rhytidoplasty. Annals of Plastic Surgery. May 2005;54:(4): 511–514. 14. Litner JA, Rotenberg BW, Dennis M, Adamson PA. Impact of cosmetic facial surgery on satisfaction with appearance and quality of life. Arch Facial Plastic Surg. 2008;10(2):79–83. 15. Gupta MA, Goldfarb MT, Schork NJ, Weiss JS, Gupta AK, Ellis CN, Voorhees JJ. Treatment of mildly to moderately photoaged skin with
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topical tretinoin has a favorable psychosocial effect: a prospective study. J Am Acad Dermatol. 1991;24:780–781. Gupta MA, Schork NJ, Ellis CN. Psychosocial correlates of the treatment of photodamaged skin with topical retinoic acid: a prospective controlled study. J Am Acad Dermatol. 1994;30:969–972. Dayan SH, Lieberman ED, Thakkar NN, Larimer KA, Anstead A. Botulinum toxin A can positively impact first impression. Dermatol Surg. 2008;34:S40–S57. Finzi E, Wasserman E. Treatment of depression with botulinum toxin A: a case series. Dermatol Surg. 2006;32:645–649. American Psychiatric Association. Diagnostic and Statistical Manual for Mental Disorders (DSM IV-TR), 4th ed, Text Revision. Washington: American Psychiatric Association, 2000. Edgerton MT, Langman MW, Pruzinsky T. Plastic surgery and psychotherapy in the treatment of 100 psychologically disturbed patients. Plastic Reconstr Surg. October 1991;88(4):594–608. Sarwer DB, Brown GK, Evans DL. Cosmetic breast augmentation and suicide. Am J Psychiatry. 2007;164(7):1006–1013. Crerand CE, Franklin ME, Sarwer DB. Body dysmorphic disorder and cosmetic surgery. Plast Reconstr Surg. 2006;118(7): 167e–180e. Gupta MA. Fear of aging: a precipitating factor in late onset anorexia nervosa. Int J Eat Disord. 1990;9:221–224. Gupta MA, Schork NJ. Aging-related concerns and body image: possible future implications for eating disorders. Int J Eat Disord. 1993;14:481–486. Gupta MA. Concern about aging and drive for thinness: a factor in the biopsychosocial model for eating disorders? Int J Eat Disord. 1995;18:351–357. Morgan E, Froning ML. Child sexual abuse sequelae and body-image surgery. Plast Reconstr Surg. September 1990;86(3):475–478. Summit RC. Child sexual abuse and body-image surgery. Discussion. Plast Reconstr Surg. September 1990;86(3):479–480. Gupta MA. Severe psychiatric decompensation after panniculectomy in a previously morbidly obese patient with dissociative identity disorder. Manuscript in preparation. Schweitzer I, Hirschfeld JJ. Postrhytidectomy psychosis: a rare complication. Plast Reconstr Surg. September 1984;74(3):419–422.
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103 Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine Wipes in Postmenopausal Women Miranda A. Farage . Kenneth W. Miller . William J. Ledger
Introduction Vulvar skin changes over time. The most significant changes, which are hormonally mediated, are linked to the onset of puberty, the menstrual cycle, pregnancy, and menopause. Postmenopausal estrogen deficiency can lead to vulvar atrophy and irritation. This chapter reviews the morphology and physiology of the vulva over a woman’s lifetime, vulvar irritation associated with menopause, and studies on the benefits of feminine wet wipes in postmenopausal women.
Effects of Aging on Vulvar Morphology, Physiology, and Susceptibility to Irritation The female lower urogenital tract is unique in that it is derived from all three embryonic germ cell layers: the ectoderm, the endoderm, and the mesoderm [1, 2]. Like the skin at other anatomical sites, the cutaneous epithelia of the mons pubis, labia, clitoris, and perineum originate from the embryonic ectoderm and exhibit a keratinized, stratified squamous structure with sweat glands, sebaceous glands, and hair follicles [3, 4]. The degree of keratinization is greatest on the mons pubis and labia majora; it decreases over the clitoris and the outer surface and inner two thirds of the labia minora, the epidermis of the labia minora being thinner than that of the labia majora. From the inner one third of the surface of the labia minora through the vestibule, the vulvar epithelium is non-keratinized and is composed of mucosal tissue originating from the embryonic endoderm [2, 5]. The vagina originates from the embryonic mesoderm and bears a non-keratinized squamous epithelium that is responsive to estrogen cycling [1].
> Table 103.1 summarizes the changes in the morphology and physiology of the vulva over a woman’s lifetime. The most significant changes are hormonally mediated and linked to the onset of puberty, the menstrual cycle, pregnancy, and menopause. From birth until about 4–6 weeks of age, the effects of residual maternal estrogens on the vulva are evidenced by swollen labia majora, well-developed labia minora, and a relatively large clitoris [3, 6]. During early childhood, the female genitalia receive little estrogen stimulation, resulting in flattened labia majora and thin labia minora and hymen [6]. Although vulvar hair follicles and sebaceous glands are present at birth, these structures do not mature until the adrenal glands are activated at puberty. Prior to puberty, the labia minora has barely discernible vellus hair follicles; these disappear when the follicles of the labia majora and mons pubis terminally differentiate at puberty [7]. At puberty, adrenal and gonadal maturation induce further changes in vulvar skin. Follicular development causes estrogen production to rise: as estrogen stimulation increases, the vulvar epithelium thickens, the labial skin becomes rugose, and the clitoris becomes more prominent [4]. During the reproductive years, vulvar changes are linked to the menstrual cycle and pregnancy. The vulvar epidermis and dermis reach their fullest thickness during the reproductive years [3, 4] (> Fig. 103.1). While vulvar epithelial thickness remains constant throughout the menstrual cycle, cytologic changes associated with sex hormone cycling have been observed [8]: Orthokeratosis predominates at the beginning of the menstrual cycle; parakeratosis increases at mid-cycle; then orthokeratosis rises once again by the end of the cycle. During pregnancy, an increase in total blood volume heightens the coloration of the vulva and the vagina.
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_103, # Springer-Verlag Berlin Heidelberg 2010
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. Table 103.1 Vulvar physiology and characteristics from birth to postmenopause (Farage et al. [3]. Adapted and reprinted with permission from Springer Science and Business Media) Life stage
Pertinent physiology
Newborn
Effects of residual, trans-placental maternal estrogens
● Plump labia majora ● Well-developed labia minora ● Immature hair follicles and sebaceous glands
Early childhood
Lack of stimulation by adrenal or gonadal steroid hormones
● Mons pubis and labia majora lose fat ● Benign labial adhesions, if present, normalize without treatment [44]
Puberty
Adrenal and gonadal maturation ensues. Secondary sex ● Subcutaneous fat is deposited in the mons pubis characteristics are acquired and menstruation and labia majora begins [45] ● Vulvar epithelium thickens ● The labia minora and clitoris become more prominent ● Pubic hair emerges
Reproductive years
Menstruation
● The morphology of the vulva is mature ● Vulvar skin thickness remains constant throughout the menstrual cycle [1] ● Parakeratosis of the vulvar stratum conreum rises at mid-cycle [1, 46]
Pregnancy: blood volume increases; menstrual cycle ceases during gestation
● Hair may darken along the midline of the abdomen ● Increased blood flow heightens vulvar coloration ● Susceptibility to vulvar varicose veins increases [9, 10] ● Flattening of the fourchette and perineal trauma may occur during delivery
Follicular function and menstrual cycle cease. The prevalence of urinary and fecal incontinence rises. Physical health, immune function, tissue regeneration capacity, and cognition may be compromised with increasing age
● Pubic hair becomes sparse ● Subcutaneous fat is lost ● Vulvar tissue atrophies ● Risk of perineal dermatitis rises in older women with incontinence
Menopause
Saphenous, vulvar, and hemorrhoidal swelling also occur due to increased blood volume and venous pressure in femoral and pelvic vessels from the enlarging uterus [9]. Increases in progesterone levels also elevate venous distensibility; this may lead to vulvar varices that typically regress postpartum [3, 9, 10]. Following menopause, circulating estrogen levels (primarily estradiol) are dramatically reduced (from >120 pg/mL to around 18 pg/mL) [11]. Concurrent with this change, levels of gonadotropins increase: folliclestimulating hormone (FSH) rises earlier and to a greater extent than luteinizing hormone (LH). Sex hormone levels premenopause and postmenopause are shown in > Fig. 103.2. Postmenopausal estrogen depletion induces further changes. At a cytological level, estrogen-linked parakeratosis decreases during menopause and is essentially
Vulvar characteristics
nonexistent by the eighth decade [12]. On the morphological level, connective tissue proliferates, elastin fragmentation rises, and hyalinization of collagen occurs. The endometrium becomes thinner, the labia majora loses subcutaneous fat, and the labia minora, vulvar vestibule, and vaginal epithelium progressively atrophy. [4, 13] Vaginal secretions diminish and vaginal pH rises. These effects make the vulvovaginal epithelium more susceptible to inflammatory changes such as atrophic vaginitis [3, 14].
Characteristics of Aging Vulvar Skin Vulvar skin differs from exposed (forearm) skin in several characteristics: skin hydration, skin friction, permeability, and visually discernible irritation [3, 15]. However, aging does not appear to induce significant changes in the
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103
. Figure 103.1 Changes in epithelial thickness of the labia majora with age [48] (Farage et al. [47]. Reprinted with permission from Informa Healthcare, New York)
. Figure 103.2 Differences in hormone concentration premenopause (shaded bar) and postmenopause (solid bar). E2 = estradiol; E1 = estrone; FSH = follicle-stimulating hormone; LH = luteinizing hormone (From [49]; Phillips et al. [48]. Reprinted with permission from Elsevier)
vulvar skin, as evidenced by studies of premenopausal and postmenopausal women (> Table 103.2). For example, vulvar skin is more hydrated than forearm skin (as measured by transepidermal water loss [TEWL]) [16, 17] and has a higher friction coefficient (p < 0.001) [16]. Small age-related differences in these two parameters were observed in forearm skin, but not in vulvar skin.
Effects on skin permeability depend on the nature of the penetrant. For example, vulvar skin was more permeable to hydrocortisone than forearm skin, but both regions exhibited comparable permeability to testosterone [18]. Age-related changes depended on the penetrant. Hydrocortisone absorption from the vulva was significantly higher (p < 0.01) in premenopausal women compared to postmenopausal women; a trend was also observed without significance for forearm skin. However, no significant differences (p > 0.05) in percutaneous testosterone absorption from vulvar or forearm skin were observed in premenopausal or postmenopausal women. Studies with a model irritant (1% aqueous sodium lauryl sulfate [SLS]) suggest a lower erythematous response on the vulva than on the forearm [19]. Following patch testing with SLS, more intense erythema was observed on the forearms of premenopausal women compared to postmenopausal women; however, no visually discernible erythema was observed in the vulvar region of either premenopausal or postmenopausal women.
Vulvar Irritation in Older Women As women age, the skin of the vulva becomes less elastic and the underlying fat and connective tissues can break down, leading to a variety of disorders and symptoms
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. Table 103.2 Physiologic skin parameters in premenopausal and postmenopausal women (Farage et al. [3]. Reprinted with permission from Springer Science and Business Media) Parameter Water barrier function (TEWL, g/m2 h)
Site
Age group
Forearm Premenopausal
N
Measured value
Significancea Reference
34
3.7 0.4
p < 0.05
Postmenopausal 10 Vulva
Premenopausal
34
Postmenopausal 10 Skin hydration (capacitance, AU)
Vulva
2.6 0.3 14.8 1.5 93.3 2.3
Postmenopausal 10
91.9 2.8
Premenopausal
n.s.
13.5 1.8
34
Forearm Premenopausal
[16]
34 116.8 4.1
n.s.
[16]
n.s.
Postmenopausal 10 118.0 8.2 Friction coefficient (m)
Forearm Premenopausal Vulva
34
Postmenopausal 10
0.45 0.01
Premenopausal
0.60 0.04 n.s.
34
Postmenopausal 10 Hydrocortisone penetration (% dose absorbed)
Forearm Premenopausal Postmenopausal Vulva
Testosterone penetration (% dose absorbed)
9
2.8 2.4
9
1.5 1.1
Premenopausal
9
8.1 4.1
9
4.4 2.8
9
20.2 8.1
Postmenopausal
9
14.7 4.2
Premenopausal
9
26.7 8.0
Postmenopausal
9
24.6 5.5
Visual erythema scores (scored on day 2 after 24 h Forearm Premenopausal 10 exposure to 1% sodium lauryl sulfate [SLS]) Postmenopausal 10 Vulva
Premenopausal
[16]
0.60 0.06
Postmenopausal Forearm Premenopausal Vulva
0.49 0.02 p < 0.05
9
n.s.
[18]
p < 0.01 n.s.
[18]
n.s. p = 0.03
[19]
5
10
0
Postmenopausal 10
0
n.s.
n.s. = not significant; TEWL = transepidermal water loss a Level of statistical significance of age-group differences
which result in vulvar irritation. A summary of the most common inflammatory vulvar disorders in older women follows.
Atrophic Vaginitis Up to 40% of postmenopausal women have symptoms of atrophic vaginitis, an inflammatory condition of the vulva and vagina linked to estrogen depletion [20, 21]. Estrogen receptors of the vagina, vulva, urethra, and bladder trigone atrophy as postmenopausal estrogen levels diminish. Concurrently, the vaginal epithelium thins, vaginal secretions decline, and vaginal pH rises. In total, such changes
weaken the lining of the vagina and urinary tract and increase susceptibility to inflammation and urinary tract infections [20, 21]. Symptoms of atrophic vaginitis include vaginal and vulvar discomfort, dryness, burning, itching, and dyspareunia (painful intercourse). Inflammation of the vaginal epithelium also can contribute to urinary symptoms, including increased frequency, urgency, dysuria, stress incontinence, and/or recurrent infection [21]. Atrophic vaginitis is usually treated with low-dose, hormone replacement therapy (HRT). Studies have compared a variety of treatment regimens including creams, tablets, suppositories, pessaries, and rings, with no treatment being demonstrated as superior to another [21].
Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine
Due to the nature of the atrophic epithelia, initial systemic absorption is low; however, as vascularity improves, absorption increases [22]. In addition, it has been demonstrated through cytology that low doses of estrogen are needed to maintain vaginal elasticity [23]. It is therefore recommended that initial estrogen treatment consists of low doses [21, 24].
Incontinence Dermatitis Women often experience greater urinary urgency as they age; bladder capacity and voiding efficiency also may decline [25]. When accompanied by illness, obstetrical injury, changes in nutrition or hormonal status, side effects of medication, and/or decreased cognitive function, such changes can contribute to urinary incontinence [25, 26]. While prevalence rates vary, urinary incontinence rises among older people and is relatively common after the age of 50 [27]. Although research studies found no significant agerelated decrease in the barrier function and hydration
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of vulvar skin or any significant difference in susceptibility to model irritants, older women with incontinence, nevertheless, are at increased risk for developing incontinence dermatitis. This risk results from a combination of factors, including tissue atrophy, delayed dissipation of excess skin moisture, shear forces of bone against skin due to limited mobility, and lower tissue regeneration capacity. In addition, skin occlusion associated with incontinence garments can have an irritating effect on the skin [28]. Skin occluded by incontinence garments or pads is wetter, and with a higher pH, bacterial count, and susceptibility to erosion [29]. Barrier permeability and molecular and cellular homeostasis are also affected [30]. Factors contributing to the morbidity of incontinence dermatitis in the elderly are displayed in > Fig. 103.3. Incontinence dermatitis begins with mild skin erythema, which, if untreated, may intensify and be accompanied by blisters and erosions in severe cases [27]. In women, urinary dermatitis first appears between the labial folds; signs of fecal dermatitis begin in the perianal area and progresses to the posterior aspect of the upper thighs [31]. Secondary infections can also occur (see below).
. Figure 103.3 Factors contributing to the morbidity of incontinence dermatitis in the elderly population (Farage et al. [27]. Reprinted with permission from Informa Healthcare, New York)
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Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine
Treatment of urinary and fecal incontinence dermatitis must take into account the fragility of the older skin as well as concurrent cutaneous damage due to secondary infections or cleansing. Limited systematic research exists on specific cleansing regimens and their impact on the prevention or treatment of incontinence dermatitis. One prospective trial of the impact of preventative care was a preliminary examination of structured intervention in 15 institutionalized patients with dementia [32]. In this study, the number of patients who developed dermatitis was unchanged regardless of whether or not preventative treatments were used. Dermatitis developed only in patients with urofecal incontinence with more than four episodes in a 24-h period. None of the patients were able to inform caregivers of incontinent episodes. While the small number of subjects and their poor mental health limit the conclusions that can be drawn from this study, the results suggest that patients incapable of reporting incontinent episodes should be monitored closely.
Vulvar Intertrigo Vulvar intertrigo is an inflammation of the genitocrural folds, the labia, and the perineum caused by skin-on-skin friction. It is characterized initially by mild erythema that may progress to more intense inflammation. While most commonly seen in morbidly obese women, the condition can also ensue from urinary and fecal incontinence that traps moisture in folds of the skin [27, 33].
Fungal Infections Secondary to Urinary Incontinence Fungal infections of vulvar and perineal skin related to urinary incontinence generally involve two species: Candida albicans and Tinea cruris. C. albicans infections of the vagina often manifest as an itchy erythema with a creamy white discharge. Infections that involve the vulva or perineum create erythematous patches with satellite pustules. Candida intertrigo can develop in moist skin folds [28, 34]. Because the gastrointestinal tract is a primary reservoir for C. albicans, fecal incontinence increases the risk of secondary infection [35]. Routine antibiotic use may also increase the risk for secondary infections of the vulva with Candida [28]. Treatment of candidiasis consists of keeping the skin dry and use of topical antifungal treatments containing nystatin or clotrimazole [26, 34].
T. cruris is a fungal infection of the inguinal folds, perineum, and buttocks [34]. Although rare and more commonly seen in men, its prevalence increases in older women due to the diminished cellular immune response [36]. T. cruris usually presents as a ring-shaped eruption with an actively advancing border and a scaly, healing center. It often occurs in patients with T. pedis and onychomycosis and is thought to spread to the groin from contaminated clothing. Most patients with T. cruris can be treated easily with topical antifungal agents; maintaining dry skin helps prevent this condition.
Bacterial Infections Aging skin that has been compromised by incontinence dermatitis may facilitate the proliferation and invasion of microbials such as Staphylococcus in elderly subjects [35]. Vulvar folliculitis, characterized by red, tender pus-filled papules surrounding the hair follicles, often involves infection by staphylococci and Streptococci species [28]. Some surgical incontinence therapies, such as transobturator and tension-free vaginal tape (TVT) have resulted in serious subdermal infections by these organisms, progressing to cellulitis and potentially fatal, necrotizing fasciitis, which are rare but serious [37, 38].
Pruritis Ani and Perianal Inflammation Anogenital pruritus, characterized by perianal itching, erythema, and/or lesions, can stem from incontinence, but also may result from cleansing with harsh soaps. Chronic scratching exacerbates perianal inflammation and may compromise the skin. This condition is more common among with people with impaired mental function or dementia [39].
Decubitus Ulcers Pressure or decubitus ulcers, commonly known as bedsores, occur when soft tissues become compressed between bony protuberances and contact surfaces. Under these conditions, friction or shearing forces contribute to tissue ischemia, infarction, and tissue erosion [40, 41]. Pressure ulcers occur most often in patients with cognitive impairment, immobility, or both. Over 60% of decubitus ulcers occur in patients over 70 years; the prevalence rate in nursing homes is estimated to be 20–40% [40]. Excessive skin hydration associated with urinary incontinence
Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine
increases the susceptibility to pressure ulcers: the frequency of pad changes for incontinent patients correlates directly to the risk of Stage II pressure ulcers (defined as partial thickness skin loss involving the epidermis, dermis, or both) [28, 42].
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. Table 103.3 Subjective product ratings following 28 days use of wet wipes or dry toilet tissue by postmenopausal women (Farage et al. [43]. Reprinted with permission from Science Printers and Publishers) Treatment
Benefits of Feminine Wet Wipes in Postmenopausal Women Assessment
Clinical studies suggest a potential benefit of feminine wet wipes to vulvar skin of postmenopausal women. A 28-day, examiner-blind, randomized, prospective trial compared skin effects of a prototype wet wipe to dry toilet tissue in 120 premenopausal and 60 postmenopausal women [43]. Results in the postmenopausal group are discussed here. These participants were women aged 55–80 years, free of vulvovaginal infection (e.g., candidiasis, genital herpes, chlamydia, Trichomonas vaginalis, and bacterial vaginosis), who were not on hormone replacement therapy (HRT). Women with stress or urge urinary incontinence were included; approximately 30% of the participants reported a history of stress incontinence. Thirty-one women were randomly assigned to the treatment group and 29 to the comparison group. The treatment group used individual sheets of tissue moistened with a lotion that contained skin cleansers and moisturizing agents (>90% water with low levels of humectants, surfactants, emulsifiers, fragrance, and preservatives). This product was used in lieu of toilet tissue to clean the vulva, perineum, and/or anal area after every episode of urination or defecation during the 28-day study period. The comparison group (controls) used a commercially available dry toilet tissue. Skin condition was assessed after 14 2 and 28 2 consecutive days. Participants also reported subjective symptoms and product preferences. In general, mild to barely discernible irritation was present at study inception. Erythema of the mons pubis, labia majora, labia minora, vestibule, perineum, buttocks, or upper thighs, when present, improved over the course of the study in both groups. Genital moisture on the labia majora and the perineum significantly increased in wet-wipes users (p = 0.01) compared to dry-tissue users. Subjective reports from participants were instructive (> Table 103.3). Wet wipes were significantly preferred to dry toilet tissue by postmenopausal women: 26 of 31 (84%) of wet-wipes users rated their assigned product excellent to very good for skin comfort compared to 19 of 29 (66%) of dry-tissue users. However, 4 of 31
Wet wipes
Dry toilet tissue
N = 31
N = 29
N (%)
N (%)
Comfort ratings Excellent
10 (32)
8 (28)
Very good
16 (52)
11 (40)
Good
3 (10)
8 (28)
Fair
2 (6)
1 (3)
Poor
0 (0)
1 (3)
Severity of subjective comments about sensory effects Dryness
1 (3.2)
3 (10)
Slight
Slight
Sticky sensation
3 (9.7)
0 (0)
Slight
None
Burning
4 (12.9)
1 (3.4)
Slight
Slight
Itching
1 (3.2)
0 (0)
Slight
None
3 (9.7)
1 (3.4)
Slight
Slight
Stinging
The first evaluation occurred at day 14 ( 2) of product use
(13%) of women using wet wipes reported more burning and 3 of 31 (10%) experienced more stinging compared to the product they typically used. In addition, 3 of 31 (10%) reported a more of a ‘‘sticky’’ sensation with the wet product. Overall, 3 of the 31 (10%) postmenopausal participants considered the wipes to be too wet [43]. Hence, a significant majority of postmenopausal users favored wet wipes over conventional products, although some did not care for the associated sensations of wetness. However, postmenopausal wipes users were less likely to be bothered by skin wetness than premenopausal women (see original report) and were more likely to favor wet wipes over dry tissue for genital cleaning. Taken together, the results suggest that the hydrating effect of the wet wipes on genital skin was perceived to be beneficial by most postmenopausal women in the study, a group more likely to experience vulvar atrophy and skin dryness.
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Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine
Conclusion The vulva undergoes morphological and physiological changes over the course of a woman’s lifetime. Many of these changes are mediated by sex hormones. Estrogen depletion alters the structure of vulvovaginal tissues, making them more susceptible to inflammation. Vulvovaginal skin disorders associated with aging include atrophic vaginitis, incontinence dermatitis, fungal and bacterial infections secondary to incontinence, anogenital pruritus, perianal inflammation, and decubitus ulcers. Clinical trials indicate that feminine hygiene wet wipes may improve skin comfort in postmenopausal women. Four weeks of exclusive use of wet wipes in lieu of dry toilet tissue caused negligible effects on vulvar skin irritation, but a clinically observable increase in genital skin moisture. Most postmenopausal women in the study favored wet wipes over dry toilet tissue, which may suggest improved comfort of atrophied vulvar skin and amelioration of dryness associated with postmenopausal estrogen depletion.
Cross-references > Aging
Genital Skin and Hormone Replacement Therapy Benefits > Solutions and Products for Managing Female Urinary Incontinence > Unique Skin Immunology of the Lower Female Genital Tract with Age > Vaginal Secretions with Age
References 1. Nauth H. Anatomy and physiology of the vulva. In: Elsner P, Martius J (eds) Vulvovaginitis. New York: Marcel Dekker, 1993. 2. Sargeant P, Moate R, Harris JE, et al. Ultrastructural study of the epithelium of the normal human vulva. J Submicrosc Cytol Pathol. 1996;28:161–170. 3. Farage M, Maibach H. Lifetime changes in the vulva and vagina. Arch Gynecol Obstet. 2006;273:195–202. 4. Jones IS. A histological assessment of normal vulval skin. Clin Exp Dermatol. 1983;8:513–521. 5. Woodruff JD, Friedrich EGJ. The vestibule. Clin Obstet Gynecol. 1985;28:134–141. 6. Chang L, Muram D. Pedatric and adolescent gynecology. In: DeCherney AH, Nathan L (eds) Current Obstetric and Gynecological Diagnosis & Treatment. New York: McGraw-Hill, 2002. 7. Harper WF, McNicol EM. A histological study of normal vulval skin from infancy to old age. Br J Dermatol. 1977;96:249–253. 8. Nauth HF, Schilke E. Cytology of the exfoliative layer in normal and diseased vulvar skin: correlation with histology. Acta Cytol. 1982;26:269–283.
9. Torgerson RR, Marnach ML, Bruce AJ, et al. Oral and vulvar changes in pregnancy. Clin Dermatol. 2006;24:122–132. 10. Wong RC, Ellis CN. Physiologic skin changes in pregnancy. J Am Acad Dermatol. 1984;10:929–940. 11. Pandit L, Ouslander JG. Postmenopausal vaginal atrophy and atrophic vaginitis. Am J Med Sci. 1997;314:228–231. 12. Nauth HF, Bo¨ger A. New aspects of vulvar cytology. Acta Cytol. 1982;26:1–6. 13. Erickson KL, Montagna W. New observations on the anatomical features of the female genitalia. J Am Med Wom Assoc. 1972;27:573–581. 14. Farage MA, Miller KW, Elsner P, et al. Intrinsic and extrinsic factors in skin ageing: a review. Int J Cosmet Sci. 2008;30:87–95. 15. Oriba HA, Elsner P, Maibach HI. Vulvar physiology. Semin Dermatol. 1989;8:2–6. 16. Elsner P, Maibach HI. The effect of prolonged drying on transepidermal water loss, capacitance and ph of human vulvar and forearm skin. Acta Derm Venereol. 1990;70:105–109. 17. Elsner P, Wilhelm D, Maibach HI. Frictional properties of human forearm and vulvar skin: influence of age and correlation with transepidermal water loss and capacitance. Dermatologica. 1990;181:88–91. 18. Oriba HA, Bucks DA, Maibach HI. Percutaneous absorption of hydrocortisone and testosterone on the vulva and forearm: effect of the menopause and site. Br J Dermatol. 1996;134:229–233. 19. Elsner P, Wilhelm D, Maibach HI. Effect of low-concentration sodium lauryl sulfate on human vulvar and forearm skin. age-related differences. J Reprod Med. 1991;36:77–81. 20. Bachmann GA, Nevadunsky NS. Diagnosis and treatment of atrophic vaginitis. Am Fam Physician. 2000;61:3090–3096. 21. Castelo-Branco C, Cancelo MJ, Villero J, et al. Management of postmenopausal vaginal atrophy and atrophic vaginitis. Maturitas. 2005;52(Suppl 1):S46–52. 22. Heimer GM, Englund DE. Effects of vaginally-administered oestriol on post-menopausal urogenital disorders: a cytohormonal study. Maturitas. 1992;14:171–179. 23. Fraser IS, Ayton R, Farrell E, et al. A multicentre Australian trial of low dose estradiol therapy for symptoms of vaginal atrophy using a vaginal ring as delivery system. Maturitas. 1995; 22(Suppl):S41. 24. Santen RJ, Pinkerton JV, Conaway M, et al. Treatment of urogenital atrophy with low-dose estradiol: preliminary results. Menopause. 2002;9:179–187. 25. Millard RJ, Moore KH. Urinary incontinence: the cinderella subject. Med J Aust. 1996;165:124–125. 26. Resnick NM. Geriatric medicine. In: Tierney L, McPhee S, Papadakis M (eds) Current Medical Diagnosis and Treatment, 39th ed. New York: McGraw Hill, 2000, p. 62. 27. Farage M, Bramante M. Genital hygiene: culture, practices, and health impact. In: Farage M, Maibach H (eds) The Vulva: Anatomy, Physiology, and Pathology. New York: Informa Healthcare, 2006. 28. Farage MA, Miller KW, Berardesca E, et al. Incontinence in the aged: contact dermatitis and other cutaneous consequences. Contact Dermatol. 2007;57:211–217. 29. Aly R, Shirley C, Cunico B, et al. Effect of prolonged occlusion on the microbial flora, ph, carbon dioxide and transepidermal water loss on human skin. J Invest Dermatol. 1978;71:378–381. 30. Zhai H, Maibach HI. Skin occlusion and irritant and allergic contact dermatitis: an overview. Contact Dermatitis. 2001;44:201–206. 31. Gray M. Preventing and managing perineal dermatitis: a shared goal for wound and continence care. J Wound Ostomy Continence Nurs. 2004;31:S2–9, quiz S10–2.
Changes in Vulvar Physiology and Skin Disorders with Age and Benefits of Feminine 32. Lyder CH, Clemes-Lowrance C, Davis A, et al. Structured skin care regimen to prevent perineal dermatitis in the elderly. J ET Nurs. 1992;19:12–16. 33. Mistiaen P, Poot E, Hickox S, et al. Preventing and treating intertrigo in the large skin folds of adults: a literature overview. Dermatol Nurs. 2004;16:43–6, 49–57. 34. Martin ES, Elewski BE. Cutaneous fungal infections in the elderly. Clin Geriatr Med. 2002;18:59–75. 35. LeLievre S. Skin care for older people with incontinence. Elder Care. 2000;11:36–38. 36. Shenefelt PD, Fenske NA. Aging and the skin: recognizing and managing common disorders. Geriatrics. 1990;45:57–9, 63–66. 37. Caquant F, Collinet P, Deruelle P, et al. Perineal cellulitis following trans-obturator sub-urethral tape uratape. Eur Urol. 2005;47: 108–110. 38. Connolly TP. Necrotizing surgical site infection after tension-free vaginal tape. Obstet Gynecol. 2004;104:1275–1276. 39. Fiers S, Thayer D. Management of intractable incontinence. In: Doughty D (ed) Urinary and Fecal Incontinence: Nursing Management, 2nd edn. St. Louis: Mosby, 2000. 40 Professional Guide to Diseases, 9th. Ambler, PA: Springhouse Division, Lippincott, Williams and Wilkins, 2007, pp. 775–781. 41. Edlich RF, Winters KL, Woodard CR, et al. Pressure ulcer prevention. J Long Term Eff Med Implants. 2004;14:285–304.
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42. Fader M, Clarke-O’Neill S, Cook D, et al. Management of night-time urinary incontinence in residential settings for older people: an investigation into the effects of different pad changing regimes on skin health. J Clin Nurs. 2003;12:374–386. 43. Farage MA, Stadler A, Chassard D, et al. A randomized prospective trial of the cutaneous and sensory effects of feminine hygiene wet wipes. J Reprod Med. 2008;53:765–773. 44. Williams TS, Callen JP, Owen LG. Vulvar disorders in the prepubertal female. Pediatr Ann. 1986;15:588–589, 592–601, 604–605. 45. Marshall W, Tanner J. Puberty. In: Davis J, Dobbing J (eds) Scientific Foundations of Paediatrics. London: Heinemann, 1981. 46. Nauth HF, Haas M. Cytologic and histologic observations on the sex hormone dependence of the vulva. J Reprod Med. 1985; 30:667–674. 47. Farage MA, Maibach HI, Deliveliotou A, et al. Changes in the vulva and vagina throughout life. In: Farage A, Maibach HI (eds) The Vulva: Anatomy, Physiology, and Pathology. New York: Informa Healthcare, 2006. 48. Phillips TJ, Demircay Z, Sahu M. Hormonal effects on skin aging. Clin Geriatr Med. 2001;17:661–672, vi. 49. Yen SS. The biology of menopause. J Reprod Med. 1977;18:287–296.
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99 Cosmetic Anti-aging Ingredients Donald L. Bissett . Mary B. Johnson
Introduction Many cosmetic materials claim to have anti-aging effects when applied topically. As there are many such materials and also because ‘‘anti-aging’’ encompasses many definitions (e.g., prevention vs. improvement; wide array of benefit areas such as wrinkling, sagging, texture, sallowness, and hyperpigmentation), this relatively short chapter must be very selective regarding the materials discussed. Thus, the focus will be on only a few classes of cosmetic agents that are reported to provide improvement in the wrinkling, firming and/or sagging appearance of the skin. Particular attention will be directed to those materials within these classes for which they are readily available or published clinical data to support the reported benefits in improving skin appearance.
Vitamin A Forms There are several forms of vitamin A used cosmetically, in particular retinol, retinyl esters (e.g., retinyl acetate, retinyl propionate, and retinyl palmitate), and retinaldehyde. Through endogenous enzymatic reactions in the skin, these forms are all converted ultimately to trans-retinoic acid, which is the active form of vitamin A in skin. Specifically, retinyl esters are converted to retinol via esterases. Retinol is then converted to retinaldehyde by retinol dehydrogenase. Finally, retinaldehyde is oxidized to retinoic acid by retinaldehyde oxidase [1].
Mechanisms The active form of vitamin A in skin is trans-retinoic acid (t-RA). t-RA interacts with nuclear receptor proteins, which then interact with specific DNA sequences to either increase or decrease expression of many specific proteins/ enzymes [1]. Some specific changes that could be relevant to skin anti-aging effects are those that result in thicker skin (epidermis and dermis) to diminish the appearance
of fine lines and wrinkles, such as increased epidermal proliferation and differentiation, increased production of epidermal ground substance (glycosaminoglycans or GAGs, which bind water), and increased dermal production of extracellular matrix components such as collagen. On the inhibitory side, retinoids are reported to inhibit production of collagenase [2] to reduce loss of dermal collagen. While retinoid will stimulate production of GAGs in epidermis, it will inhibit production of excess ground substance in photoaged dermis. Excess dermal GAGs are associated with altered dermal collagen structure and wrinkled skin appearance [3, 4]. As at least some of the epidermal effects of topical retinoid (e.g., epidermal thickening) [5] occur relatively rapidly (days) after initiation of treatment, diminution of appearance of fine lines may be realized quickly. The dermal effects likely occur on a much longer time frame (weeks to months), so that reduction in appearance of deep wrinkles requires much longer time frames.
Efficacy While much of the substantial literature on the improvement in the appearance of skin wrinkles by topical retinoids is focused on t-RA, data is also available on the vitamin A compounds that are used cosmetically. As retinoids are irritating to the skin, defining skin-tolerated doses clinically is a key step in working effectively with these materials. Retinol is better tolerated by the skin than t-RA [2], and the ester retinyl propionate is milder to the skin than retinol and retinyl acetate (> Fig. 99.1). As retinoids in general tend to be fairly potent, cosmetic moisturizer formulations containing less than 1% are generally sufficient to obtain significant improvement in appearance. At low levels, both retinol and retinyl propionate have been shown [1] to be significantly effective in reducing the appearance of facial wrinkles (> Fig. 99.2). The effectiveness of 0.05% retinaldehyde on facial skin has also been reported [6], although it has irritation potential similar to retinol [7]. Retinyl palmitate has very low irritation potential, and is effective if tested at a high dose, such as at 2% [8].
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_99, # Springer-Verlag Berlin Heidelberg 2010
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. Figure 99.1 Retinoid irritation in cumulative human back irritation testing (double-blind, vehicle-controlled, randomized study; daily patching for 20 days, under semi-occluded patch, n = 45; 0–3 irritation grading). Doses and abbreviations used were: 0.09% RP (retinyl propionate), 0.086% RA (retinyl acetate), and 0.075% ROH (retinol). RP and RA were significantly (p < 0.05) less irritating than ROH, and RP was less irritating than RA (p < 0.10)
Product/Formulation Challenges A challenge in working with retinoids is their tendency to induce skin irritation, which negatively affects skin barrier properties and consumer acceptance. Mitigation of the irritation may be managed to some extent with appropriate formulation to meter delivery into the skin, use of retinyl esters that are less irritating than retinol, or inclusion of other moisturizing ingredients to counter this issue. Retinoids are unstable to oxygen and light. Thus, formulation and packaging should be done in an environment that minimizes exposure to oxygen and light. The final product packaging should be opaque and oxygen-impermeable, and should also include use of a small package orifice to reduce oxygen exposure once the container is opened. In addition, other strategies can be employed, such as encapsulation of the retinoid and inclusion of stabilizing antioxidants.
Vitamin B3 . Figure 99.2 Reduction in the appearance of wrinkles in a 12-week clinical study (double-blind, left–right randomized, splitface, placebo vehicle-controlled study with once daily application, n = 52–56 per product. Evaluation for reduction vs. baseline in wrinkling and hyperpigmentation was done by three independent expert graders (0–4 grading scale) on blind-coded images after 4, 8, and 12 weeks of treatment. The grader scores at each time point were averaged. There were significant effects for both treatments across the study. The data presented here are averages for all three time points. S indicates significant at p 0.05. The low irritation of retinyl propionate (RP) vs. retinol (ROH) permits use of higher levels to achieve greater effects without significant negative aesthetic issues
Forms There are three primary forms of vitamin B3 that have found utility in skin care products: niacinamide (aka nicotinamide), nicotinic acid, and nicotinate esters (e.g., myristoyl nicotinate, benzyl nicotinate).
Mechanisms Vitamin B3 serves as a precursor to a family of endogenous enzyme co-factors, specifically nicotinamide adenine dinucleotide (NAD), its phosphorylated derivative (NADP), and their reduced forms (NADH, NADPH), which have antioxidant properties. These co-factors are involved in many enzymatic reactions in the skin, and thus have potential to influence many skin processes [9]. This precursor role of vitamin B3 may be the mechanistic basis for the diversity of clinical effects observed for niacinamide [10]. The anti-aging effects relevant to this discussion include: ● Inhibition of sebum production, specifically affecting the content of triglycerides and fatty acids, likely contributing to reduction in appearance of skin pore size and thus improved skin texture
Cosmetic Anti-aging Ingredients
● Increase in epidermal production of skin barrier lipids (e.g., ceramides) and also skin barrier layer proteins and their precursors (keratin, involucrin, filaggrin), leading to the observed enhancement of barrier function as determined by reduced transepidermal water loss (TEWL) ● Increase in production of collagen, based on in vitro measurements, which may contribute to the observed reduction in the appearance of fine lines/wrinkling ● Reduced production of excess dermal GAGs (glycosaminoglycans) in culture. As nicotinic acid and its esters are also precursors to NAD(P), they would be expected to provide these same benefits to skin. Nicotinic acid and many (if not all) of its esters (following in-skin hydrolysis to free nicotinic acid) also stimulate blood flow, leading to increased skin redness or a flush response [11].
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and pore size, which likely contribute at least in part to improved skin texture (> Fig. 99.4). Fairly high doses (2–5%) of vitamin B3 have been used to achieve desired benefits. However, since there is very high tolerance of the skin to niacinamide even with chronic usage, high doses can be used acceptably. Some data on myristoyl-nicotinate has been presented [12] to suggest that a similar broad array of benefits occurs with this agent when used topically (1–5% doses). Clinical anti-aging data for topical nicotinic acid and other esters is not available.
Product/Formulation Challenges
As representative of the vitamin B3 family of compounds, topical niacinamide [10] reduces the appearance of fine lines/wrinkling (> Fig. 99.3). The effect increases over time, and is significant after 8–12 weeks of treatment. Topical niacinamide also improves other aspects of aging skin, such as reduction in sebaceous lipids (oil control)
The key challenge for formulating with niacinamide and nicotinate esters is avoiding hydrolysis to nicotinic acid. Nicotinic acid, even at low doses, can induce an intense skin reddening (flushing) response [11]. While a little skin redness (increased skin ‘‘pinkness’’) may be a desired effect, the flushing response among individuals is highly variable in terms of dose to induce it, time to onset of the response, and duration of response. Additionally, the flushing can also have associated issues such as burning, stinging, and itching, particularly under cold and/or dry conditions. To avoid hydrolysis, formulating in the pH range of 5–7 is preferred. This flushing issue also requires
. Figure 99.3 Topical moisturizer formulation containing 5% niacinamide reduces appearance of fine lines/wrinkling in facial skin. Subjects were female Caucasians (n = 50) who applied placebo control vs. 5% niacinamide formulations to their faces (12-week, double-blind, split-face, left–right randomized clinical trial)
. Figure 99.4 Topical moisturizer formulation containing niacinamide improves skin surface texture. Subjects were female Caucasians (n = 50) who applied placebo control vs. 5% niacinamide formulations to their faces (12-week, doubleblind, split-face, left–right randomized clinical trial)
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that the purity of the raw material (e.g., niacinamide) be very high to minimize any contaminating free acid. There are many commercial options for nicotinate esters. Unfortunately, many of them are readily hydrolyzed to nicotinic acid on or in the skin, such that flushing responses occur rapidly (within seconds to minutes) even at very low concentrations ( Fig. 99.5). The doses employed were fairly high (3–10%). At the high dose, there may be skin irritation
issues, as evidenced by the high subject drop-out rate in such testing [18]. Ascorbyl phosphate [15] and tetrahexyldecyl ascorbate [18] have also been reported to be effective.
Product/Formulation Challenges The key challenge with vitamin C compounds in general is stability (oxygen sensitivity), particularly with ascorbic acid. Not only does oxidation lead to loss of the active material, there is also rapid product yellowing (a likely aesthetic negative for the cosmetic user). Various stabilization strategies can be attempted to address the issue, such as exclusion of oxygen during formulation, oxygen impermeable packaging, encapsulation, low pH, minimization of water, and inclusion of other antioxidants. In spite of all these approaches, in general ascorbate stability remains a challenge, and some of these approaches (e.g., very low pH) can lead to unwanted effects of skin irritation. For the ascorbyl phosphates (Mg and Na salts), the resulting high content of salt in the product can dramatically impact the thickener system, requiring increased thickener ingredient concentration. These ascorbate derivatives are also considerably more expensive than other ascorbate compounds.
Cosmetic Anti-aging Ingredients
Peptides Forms There is a limitless array of possible peptides, based on amino acid sequence, number of amino acids, use of amino acids not normally found in proteins, and use of derivatives/isomers of amino acids. A few pure peptides with well-characterized sequences that have received particular focus in the cosmetic industry are palmitoyl-lysine-threonine-threonine-lysine-serine (pal-KTTKS; Matrixyl1), acetyl-glutamate-glutamatemethionine-glutamine-arginine-arginine (Ac-EEMQRR; Argireline1), and the tripeptide copper-glycine-histidinelysine (Cu-GHK).
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. Figure 99.6 Pal-KTTKS reduces excess dermal GAGs. In cell culture fibroblasts from an old donor (57 years old), there was a two- to threefold increase in GAGs (measured as hyaluronic acid) vs. from a young donor (neonatal). Pal-KTTKS was effective in reducing the excess GAG level in old fibroblasts. Abbreviation: t-RA (trans-retinoic acid)
Mechanisms KTTKS is a fragment of dermal collagen and has been shown to stimulate production of collagen in vitro, and has thus been discussed with regard to wound healing [19]. Incorporation of long-chain lipophilic residues such as palmitoyl onto peptides can dramatically improve their delivery into skin [20]. Thus, palmitoyl-KTTKS was synthesized for topical use. Like the underivatized peptide, the palmitate derivative (pal-KTTKS) is also active in stimulating collagen production in vitro [21, 22]. In addition, at extremely low levels (ppb) in culture, pal-KTTKS reduces excess dermal GAGs (> Fig. 99.6). As discussed above, this effect may also contribute to an improvement in the appearance of wrinkles. Like KTTKS, GHK is also a fragment of dermal collagen [23]. Copper is a required factor for activity of lysyl oxidase, an enzyme involved in collagen synthesis [24]. The complex of these two (Cu-GHK) has been shown to stimulate wound-healing processes by increasing production of dermal matrix components such as collagen and specific matrix remodeling MMPs (matrix metalloproteinases) [25, 26]. Ac-EEMQRR is described as a mimic of botulinum neurotoxin (Botox1), which functions by inhibiting neurotransmitter release, thus ‘‘relaxing’’ the muscles involved in defining facial wrinkles [27]. As the reported mechanisms of pal-KTTKS and Cu-GHK involve matrix production and remodeling, their appearance benefits would be expected to require chronic treatment. In contrast, Ac-EEMQRR should have acute benefit effects based on its reported Botox1-like mechanism.
Efficacy The peptide pal-KTTKS has been shown to be quite potent clinically, providing a significant improvement in the appearance of wrinkles from the very low topical dose of 3 ppm (> Figs. 99.7) [21]. This low dose for clinical activity is consistent with the very low concentration (as low as ppb) required to observe in vitro effects (> Fig. 99.6). This topical peptide was extremely well tolerated by test subjects, i.e., it did not induce skin irritation responses (no redness, dryness, burning, stinging, or itching responses) and did not affect skin barrier function [21]. In contrast to the potency of pal-KTTKS, the reported skin anti-aging effects of other peptides require much higher doses, such as 2% for Cu-GHK [28] and as high as 10% for Ac-EEMQRR [27].
Product/Formulation Challenges An important challenge is delivery into skin, as peptides are poorly penetrating, especially as the number of amino acid residues, and thus peptide size, increases. An approach to this problem is addition of a lipophilic chain (e.g., palmitate), which, in the case of KTTKS, increases
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. Figure 99.7 Topical moisturizer formulation containing pal-KTTKS improves the appearance of facial skin wrinkles (quantitative computer image analysis; female Caucasian subjects, n = 93). Smaller numbers indicate fewer fine lines/wrinkles
Efficacy Several topical studies have been discussed and overviewed [29–32]. Significant improvements were reported in the appearance of skin lifting and skin firming (e.g., under-eye firming, cheek area firming, jaw line lifting and firming, increased elasticity). The topical treatment (3% DMAE) was well tolerated by test subjects. The interesting aspect of the clinical effects is that while some testing has been for a duration of weeks to months, the onset of the benefit was reported to be very rapid, within minutes of topical application [29–32]. This seems consistent with the suggested mechanisms.
Product/Formulation Challenge DMAE, a base, has historically been used as a formula pH adjusting agent. In the unneutralized state, its pH is approximately 10. Thus, pH adjustment to the desired value appears to be sufficient for its use in formulation.
Plant Growth Factors peptide skin penetration several fold over the underivatized version. An additional challenge is cost. As the number of amino acid residues increases, the cost of peptide synthesis can increase dramatically. The consequences are that only low levels of peptide can be used in a product (which is acceptable if the peptide is potent, as in the case of pal-KTTKS), or the finished product cost to the consumer must be high.
Dimethylaminoethanol (DMAE) Mechanism DMAE (also known as deanol) is a precursor to acetylcholine, a neurotransmitter involved in increased muscle tone. There could thus be firming of the skin via effects on the facial musculature. In addition, acetylcholine may affect the keratinocytes (specifically their proliferation, adhesion, and motility), leading to ‘‘epidermal contractility,’’ resulting in a firming/tightening/anti-sagging effect on the skin [29]. DMAE also has anti-oxidant properties, which may contribute to its anti-aging effects [30]. This agent additionally has been shown to induce vacuolar cell expansion, which may contribute to a skin tightening or fullness effect [31].
Forms There are three forms discussed in the literature: kinetin (N6-furfuryladenine), zeatin, and pyratine 6.
Mechanisms These materials are plant growth hormones. While their specific mechanisms have not been elucidated, they have been observed to promote growth and have anti-senescence effects in plants. They have powerful natural antioxidant effects in protecting DNA and protein from oxidative damage. In human fibroblast cell culture, even very low levels (ppm) delay the onset of changes associated with cell aging, such as appearance of lipofuscin, appearance of multinucleate cells, microtubule disorganization, and more youthful phenotype [33, 34].
Efficacy In clinical tests, topical 0.1% kinetin was reported to improve the appearance of several problems associated with aging skin, such as wrinkling and poor texture [33, 35]. The 0.1% dose is well tolerated by the skin, with no significant irritation issues described. Zeatin and
Cosmetic Anti-aging Ingredients
pyratine-6 have similarly been shown to provide anti-aging effects [34, 36], including improved appearance of facial skin wrinkling.
Product/Formulation Challenge The limitation with these materials is their fairly low solubility in formulation, thus restricting the upper dose to a low level (e.g., 0.1%) for an aesthetically elegant formulation. This also impacts delivery into skin, although even from this relatively low dose sufficient material does enter skin to provide beneficial skin effects.
Triterpenoids Forms There are numerous plant-derived triterpenoid compounds and derivatives of them, with a few receiving attention in the cosmetic area, e.g., asiatic acid, ursolic acid, medacassic acid, oleanolic acid, betulinic acid, and boswellic acid. There are also naturally occurring saccharide esters of these, such as asiaticoside, which is the ester of asiatic acid.
Mechanisms There are many reported mechanisms for triterpenoids, for example, antioxidant, anti-inflammatory, elastase inhibition, wound healing, and promotion of collagen and ceramide production [37, 38]. As triterpenoids share some structural similarity to steroidal compounds such as hydrocortisone, that is consistent with the reported mechanistic properties and potency of such compounds (e.g., anti-inflammatory effects).
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Product/Formulation Challenge The key issue with triterpenoids is poor solubility, which also results in limited skin delivery. Formulation in liposomes has been employed to improve both delivery and formula solubility, although the resulting increase in oil content of the formulations may negatively impact aesthetics.
Ubiquinone (Co-Enzyme Q10) Mechanism Ubiquinone is an endogenous antioxidant present throughout the body, including the skin. The levels decrease with age. Topical ubiquinone replenishes the level and increases the antioxidant capacity of the skin [40, 41].
Efficacy In a brief summary [42], topical treatment of facial skin with 0.3% ubiquinone was observed to reduce apparent wrinkle depth in the eye area. The corneocyte area, which increases with aging due at least in part to slower stratum corneum turnover, was also reported to be reduced by this topical treatment of skin, suggesting increased turnover. In this testing, topical ubiquinone was well tolerated by the skin.
Product/Formulation Challenge Ubiquinone is yellow-orange in color. Thus, only relatively low doses ( Fig. 99.8) leads to greater benefits than the individual materials. As a further example, the combination of kinetin with niacinamide has also recently been shown to provide what the authors described as synergistic effects above what niacinamide alone provided [35]. Thus, combinations of ingredients have great potential to drive cosmetic effects higher.
Cross-references > Aging
and Anti-aging Strategies and Aging Skin > Topical Growth Factors for Skin Rejuvenation > Topical Peptides and Proteins for Aging Skin > Cosmetics
References 1. Oblong JE, Bissett DL. Retinoids. In: Draelos ZD (ed) Procedures in Cosmetic Dermatology, Cosmeceuticals. Philadelphia: Elsevier Saunders, 2005:35–42. 2. Kang S, Duell EA, Fisher GJ, et al. Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels of irritation. J Invest Dermatol. 1995; 105:549–556. 3. Dunstan RW, Kennis RA. Selected heritable skin diseases of domesticated animals. In: Sundberg JP (ed) Handbook of Mouse Mutations with Skin and Hair Abnormalities. Boca Raton: CRC Press, 1994:524–525. 4. Kligman AM, Baker TJ, Gordon HL. Long-term histologic follow-up of phenol face peels. Plast Reconstructr Surg. 1975;75:652–659. 5. Griffiths CE, Finkel LJ, Tranfaglia MG, et al. An in vivo experimental model for effects of topical retinoic acid in human skin. Br J Dermatol. 1993;129:389–394. 6. Creidi P, Humbert P. Clinical use of topical retinaldehyde on photoaged skin. Dermatology. 1999;199S:49–52. 7. Fluhr JW, Vienne MP, Lauze C, et al. Tolerance profile of retinol, retinaldehyde, and retinoic acid under maximized and long-term clinical conditions. Dermatology. 1999;199S:57–60.
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8. Erling T. Skin treatment with two different galenical formulations of retinyl palmitate in humans. J Appl Cosmetol. 1993;11:71–76. 9. Matts PJ, Oblong JE, Bissett DL. A review of the range of effects of niacinamide in human skin. Int Fed Soc Cosmet Chem Mag. 2002;5:285–289. 10. Bissett DL, Miyamoto K, Sun P, et al. Topical niacinamide reduces yellowing, wrinkling, red blotchiness, and hyperpigmented spots in aging facial skin. Int J Cosmet Sci. 2004;26:231–238. 11. Andersson RG, Aberg G, Brattsand R, et al. Studies on the mechanism of flush induced by nicotinic acid. Acta Pharmacol Toxicol. 1977;41:1–10. 12. Jacobson MK, Kim H, Kim M, et al. Modulating NAD-dependent DNA repair and transcription regulated pathways of skin homeostatis: evaluation in human subjects. Poster, 60th Annual Meeting of the American Academy of Dermatology, New Orleans, 2002, Feb. 22–27. 13. Tajima S, Pinnell SR. Ascorbic acid preferentially enhances type I and III collagen gene transcription in human skin fibroblasts. J Dermatol Sci. 1996;11:250–253. 14. Geesin JC, Darr D, Kaufman R, et al. Ascorbic acid specifically increases type I and type III pro-collagen messenger RNA levels in human skin fibroblasts. J Invest Dermatol. 1988;90:420–424. 15. Raschke T, Koop U, Dusing HJ, et al. Topical activity of ascorbic acid: From in vitro optimization to in vivo efficacy. Skin Pharmacol Physiol. 2004;17:200–206. 16. Fitzpatrick RE, Rostan EF. Double-blind, half-face study comparing topical vitamin C and vehicle for rejuvenation of photodamage. Dermatol Surg. 2002;28:231–236. 17. Humbert PG, Haftek M, Creidi P, et al. Topical ascorbic acid on photoaged skin: clinical, topographical and ultrastructural evaluation: Double-blind study vs. placebo. Exp Dermatol. 2003;12: 237–244. 18. Traikovich SS. Use of topical ascorbic acid and its effects on photodamaged skin topography. Arch Otolaryngol Head Neck Surg. 1999; 125:1091–1098. 19. Katayama K, Armendariz-Borunda J, Raghow R, et al. A pentapeptide from type procollagen promotes extracellular matrix production. J Biol Chem. 1999;268:9941–9944. 20. Foldvari M, Attah-Poku S, Hu J, et al. Palmitoyl derivatives of interferon alpha: potent for cutaneous delivery. J Pharm Sci. 1998; 87:1203–1208. 21. Robinson L, Fitzgerald N, Doughty DG, et al. Topical Palmitoyl Pentapeptide provides improvement in photoaged human facial skin, Int J Cosmet Sci. 2005;27:155–160. 22. Lintner K, Mas-Chamberlin C, Mondon P. Pentapeptide facilitates matrix regeneration of photoaged skin. Ann Dermatol Venereol. 2002;129:1S401. 23. Pickart L. Copperceuticals and the skin. Cosmet Toilet. 2003;118: 24–28. 24. Smith-Mungo LL, Kagan HM. Lysyl oxidase: Properties, regulation and multiple functions in biology. Matrix Biol. 1998;16:387–398. 25. Canapp SO, Farese JP, Schulz GS, et al. The effect of topical tripeptidecopper complex on healing of ischemic open wounds. Vet Surg. 2003;32:515–523. 26. Buffoni F, Pino R, Dal Pozzo A. Effect of tripeptide-copper complexes on the process of skin wound healing and on cultured fibroblasts. Arch Int Pharmacodyn Ther. 1995;330:345–360. 27. Blanes-Mira C, Clemente J, Jodas G, et al. A synthetic hexapeptide (Argireline) with antiwrinkle activity. Presentation, 37th Annual Conference of the Australian Society of Cosmetic Chemists, Queensland, 2003, March 13–16.
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28. Kruger N, Fiegert L, Becker D, et al. For the treatment of skin aging: Trace elements in form of a complex of copper tripeptide. Cosmet Med. 2003;24:31–33. 29. Cole CA, Bertin C. Dimethylaminoethanol: A new skin-care ingredient for aging skin. In: Baran R, Maibch HI (eds) Textbook of Cosmetic Dermatology, 3rd ed. Abingdon: Taylor & Francis, 2005, pp. 95–101. 30. Nagy I, Floyd RA. Electron spin resonance spectroscopic demonstration of the hydroxyl free radical scavenger properties of dimethylaminoethanol in spin trapping experiments confirming the molecular basis for the biological effects of centrophenoxine. Arch Gerontol Geriatr. 1984;3:297–310. 31. Morissette G, Germain L, Marceau F. The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology. Br J Derm. 2007;156:433–439. 32. Uhoda L, Faska N, Robert C, et al. Split-face study on the cutaneous tensile effect of 2-dimethylaminoethanol (deanol) gel. Skin Res Technol. 2002;8:164–167. 33. Levy SB. Kinetin. In: Baran R, Maibach HI (eds) Textbook of Cosmetic Dermatology, 3rd ed. Abingdon: Taylor & Francis, 2005, pp. 129–132. 34. Rattan SI, Sodagam L. Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro. Rejuvenation Res. 2005;8:46–57. 35. Chiu P-C, Chan C-C, Lin H-M, et al. The clinical anti-aging effects of topical kinetin and niacinamide in Asians: a randomized
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double-blind, placebo-controlled, split-face comparative trial. J Cosmet Derm. 2007;6:243–249. McCullough JL, Garcia RL, Reece B. A clinical study of topical pyratine 6 for improving the appearance of photodamaged skin. J Drugs Derm. 2008;7:131–135. Lu L, Ying K, Wie SM, et al. Asiaticoside induction for cell-cycle progression, proliferation and collagen synthesis in human dermal fibroblasts. Int J Dermatol. 2004;43:801–807. Yarosh DB, Both D, Brown D. Liposomal ursolic acid (Merotaine) increases ceramides and collagen in human skin. Horm Res. 2000;54: 318–321. Martelli L, Berardesca E, Martelli M. Topical formulation of a new plant extract complex with refirming properties: Clinical and noninvasive evaluation in a double-blind trial. Int J Cosmet Sci. 2000;22: 201–206. Passi S, DePita O, Grandinetti M, et al. The combined use of oral and topical lipophilic antioxidants increases their levels both in sebum and stratum corneum. Biofactors. 2003;18:289–297. Stab F, Wolber R, Blatt T, et al. Topically applied antioxidants in skin protection. Methods Enzymol. 2000;319:465–478. Hoppe U, Bergemann J, Diembeck W, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. Biofactors. 1999;9:371–378. Yu RJ, Van Scott EJ. Alpha-hydroxyacids, polyhydroxyacids, aldobionic acids and their topical actions. In: Baran R, Maibach HI (eds) Textbook of Cosmetic Dermatology, 3rd ed. Abingdon: Taylor & Francis, 2005, pp. 77–93.
105 Cosmetic Surgery in the Elderly Dwight Scarborough . Kimberly M. Eickhorst . Emil Bisaccia
Introduction Aging is a dynamic, biological process of tissue involution and evolution. In the skin, these physiologic and morphologic changes occur throughout many tissue layers. With time, both the epidermis and the dermis become thin. However, in the facial region, changes in the dermis, supporting tissues, adipose, and superficial muscular aponeurotic system (SMAS) are the most marked ones. It is specifically the alterations in the reticular cutis, the structures that lie between the skin and the SMAS (unique to the facial and neck regions), that most significantly contribute to the clinical signs of facial aging. From an etiologic perspective, there are two categories of aging: extrinsic and intrinsic. Extrinsic changes result in pigmentary and textural differences that tend to leave the skin with blotchy discoloration. These types of skin changes are environmentally linked to ultraviolet radiation, oxidative damage, exposure to the elements, and smoking. In contrast, intrinsic changes are secondary to dermal, bone, and fat remodeling and are inevitable. Intrinsic changes are dependent on genetic and hormonal influences and are apparent in the sagging skin, rhytids, and subdermal atrophy seen in the elderly. Changes in the reticular cutis of the face exemplify intrinsic aging. Many fibrous septae tightly connect the SMAS to the dermis and serve as a scaffold for the overlying skin. However, with time, intrinsic alterations in collagen, elastin, and ground substance cause the reticular cutis to become more compact and the integrity of these connections is lost. As a consequence, the dermis is more lax and simply drapes the facial musculature rather than closely adhering and enveloping the underlying facial structures. These changes, in addition to the reduction in bony skeletal mass, lost facial muscular tone, and protrusion of fat pads result in less structural support for the skin. Thus, the skin tends to hang off the face creating signs of gauntness, laxity, and rhytids. Clinically, aging skin often appears ‘‘tarnished’’ with dyspigmentation, laxity, a yellow hue, wrinkles, telangiectasias, and a leathery appearance, as well as potential cutaneous malignancies and scars from their removal. But, on a microscopic level, aging skin begins to lose the
orderly maturation of keratinocytes and melanocytes, Langerhan cells decrease in number, the dermis thins as it loses glycosaminoglycans and type I and III collagens, and disorganized, abnormal collagen and elastin replace their predecessors [1]. Regardless of the exact etiologic mechanism of aging, it is complex and variable. This wide range of complexities and variabilities is echoed in the potential dermatologic interventions that can be employed to correct or reverse the signs of aging. Dermatologic options for such revitalization range from nonsurgical modalities (Botox, filler substances, and nonablative lasers) to more aggressive resurfacing procedures (chemical peels, dermabrasion, and ablative lasers)and to more traditional, surgical procedures (liposuction, blepharoplasty, and various types of face and neck lifting). The application of these revitalizing tools, along with their associated risks and expected outcomes, are explored over the course of the chapter.
Botox Surgical options for rejuvenation continue to improve with increased numbers of minimally invasive procedures that are becoming available. However, many patients, either due to preexisting medical conditions or pure concern over associated surgical risks, desire aesthetic correction from a nonsurgical approach. Aging of the upper face can be defined by varying degrees of forehead laxity, brow ptosis, horizontal creases in the mid forehead, and deepening glabellar furrows. For these patients, cosmetic injectables are simple modalities that can safely and efficaciously improve one’s appearance. Most wrinkles and undesirable facial lines are worsened by continued muscle movement. The hypertonic condition of the underlying muscular structure can render a harsh look to the face. Botox, a neurotoxin, can inhibit muscular contraction and diminish the excess, undesirable lines that communicate fatigue and/or negative expressions. Approved by the Food and Drug Administration (FDA) in 2002 for cosmetic treatment of glabellar lines, botulinum toxin type A (BTX-A), Botox, has become widely available for facial rejuvenation. By inhibiting muscular contraction,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_105, # Springer-Verlag Berlin Heidelberg 2010
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Botox helps to reduce evidence of dynamic lines and correct facial asymmetry. Its appeal is largely due to its impressive safety profile in the appropriate trained professional’s hands, lack of downtime, and relatively quick onset of action. Botulinum toxin A is a naturally occurring exotoxin produced by Clostridium botulinum, which ultimately inhibits acetylcholine release and prevents local neuromuscular transmission. Botox is also used ‘‘off-label,’’ or without the official approval of the FDA, for several other cosmetic indications. For example, horizontal forehead lines, ‘‘crow’s feet,’’ ‘‘bunny lines,’’ chin dimpling, and platysmal bands are safely corrected by Botox [2]. The toxin binds to the receptor sites on motor nerve terminals and blocks neuromuscular conduction by inhibiting the release of acetylcholine. More specifically, BTX-A facilitates the cleavage of synaptosomal-associated membraneprotein (SNAP)-25, which is required for exocytosis of acetylcholine. BTX-A has not been without competition. There are seven subtypes of botulinum neurotoxin (BTX-A through BTX-G). Type A is currently the most effective for human use, although BTX-B has also become available. Dysport (BTX-A), currently used only outside the United States, and Myobloc (BTX-B), approved by the FDA in 2000 only for the treatment of cervical dystonia, are not currently FDA-approved for cosmetic use. It should be noted that all three of these toxins have completely different profiles in relation to manufacturing, action at the muscular junction, and potency and therefore should not be used interchangeably. For example, Dysport is known to diffuse farther away from injection sites, whereas Myobloc is much more painful on injection, compared to BTX-A toxins, due to its higher acidity. Myobloc also has a shorter duration of effectiveness [3]. Each vacuum-dried vial of Botox contains 100 U of (mouse) toxin, 0.5 mg of ‘‘human’’ albumin, and 0.9 mg of sodium chloride in sterile form, without preservative. Different physicians choose to dilute the Botox to different concentrations. Preserved saline dilution tends to be less painful upon administration. Once the Botox product is reconstituted, it should be stored at 28 C. Degree of dilution can alter the manner in which the physician chooses to deliver the Botox. While higher doses of BTX-A delivered in smaller volumes (50 or 100 units/mL) keep the effects more localized and enable precise delivery of the toxin with little diffusion, smaller doses in larger volumes (5–10 units/mL) may generate more widespread effects [4, 5]. The effects of BTX-A at the neuromuscular junction are evidenced in about 1–2 weeks and last for about 3–4 months. Clinically, however, patients may reap the benefit of relaxed skin tension lines
for up to 6 months, depending on the patient and treated location [6]. Botox injection is ideal in the upper face (> Figs. 105.1 and > 105.2). Men, in comparison to women, usually require a greater amount of the toxin to achieve the same result. Most commonly used on the forehead, crow’s feet area, and glabella, Botox paralyzes the dynamic facial muscles and helps to decrease brow furrows, while widening the eyes. When used on the lower face, it can be especially helpful in correcting facial asymmetry. Other areas, such as the ‘‘bunny lines’’ and platysmal bands of the lower face, have also been treated with success; however, even the well-trained and experienced physicians with a thorough understanding of facial anatomy should proceed with caution in these areas. Dysphagia is a muchfeared complication of platysmal band treatment. As a Category C drug, Botox is not recommended for pregnant or breast-feeding women. Other contraindications include therapy with aminoglycosides or acetylsalicylic acid prior to treatment, as well as certain neuromuscular conditions (i.e., Eaton-Lambert, myasthenia gravis, amyotrophic lateral sclerosis, etc.). These conditions may potentiate the effects of Botox. Temporary side effects include bruising, headache, pain at injection
. Figure 105.1 Pre-Botox
. Figure 105.2 Post-Botox
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site, asymmetry, muscle twitching, numbness, eyebrow or eyelid ptosis, and double vision. The most commonly encountered complications from Botox include an overtreated frontalis, dropped brow, asymmetry, and bruising, particularly in the lateral canthus [3]. These complications are best avoided by injecting 1 cm or more from the bony orbital rim and injecting at or above the mid brow. Requesting that the patient should remain upright for about 2 h after the procedure, while avoiding any manipulation to the treated areas, may also help in minimized unwanted side effects. Newer trends with Botox emphasize the need for decreased number of toxin units in the forehead and greater amounts in the crow’s feet region. These new recommendations are based on a finding that 20 or more units of Botox in the forehead is more likely to lead to suboptimal outcomes [3]. There has also been an increased desire to couple Botox with other nonsurgical fillers to achieve not only a more relaxed look, but also a smoother, fuller, and more youthful appearance.
Fillers BoNTA remains the cornerstone of treatment in rejuvenation of the upper face. But its effects are only twodimensional. By ‘‘volumizing’’ the face with injectable fillers a more three-dimensional outcome can augment results achieved from Botox and maximize cosmesis. Studies have shown that BoNTA and hyaluronic acid filler, in combination, synergistically improve the appearance of the lower face, in comparison to use of either injectable alone [3]. Fillers are therefore central to the midface revitalization, due to their ability to restore lost volume throughout the aging process. As another nonsurgical modality, fillers are also very attractive to the aging population. Instantly gratifying results and minimal downtime after administration make these product a win-win among patients. The ‘‘ideal’’ filler that maximizes longevity and ease of delivery and minimizes downtime and side effects has yet to be developed. But the discussion that follows highlights some of the more commonly used fillers and their associated indications and challenges. Fillers are either biodegradable (12–18 months), slowly biodegradable (2–5 years), or permanent. Some of the more commonly used injectables can be broadly categorized into three main groups based on their duration: (1) temporary biodegradables (autologous fat, human collagen, bovine collagen, and hyaluronic acid), (2) longlasting biodegradables (calcium hydroxylapatite, poly-Llactic acid), and (3) permanent (polymethylmethacrylate).
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While dermal fillers tend to correct fine lines, more permanent injectables are placed in the subdermis and reserved for deeper furrows and longer-lasting volume restoration. Some fillers also have a secondary effect on volume enhancement, in that they additionally stimulate collagen neogenesis. Unfortunately, the advantage of longer-lasting fillers is often offset by their tendency to create tissues reactions, such as granulomas and extrusion. The permanency of these products may also not adequately address changes in the dynamically aging face, resulting in an unnatural look with the progression of time. In choosing the appropriate filler for a given individual there are many considerations. These include antigenicity of the injectable material and need for prior skin testing, duration of action, ease of delivery, number of sessions required to achieve desired degree of correction, product longevity, and overall side-effect profile. In choosing the appropriate filler, the patient’s skin quality and age must also be taken into account; older patients may require a greater amount of a particular injectable product to attain results similar to those of a younger patient.
Temporary Biodegradable Fillers Historically, autologous fat has provided safe, inexpensive volume replacement. Cosmetic areas that lend themselves to treatment with fat transplantation included deepened nasolabial folds, the fallen and receding upper lip with radial perioral lines, sunken cheeks, and transverse forhead creases. The dorsal hands also are an ideal location for use of autologous fat (> Figs. 105.3 and > 105.4). However, due to the multistep nature of tissue harvest and implantation, as well as controversy over the effective, longevity of fat transplantation, this filler has become a less ideal option for controlled volume replacement. Recent trends in temporary biodegradable fillers include use of either collagen derivatives (human or bovine) or hyaluronic acid. These products are injected into the superficial and mid-dermis to treat finer rhytides. Bovinederived injectables (Zyderm1 and Zyplast1) require two pretreatment allergy tests to screen for hypersensitivity reactions, whereas, human-derived collagens (Cosmoderm1 and Cosmoplast1) require none. The reported rate of bovine-collagen sensitivity reactions ranges from 1% to 2% and injection-related events include swelling, redness, itching, nodule formation, granulomas, and abscesses [7]. Therefore, the type III and I collagens that compose Cosmoderm and Cosmoplast, approved by the FDA in 2003, clearly offer a safety benefit
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. Figure 105.3 Pre-fat transfer
. Figure 105.5 Pre-collagen
. Figure 105.4 Post-fat transfer
. Figure 105.6 Post-collagen
over bovine collagens. All of these products also contain some amount of local anesthetic as part of their suspension. The first generation collagens (Zyderm1 and Cosmoderm1) are quickly degraded. So a second generation of collagen products (i.e., Zyplast1, Cosmoplast1) was developed to increase longevity and allow for deeper implantation. These second-generation fillers are crosslinked to compounds such as glutaraldehyde, to produce a more durable product, ‘‘erasing’’ mild wrinkles for up to 3–12 months [7] with no difference in duration between bovine and human products [8] (> Figs. 105.5 and > 105.6). But, most recent to arrive in the temporary filler market is the newest generation of collagen-based injectables, Evolence1. Evolence1 is a porcine tendon, collagen-derived, highly purified dermal filler (Dermicol-P35) that has low immunogenicity and promises increased durability for up to 1 year [9]. Glycation, a unique, ribose-induced
collagen cross-linking step, is used to strengthen the porcine collagen, without use of glutaraldehyde. Therefore, this filler more closely mimics the natural cross-linking of collagen by the body and hypersensitivity pretesting is not necessary [8]. As with the aforementioned collagen fillers, it can be injected into the mid-dermis with a linear threading technique. Use of Evolence1 is best avoided in the lips and periorbital regions due to risk of nodule formation [8]. Cross-linked or stabilized hyaluronic acid (HA) is currently the most used resorbable filler [10]. In the USA, there are currently eight HA dermal fillers approved for commercialization by the FDA [11]. HA fillers are so appealing due to their low immunogenicity and rare, limited side effects (i.e., bruising). This is largely due to HA’s biocompatibility across species. It can be produced from animal origin and rooster combs. However, it is mostly extracted from a nonpathogenic bacterium, Streptococcus equi, as in Restylane1 [10] (> Figs. 105.7 and > 105.8).
Cosmetic Surgery in the Elderly
This product must be correctly placed intradermally. If placed too high above the dermis, bumpy bluish nodules may be seen. If placed too deep into the dermis, durability is sacrificed. The most feared complication is vascular injection leading to vascular occlusion and necrosis [7]. Retrograde injection helps to minimize this
. Figure 105.7 Pre-restylane
. Figure 105.8 Post-restylane
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risk. Although the different HA products vary in longevity, one of the newer HA’s to market, Juvaderm1, was shown to have treatment efficacy for up to 12 months [3] (> Figs. 105.9 and > 105.10).
Long-Lasting Biodegradable Fillers Calcium hydroxylapatite (Radiesse1) and poly-L-lactic acid (Sculptra1) represent devises injected below the dermis, which are more appropriate for moderate to severe rhytides. These products offer the advantage of new collagen production, in addition to the increased volume created by the injectable itself. Radiesse1 promises improvement of deeper furrows and creases for about 1 year, whereas response to Sculptra1 can last for more than 2 years [7]. A cross-hatched threading technique is unique to the delivery of Sculptra1. Caveats regarding Radiesse1 include not placing the product in the lips due to higher reports of granulomas formation. Similarly, caution is advised with Sculptra1, which usually requires subsequent, touch-up injections. Once Sculptra1 is initially placed, dermal thickness gradually increases over a period of about 1 month. Therefore, care should be taken not to overcorrect for volume during initial administration of poly-L-lactic acid. Posttreatment massage is recommended for both products to ensure even distribution.
. Figure 105.9 Pre-juvederm
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. Figure 105.10 Post-juvederm
Permanent Fillers It should be noted that no filler will yield truly permanent correction of wrinkles, due to the ongoing forces of gravity and senescence at work in the skin. However, this class of fillers is composed of materials that are more permanent and maintained within the skin for prolonged periods. Artefill1 is a dual-acting injectable filler. Composed of 20% polymethylmethacrylate (PMMA) microspheres, and 80% purified bovine collagen with lidocaine, this suspension is injected into the sub- or deep dermis to treat deep facial rhytides. Unlike Sculptra1, which is a more optimal global volume filler, Artefill1 is reserved for treatment of defined lines or scars. Due to its bovine collagen component, allergy testing is required by the FDA. In addition to the pure volume of the injected suspension, the PMMA microspheres within the Artefill1 induce a delayed foreign body response, which results in deposition of new collagen. So, although the initial correction of the product slowly decreases as the bovine collagen is resorbed over a 1–3-month time period [8], 50–75% of the correction is maintained long term, due to the permanence of the PMMA microspheres. Two to four treatments at intervals of about 3–4 months are often needed for complete correction [7]. Disadvantages associated with Artefill’s1 PMMA predecessors included granuloma formation thought to be primarily due to material
impurities. But Artefill’s1 improved purification and reconstitution process has lowered granuloma formation [12]. The relative advantages of Artefill are (1) unique microsphere technology providing a complete smooth surface of the microspheres, (2) indications similar to those of collagen and hyaluronic acid, (3) ease of injection despite higher viscosity than collagen alone, (4) permanent stimulation of connective tissue and collagen deposition, (5) long-lasting aesthetic effect over many years, and (6) a low rate of granulomas formation similar to collagen and hyaluronic acid injections (12). However, since the consequence of granulomas formation can be severely disfiguring, caution must be exercised. So, whether the physician’s aim is to correct fine or deep wrinkles or replace lost volume in certain cosmetic units of the face, there are a variety of nonsurgical options to rejuvenate the aging face. The ‘‘art’’ in choosing the most appropriate injectable ‘‘media’’ for the composition at hand is what separates a nonsurgical outcome from an aesthetically pleasing cosmetic result. Fillers can be utilized alone or in combination with one another, as well as with other rejuvenating techniques, to achieve impressive changes in appearance.
Chemical Peels Decreasing the visibility of dynamic rhytides with Botox and replenishing facial areas of lost volume with fillers, only partially corrects the many signs of photodamaged skin. Textural and pigmentary changes also contribute to an aged look, and need to be addressed. On a histologic level, these changes are characterized by decreased microcirculation, elastosis, epidermal atrophy, cellular atypia, and preneoplastic dysplasia [13]. Chemical peels, classified by their depth of skin penetration (superficial, medium, deep) have long been used to address these changes. Depending on the chemical agent’s depth of penetration, a skin wound is created during the peeling process. Superficial peels penetrate to the epidermis. Medium depth peels create both epidermal and dermal injury. And deep peels wound the skin to the area of the deep papillary or reticular dermis. Re-epithelialization is then driven by the remaining, uninjured adnexal structures, which generate cellular division and differentiation. Within 24 h of a chemical peel, these epidermal appendages, located primarily in the remaining dermis, begin the production of ‘‘new skin’’ and the entire process is usually complete in 7–10 days [14]. Overall, chemical peels create a thinner, more compact stratum corneum, a thicker acanthotic epidermis without atypia, and a uniform
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dispersion of melanin [14]. Additionally, as the skin regrows after injury, new collagen and glycosaminoglycans are produced. Although these added components are most pronounced in deeper peels, they result in increased skin elasticity and volume, leading to tighter and firmer skin, which minimizes the appearance of wrinkles [14]. Depending on the histologic depth of the photodamaged change, the appropriate level of peel is chosen to reverse those changes; the peel must penetrate to the histologic depth of the skin abnormality. For example, superficial peels treat textural skin changes, active acne, actinic keratoses, and superficial dyschromias confined to the epidermis. Medium depth peels are reserved for solar lentigines, multiple solar keratoses, fine wrinkles, or acne scars. In addition, although the deep peel has largely fallen out of favor due to its side-effect profile, deep peels are primarily indicated for coarser wrinkles and premalignant skin tumors [15]. Of note is that nevi, dermal and mixed melasma, dermal and mixed postinflammatory hyperpigmentation, and seborrheic keratoses respond poorly to superficial and medium depth peels [14].
Superficial Peels Superficial peels exfoliate the skin and are primarily used to treat acne and its associated hyperpigmentation, as well as photodamaged (actinic keratoses, poor skin texture, superficial dyschromia) skin. Due to minimal depth of penetration, a serial number of sessions is usually needed to accomplish the desired goal. Superficial peels include alpha-hydroxy acid peels such as glycolic acid (up to 70%), Jessner’s solution (resorcinol, lactic acid, and salicyclic acid in ethanol), trichloroacetic acid (TCA) (10–30%), salicylic acid, and retin A peels [14, 15]. In contrast to the many superficial peels, which self-neutralize, the alpha-hydroxy acid peels (i.e., glycolic acid) require an alkaline neutralizing agent, such as sodium bicarbonate, to terminate the peel’s destructive action.
Medium Depth Peels Various strengths of trichloroacetic acid (TCA) peels exist, ranging from 25% to 100%. However, TCA 35% is considered a medium depth peel. Other medium depth agents include glycolic acid 70% applied for a more prolonged duration, TCA (35–50%), carbon dioxide plus TCA, Jessner solution plus TCA 35%, and glycolic acid 70% plus TCA 35%. Due to associated dermal penetration, these peels help improve mild to moderate mobile
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rhytides around the eyes and on the cheeks. Deeper, fixed, perioral, and nasolabial rhytids will not respond well. Melasma and more severe postinflammatory hyperpigmentation may improve, but results are very variable.
Deep Peels Historically, phenol-based peels were reserved for patients with dyschromia, fine and coarse wrinkles, premalignant skin tumors, and severe acne scars. Phenol-based agents not only penetrate to the midreticular dermis, but also create significant production of new collagen [15]. Unfortunately, phenol is directly toxic to the myocardium. Cardiac arrhythmias have been recorded in up to 23% of patients who underwent full-face peel in less than 30 min [16]. Additional side effects include prolonged or permanent erythema, hypopigmentation, and porcelain scarring. Therefore, due to its toxicity, need for full cardiopulmonary monitoring with intraveneous hydration throughout the procedure, and prolonged recovery period with variable outcome, deep peeling has fallen out of favor. In instances in which deep peeling is required, it is the author’s preference to turn to the carbon dioxide (CO2) laser. The CO2 laser allows for less risk and more controlled results. Overall, the risks associated with chemical peeling increase proportionately to the increased depth of the peel. Broadly categorized, postpeel complication include infection, altered pigmentation, and scarring. Bacterial, viral, and candidal infections are also possible, especially if skin peels prematurely and exposes non-re-epithelialized skin. Acneiform eruptions and milia may also follow a peel. Prior to the peel, patients should always be asked about a history of herpes simplex. Luckily, all of these infections can be easily treated with the appropriate topical and oral regimens. Postinflammatory pigmentation, most common in darker skin types, can occur immediately or months following a peel. Pigmentation changes are often precipitated by premature sun exposure. Bleaching agents such as hydroquinone and retinoids may aid in improving unwanted pigmentary alterations. However, prolonged postpeel erythema (>3 months) is a rare complication that can occur with any depth of peel. Associated scarring can be either hypertrophic or atrophic. The most common area of scarring is the lower face [15], especially the mandible. But, the periorbital region is also at risk. To avoid this complication, it is best to inquire pre-procedure as to whether the patient has a history of scar formation or isotretinoin use in the last 18 months. Regardless, should scarring result after a
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peel, it is imperative to quickly intervene with high potency topical or intralesional steroids. Q-switched pulse dye laser treatments may also serve as a necessary adjunct. Chemical peels are an economical, readily available, and safe alternative to many other types of resurfacing modalities. The choice of peel is customized based on the desired treatment skin ‘‘target’’ and offers the advantage of minimal downtime. Used alone or in combination with other resurfacing and rejuvenating technique, peels are another great option in helping to refresh the skin’s appearance.
Microdermabrasion An alternative to chemical resurfacing of the skin is microdermabrasion. On par with a superficial chemical peel, this revitalizing tool enables chemical-free, partial skin ablation, or ‘‘skin-polishing’’ with a negative pressure device, which delivers debrading particles. A compressor and an aspirator are the essential components of the system. Aluminum oxide crystals or sodium chloride salt are projected from a reservoir via a tubing system and handpiece onto the patient’s skin. These crystals and loosened skin debris are evacuated from the treatment surface into a second tubing system and finally deposited in another closed container, thereby preventing contamination. In comparison to the open microdermabrasion techniques of the past, today’s dermabrasion is a closed-loop vacuum-assisted abrasive procedure, harnessing the physical action of inert crystals to exfoliate the skin. The closedloop system helps to ensure operator safety from possible viral released particles like herpes simplex virus and HIV. The operator can also adjust the device settings including vacuum pressure, speed of crystals, particle size, angle of impaction, number of passes, and speed of movement of the probe. These adjustments allow for ablation of different depths, as needed to accomplish the cosmetic goal. Because microdermabrasion creates partial skin ablation, the skin responds to this injury by stimulating epidermal turnover and producing new collagen. Various studies reviewing the resultant histologic changes have reported stratum corneum thinning and homogenization immediately following microdermabrasion. More chronic changes demonstrate an increase in epidermal thickness, flattening of the rete pegs, improvement in loss of polarity, liquefaction of basal cells, and hyalinization of papillary dermis [17]. Indications for microdermabrasion run the gamut: fine rhytids, photoaging, active acne, mild acne scarring, comedones, dyschromia, correction of enlarged pores,
stretch marks, tattoo removal, and scar revision. Microdermabrasion has also become particularly useful in blending cosmetic transition zones in areas previously treated with laser. Treating the photoaged neck in patients who have previously undergone facial laser resurfacing helps diminish the often pronounced contrast between the rejuvenated facial skin and the untreated neck without adverse sequela. It may take 5–7 monthly microdermabrasion treatments to blend an area on the lower two-thirds of the neck with an area already treated by CO2 laser [18]. The overall effect of the procedure is a subtle, smoothing of the skin. Advantages of microdermabrasion include that it is chemical-free, and in contrast to peels, lasers, and dermabrasion, it can be used on any skin type with few complications or morbidity. Additionally, the technique is relatively bloodless, does not require anesthesia, requires very little downtime, and can be customized to individual patients due to its high degree of operator control. On the other hand, microdermabrasion should be avoided in those with active skin infection or rosacea. Microdermabrasion can trigger a rosacea flare, which may result in permanent, unfavorable change to the skin. Furthermore, the procedure is not without risk of pigment streaking and hyperpigmentation, persistent erythema, herpes simplex activation, or ocular complications due to the entrance of crystals into the cornea. Prior to the procedure it is important to specifically ask the patient about his/her use of antioxidants like vitamin E and ginko biloba, as well as anticoagulants or any other antiplatelet agent (including aspirin). These medications can increase posttreatment edema and erythema, and even lead to petechiae. Patients should also be advised to discontinue any alpha-hydroxy acid or retinoid-containing products about 2 days prior to the procedure. Continued use of these topical regimens can lead to a brisk and more unpredictable response to treatment. During microdermabrasion a patient can expect to experience mild and limited discomfort. If too great, the level of discomfort can easily be decreased by adjusting the apparatus settings. Mild erythema, the usual procedure endpoint, and tingling may be experienced after the procedure. In general, most patients feel their skin to be smoother and less mottled after microdermabrasion [17]. However, deep rhytids, melasma, deep scars, telangiectasias, and actinic keratoses will not disappear. Microdermabrasion is an effective and nonsurgical solution for younger, smoother looking skin. Although its results may not be as dramatic as other resurfacing techniques, it has the advantages of little to no discomfort, no required anesthesia, few complications, and minimal to no recuperation time. It is a simplistic resurfacing tool,
Cosmetic Surgery in the Elderly
whether used alone or in conjunction with other resurfacing techniques, which can grant facial rejuvenation to the patient.
Dermabrasion Dermabrasion differs from microdermabrasion in that a wire brush or diamond fraise creates a wound that penetrates more deeply into the papillary dermis. Subsequently, re-epithelialization occurs via the still vital underlying adnexal structures. Thus, dermabrasion supersedes microdermabrasion, when attempting to treat deeper, facial defects. Due to decreased vascular supply and decreased numbers of adnexal structures on nonfacial areas, dermabrasion has an increased tendency to leave a scar if used in areas other than the face. Dermabrasion alters primary scar formation by creating a repair zone of new, more organized collagen within the papillary dermis. New collagen exhibits an increase in collagen bundle density and size, with unidirectional orientation of collagen fibers that tend to be more parallel to the epidermal surface. Histologically, there appears to be an upregulation of tenascin expression throughout the papillary dermis and the expression of alpha-6/beta-4 integrin subunit on the keratinocytes throughout the stratum spinosum [19]. Clinically, these changes result in more evenly contoured skin (> Figs. 105.11 and > 105.12). In contrast to lasers that thermally ablate the skin, dermabrasion is a ‘‘cold’’ form of ablation that minimizes vascular stimulation throughout the healing phase, allowing for less intense, postoperative erythema that is quicker . Figure 105.11 Pre-dermabrasion
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to resolve in some cases [20]. Although there are numerous uses for this resurfacing technique (> Table 105.1), actinically photodamaged skin, wrinkles, superficial scarring (acne and variolliform), and surgical scar revision are . Figure 105.12 Post-dermabrasion
. Table 105.1 Indications for dermabrasion Indications for dermabrasion Scarring Acne scarring Surgical scars/posttraumatic scars Pigmentary lesions Congenital pigmented nevi Lentigines Tattoo Epithelial-derived growths Actinic keratoses Seborrheic keratoses Growths with dermal components Angiofibromas Neurofibromas Syringomas Trichoepitheliomas Xanthelasma Others Photoaging-solar elastosis-rhytids Resistant acne Rhinophyma
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the more common indications. Of note is that burn scars do not respond to dermabrasion, as they lack the necessary revitalizing adnexal structures. Additionally, patients with pigmentary conditions such as melasma, encounter problems with recurrence, just as with many other resurfacing procedures. Interestingly, despite major resurfacing advances in the realm of lasers, many still find dermabrasion to be an excellent option for treating acne scars. However, others will argue that the CO2 ablative laser, which is operated in a bloodless field, is superior. Regardless, assessing depth of acne scarring is critical as to whether either technique will result in substantial improvement. Pigmentary irregularities, tattoos, and actinic keratoses may also improve with dermabrasion. But more efficacious lasers and topical treatments have largely replaced the use of dermabrasion in treating these entities. Clearly, dermabrasion (as with aggressive laser resurfacing discussed in the next section) should be contraindicated for any patients with decreased dermal appendageal structures, such as those who have recently taken isotretinoin, or with a history of radiation to the treatment area. These predisposing conditions significantly increase the potential for scarring. With specific regard to isotretinoin, the recommended delay before undergoing dermabrasion is 6–18 months. However, poor wound healing has occurred in isotretinoin patients even 38 months post-drug. Therefore, it is recommended to delay dermabrasion after isotretinoin for as long as possible. Other concerns regarding the treatment are activation of herpes simplex, spread of existing verruca vulgara, keloidal, or hypertrophic scarring, and post-procedure solar-induced pigmentary change. Prior to the procedure, it is also important to document bleeding time and platelet counts, as well as HIV and hepatitis status. Regardless, universal precautions should always be taken for every dermabrasion procedure with all persons present in the operating suite wearing protective gear including face masks, face shields, surgical gowns, and gloves. Dermabrasion is a valuable tool in the surgeon’s resurfacing armamentarium, when a skilled operator is paired with an optimal candidate. Although the list of resurfacing options continues to lengthen with the development of newer laser technology, dermabrasion remains particularly effective for the treatment of facial acne scarring, rhytids, and scar revision.
Lasers Laser technology dates back to Einstein’s study of quantum mechanics in the early twentieth century. But,
light amplification stimulated by emission of radiation (LASER) has drastically evolved since its inception. Today the theory of selective photothermolysis is utilized by cosmetic lasers to directly treat specific targets in the skin, resulting in a more rejuvenated appearance. By selecting a laser with the appropriate wavelength, pulse duration, and fluence, certain unwanted skin changes (i.e., increased melanin and telangiectasias) can be targeted and ‘‘erased,’’ while other favorable skin components, such as collagen, can be induced. A vast array of cosmetic lasers is currently available. However, before profiling different types of lasers, it is important to understand some key laser principles. The physical properties of laser light include the following: monochromacity, coherence, and collimation. Monochromacity refers to the ability of laser light to selectively target chromophores with a corresponding single wavelength. Melanin, hemoglobin, and water are the three intrinsic skin chromophores, or laser ‘‘targets.’’ Coherence defines the relationship of individual laser light waves as they travel next to one another with respect to both time and space. Collimation is described as the ability of lasers to transmit parallel rays of light without divergence and loss of intensity, despite increasing distance. As described by Parrish and Anderson, the theory of selective photothermolysis states that a specific chromophore can be selectively targeted (with a specific wavelength) and destroyed with minimal damage to surrounding tissues. This is usually best accomplished with delivery of energy using a pulse duration that is less than or equal to the thermal relaxation time of the chosen target tissue. Thermal relaxation time is defined as the amount of time needed for the target tissue to lose 50% of its incident heat, by diffusion to surrounding tissue. The first lasers that came to the cosmetic market in the 1980s and 1990s were ablative lasers, namely the carbon dioxide (10,640 nm) and erbium-doped yttrium aluminum garnet (Er:YAG) (2,940 nm) lasers. This class of lasers, through superficial destruction of tissue, clinically enabled resurfacing of unwanted solar damage and scarring by targeting intra and extracellular water. By vaporizing epidermis with continued destruction to the papillary dermis, extrinsic skin changes could be removed allowing for reepithelialization of the skin. Most patients with wrinkles were able to attain a 50–90% improvement after treatment with this subset of lasers [21] (> Figs. 105.13 and > 105.14). But, despite impressive postoperative results, the procedure was painful and required about 2 weeks of downtime with frequent and unpredictable risks such as erythema, dyspigmentation, acne eruptions, infections, and scarring. These ablative laser disadvantages then drove the demand
Cosmetic Surgery in the Elderly
. Figure 105.13 Pre-CO2 laser
. Figure 105.14 Post-CO2 laser
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redness and telangiectasias. The infrared lasers improve texture. Intense pulsed light devices improve both vascular imperfections and brown discoloration, as well as skin texture [22]. Regardless of these advancements, discontent arose over the lack of drastic improvement attained by the nonablative lasers, and the requirement of a series of treatments prior to any noticeable skin enhancement. Thus, a new technology was embraced: fractional resurfacing. This laser system is a ‘‘compromise’’ between the dichotomous poles of ablative and nonablative lasers. Fractional resurfacing entails thermally ablating about 15–25% of a skin surface during a treatment while sparing the remaining skin surface. The thermally ablated, vertical columns of skin, which are about 70–150 mm in width, are evenly distributed over the treatment area [21]. As these microthermal zones repair and heal after ablation, they can draw upon the neighboring spared tissue for fibroblasts and epidermal stem cells to increase the speed and ease of healing. Overall outcomes are associated decreased morbidity and healing time, and increased improvement in skin changes as compared to a nonablative approach.
Classification of Lasers Ablative
for a laser system with less risk and less downtime. Hence, a supply of nonablative lasers began to populate the marketplace. These nonablative lasers produced thermal energy, which reduced rhytids and extrinsic changes while preserving the epidermis. This new subset of rejuvenating lasers promised decreased pain upon delivery, little or no healing time, and the additional advantage of collagen stimulation. To deliver this promise, increasingly longer wavelength lasers were employed to target not only hemoglobin and melanin, but collagen structures too. In general, the nonablative lasers can be subdivided into three main types of lasers or light systems: (1) visible light lasers, (2) infrared lasers, and (3) intense pulsed light systems. Overall, visible light lasers somewhat improve texture but greatly reduce
The carbon dioxide (CO2, 10,640 nm) and erbium-doped yttrium aluminum garnet (Er:YAG, 2,940 nm) lasers classically define ablative lasers. These lasers work independent of melanin or hemoglobin because they primarily target the water chromophore. Because water makes up 80% of the skin, the wavelengths produced by these lasers are absorbed by tissue that is then vaporized. The erbium wavelength is 12–18 times more efficiently absorbed by water than the CO2 wavelength. Thus, the erbium laser’s tissue penetration is not as deep, but the laser is able to vaporize tissue with more precision and control. Conversely, due to the CO2 laser’s ability to penetrate deeper into tissue, it offers the advantage of blood vessel coagulation. This decreases intraoperative bleeding and postoperative ecchymosis and edema. Debate exists as to which of these lasers is the gold standard in ablation. The verdict often rests in the opinion of the operating surgeon. The ‘‘trade-off ’’ exists in that although the erbium may provide more precision and control, it also usually requires multiple passes. Up to ten passes can be required. The erbium has also been associated with less tissue contraction in comparison to the CO2 laser.
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Patient Selection The two most common indications for ablative lasers are photoaging (rhytides, dyspigmentation, vascularity, elastosis, actinic keratoses) and scarring [21]. While fine lines and wrinkles tend to respond quite well to ablative laser, deeper rhytides may improve slightly but are difficult to eradicate. The glabella and nasolabial fold are areas particularly resistant to laser resurfacing secondary to their dynamic movement [21]. Treating areas of scarring should also be approached with caution, as ablative results are highly dependent on the type of scar undergoing treatment. Elevated or minimally deep, distensible acne scars are much more amenable to treatment, as compared to ice-picked or bound down scarring. The latter usually require concomitant treatment with subcision and/or punch grafting to see worthwhile improvement. Although improvement in acne scarring is usually seen about 3 months postoperatively, it can take up to 1 year to realize the final healed outcome of the procedure. Of note is that more drastic improvement in scarring is seen if resurfacing is undertaken within 6–10 weeks from the inciting event [21].
Anesthesia Ablative lasers as a class are painful, though the CO2 laser is known to be more painful than the erbium. Nonetheless, choice of anesthesia is based on the location and overall surface area undergoing treatment. For localized treatment of individualized cosmetic units such as the periorbital or perioral regions local anesthesia with topical creams or nerve blocks can be employed. For more extensive or full-face laser resurfacing intravenous, conscious sedation is recommended.
Technique CO2 lasers usually require one to two passes to the resurfaced area. Feathering of edge borders can soften lines of demarcation. Treatment areas are not overlapped. Usually it is preferred that an entire cosmetic unit or the entire face be resurfaced rather than ‘‘a scar,’’ as this helps to avoid obvious lines between treated and nontreated areas. Any vaporized tissue should be removed from the surface of the treatment area between subsequent passes. Any sign of yellow or brown discoloration after wiping should cease treatment. Ideally, treatment endpoint is improvement of prior area of concern. With the erbium laser,
desired endpoint is removal of rhytides or pinpoint bleeding. Postoperative care includes application of ointments such as petroleum jelly and an ‘‘open’’ or ‘‘closed’’ technique of wound dressing. The closed technique consists of a totally occlusive or semi-occlusive dressing that promotes a moist healing environment and protects against postoperative scratches or abrasion. The potential drawback to this approach is increased rate of infection. The compromise of applying a semi-occlusive dressing for the first 24–48 h after surgery, and thereafter using a bland emollient, seems to be the ideal approach.
Side Effects Recovery period for both laser types are characterized by serous discharge, crusting, and a burning sensation. Patients treated with the erbium laser generally experience faster re-epithelialization and shorter duration of erythema than is seen with the CO2. However, these differences are dependent on the energy level settings. Accordingly, edema and pruritus are of notably shorter duration with erbium laser resurfacing. A history of keloids formation, connective tissue disease, koebnerizing conditions (psoriasis, vitiligo, etc.), ongoing sun exposure, undermining of treatment area in the last 6 months, or use of isotretinoin in the last year, should preclude treatment. Complications include erythema, dyspigmentation, milia, acne, eczematous dermatitis, infections, and scarring. The most feared complications of laser resurfacing are hypertrophic scarring and ectropion. After the procedure, the patient can expect to see re-epithelialization between 3 and 10 days. By this time epithelial oozing should have ceased. Pruritus, which may be present for the first few postoperative weeks, can be treated with antihistamines, cold packs, and topical corticosteroid creams. It is recommended that prophylactic antivirals (continued until re-epithelialization is complete), antibiotics, and potentially anti-candidal agents be given to all patients. It is important to set the patient’s expectation with regard to postoperative healing. Postoperative erythema is particularly variable. From 1 to 4 months of postoperative erythema is considered ‘‘normal,’’ but rarely erythema, especially associated with emotional or exertional flushing, may persist for up to 1 year.
Nonablative In general, nonablative lasers can be subdivided into three main categories: (1) visible light lasers, such the vascular,
Cosmetic Surgery in the Elderly
pulsed dye lasers at 585–595 nm, (2) infrared lasers, such as Nd:YAG laser at 1,320 nm (CoolTouch) or diode lasers at 1,450 nm (Smoothbeam), or the erbium:glass laser at 1,540 nm, and (3) intense pulsed light (IPL) systems. The Q-Switched Nd:YAG laser is unique among the above grouping in that it can emit either an invisible, near infrared light beam with a wavelength of 1,064 nm, or a green light with a wavelength of 532 nm when a frequencydoubling crystal is used. This Q-Switching technique allows for a large amount of energy to build up prior to its sudden, powerful release. It is this distinctive laser capability that makes the Q-Switched 532 nm Nd:YAG a favorite for targeting the superficial melanin chromophore in lentigos (> Figs. 105.15 and > 105.16). Overall, nonablative lasers spare the epidermis of injury, many with adjunct epidermal cooling modalities, as they target skin chromophores. Additionally, this subset of lasers stimulates collagen remodeling, which is the key to improvement in fine lines and wrinkles, as well as acne scarring. As there are many different lasers within the nonablative class of lasers, many different mechanisms are used to stimulate collagen. Regardless, the end result is usually a softening, rather than an eradication, of fine lines.
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the upper dermal plexus, at a subpurpuric level of energy (> Figs. 105.17 and > 105.18). With one pass, the tissue response of these vessels includes creation of low-grade inflammation and a growth response. Inflammatory mediators are released from endothelial cells, subsequently stimulating fibroblast activity and collagen neogenesis. Pulse dye laser studies have shown histological changes in collagen [21], however, clinical outcomes have been disappointing even after about five treatments at monthly intervals. Despite the US FDA approval for treating photodamage with the long-pulsed PDL, only modest results have been observed with these short wavelengths, presumably because of predominantly vascular targeting
. Figure 105.16 Post-frequency-doubled 532 nm Nd:YAG
Visible Lasers The pulsed dye laser (585 nm) employs low fluences at short enough pulse durations to selectively target a specific chromophore in the dermis – namely the vessels of
. Figure 105.15 Pre-frequency-doubled 532 nm Nd:YAG
. Figure 105.17 Pre-pulsed dye laser
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. Figure 105.18 Post-pulsed dye laser
candidates are young with minimal facial sagging. Nonablative laser systems may also serve as a supplement to augment and/or maintain the rejuvenating effect of ablative laser treatments.
Anesthesia The higher the fluence used (without blistering), the greater the degree of improvement, but the greater the discomfort of treatment. Some tolerate the procedure well with no anesthesia, but application of topical anesthesia for about 1 h before the treatment makes the procedure quite well tolerated by almost all individuals.
Technique and superficial penetration to the papillary dermis [21]. Due to lack of more drastic improvement, the vascular lasers have been largely replaced by longer wavelength infrared lasers, which more effectively target the middermis, resulting in more consistent mild improvement in rhytides [21].
Infrared Lasers With longer wavelengths the infrared (IR) lasers offer deeper penetration creating a more promising collagen tissue interaction. However, these lasers lack the benefit of improvement in epidermal changes such as dyschromia and vascular photoaging. The goal of nonablative IR laser treatments is to induce selective dermal injury while keeping the overlying epidermis intact. Clinically, the epidermis is not visibly disrupted. After thermal injury to the dermis, there is new production of type I collagen. This collagen is then deposited and reorganized into parallel arrays. Because of these collagen effects, rhytids, pore size, and acne scarring may improve during treatment. With all IR sources, epidermal protection is provided with a variety of cooling techniques. The ideal epidermal surface temperature is 40–48 C, which correlates with a dermal temperature of 70 C, the temperature required for collagen denaturation [22].
Patient Selection Patients with mild to moderate rhytid formation and possibly mild atrophic acne scarring, particularly when present in isolated cosmetic units, are ideal. Good
Usually five to six treatments are needed at 2–4 weeks intervals, using about one to three passes per each treatment session.
Intense Pulsed Light Intense pulsed light instruments (IPLs) emit nonmonochromatic and noncoherent light: specific filters allow select wavelengths (ranging from 515 to 1,200 nm) to be emitted by a single light source. The use of IPL has led to modest clinical improvement in wrinkles, with concomitant significant improvement in pigment and vascular abnormalities of photodamaged skin.
Patient Selection While the infrared lasers are a more optimal choice for patients with textural changes, IPL is a better choice for those who have varying degrees of photodamage with telangiectasias and unevenly distributed pigment. The great advantage to this modality is that a large surface area can be treated simultaneously for vascular and pigmentary changes with practically no downtime. Small improvements in texture may also be achieved.
Anesthesia A topical anesthetic cream may be applied and then removed prior to the laser treatment. However, many patients are able to tolerate the procedure without anesthesia.
Cosmetic Surgery in the Elderly
Technique All IPLs require application of a cold aqueous gel, as one to multiples passes of the handpiece are delivered. A series of five or six sessions at 4–6 week intervals is usually required to visualize clinical improvement. All of the nonablative laser choices discussed require little to no postoperative care. Although blistering, transient hyperpigmentation, and pinpoint scarring have been reported with some devices [21], overall risks of the procedure are minimal. Most patients experience slight erythema and edema for a few hours following the treatment session, but return to baseline shortly thereafter. Contraindications to the use of nonablative lasers include oral retinoid use within the past year, intake of drugs that increase light sensitivity, tanned skin, and pregnancy.
Fractional Fractional lasers provide a minimally invasive method for laser intervention, using fractional, nonablative thermal energy for creation of microthermal zones of necrosis paired with induction of dermal remodeling. This clinically results in more impressive changes in overall skin appearance at a decreased cost of downtime for the patient (> Figs. 105.19 and > 105.20).
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skin texture and pigmentation. Patients with extrinsic photoaging and dyschromia usually require about two to three treatments, in comparison to those patients with significant rhytides who require at least five treatments.
Anesthesia Topical anesthesia is usually recommended, though some patients are able to tolerate the discomfort associated with the treatment without any prior numbing. A topical numbing cream such as LMX may be applied about an hour prior to the procedure. But many of the newer fractional laser models have been developed to provide decreased pain on delivery [21]. Forced cool air, which additionally can help to decrease discomfort, is also delivered during the procedure.
Technique
These patients must be willing to sacrifice more postoperative downtime for more drastic improvements in
After the anesthetic cream is removed with a dry gauze, a blue tinted dye is then applied to the treatment area. This dye helps the laser to appropriately focus its optical scanner, though many of the newer fractionated lasers have been able to omit the need for such a dye. Subsequently, a gel is applied and multiple passes are made over the treatment area. Upon completion of the treatment session, soap and water are used to clean the treatment area and a moisturizer is applied.
. Figure 105.19 Pre-fractional laser
. Figure 105.20 Post-fractional laser
Patient Selection
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Side Effects
. Figure 105.21 Pre-liposuction of arm
The epidermis is primarily intact, with most patients experiencing only transient erythema, edema, and mild pruritus for about 2–5 days following the treatment. Scarring, dyspigmentation, or other adverse effects have not yet been reported [23]. Depending on the area to be treated, a course of prophylactic antivirals should be considered, whereas prophylactic antibiotics are generally unnecessary. Today, the options available for laser treatment seem almost boundless, with new laser technologies coming to the marketplace each day. Chronologically, laser applications have moved from strictly ablative devices, to nonablative systems, to a more fractionalized approach. But the field of cosmetic laser treatment will continue to evolve with the development of the ‘‘magic wand’’ that instantly erases both the extrinsic and intrinsic changes of aging, while minimizing associated risks and downtime.
Liposuction
. Figure 105.22 Post-liposuction of arm
The previously discussed cosmetic interventions have primarily concentrated on efforts to improve and rejuvenate the face and sun-exposed skin. However, body habitus also significantly contributes to the overall ‘‘youthfulness’’ of an individual’s appearance. As the body ages there is a tendency for fat to collect in certain, localized areas. Common sites of excessive fat collection, which can be corrected by liposuction, are the hips, flank, buttocks, abdomen, male breasts, outer and inner thighs, knees, calves, and arms (> Figs. 105.21 and > 105.22). With minimal scarring, liposuction can remove these unwanted body bulges and collections of excess fat, helping to improve body contour (> Figs. 105.23–105.26). Areas of localized fat hypertrophy on the face and neck can also be liposuctioned to improve appearance (> Figs. 105.27 and > 105.28). When considering liposuction in the elderly, the degree of skin laxity must be taken into account, as well as the underlying health status of the patient. Therefore, patient selection is critical to a successful outcome. Although, dry and wet techniques were historically used to perform the procedure, the current standard of care is tumescent liposuction. Tumescent liposuction involves subcutaneous infiltration of high volumes of crystalloid fluid containing low concentrations of lidocaine and epinephrine followed by suction-assisted aspiration of fat with small aspiration cannulas. There is a higher proportion of pure fat aspirated during tumescent liposuction, with a very small component of blood
compared with the aspirate obtained by other techniques of liposuction [24]. This method of liposuction can be performed while the patient is only under local anesthesia, thus, obviating the risk associated with general anesthesia. Additionally, the tumescent technique helps to reduce blood loss and postoperative pain, while maximizing fat harvest. Tumescent anesthesia for liposuction with dilute lidocaine has been well documented to result in peak serum levels 4–14 h after infiltration [25]. Traditional tumescent liposuction requires completed infusion time plus about 20 min for the desired vasoconstrictive effects of epinephrine. In order to decrease systemic absorption of lidocaine it is the author’s preference
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. Figure 105.23 Pre-lower abdomen, high hips, waist
. Figure 105.25 Pre-liposuction
. Figure 105.24 Post-lower abdomen, high hips, waist
. Figure 105.26 Post-liposuction
to adjunctly use intravenous conscious sedation when liposuctioning. This approach decreases total procedure time, thereby lowering systemic absorption of lidocaine, while still reaping the vasoconstrictive, anesthetic, and
tissue-expanding benefits of the infused tumescent fluid. Although the recommended maximal dose of lidocaine is 55 mg/kg, it is usually unnecessary to infuse dosages beyond 35 mg/kg [26].
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. Figure 105.27 Pre-neck jawline liposuction
. Figure 105.28 Post-neck jawline liposuction
Prior to liposuction all patients should have a thorough consultation, including preoperative photos and discussion of realistic expectations. In addition to obtaining medical clearance, physical exam with the patient in the standing position should be performed. The skin is
examined for fat distribution, skin elasticity, tone, and redundancy. Facial and neck liposuction is especially dependent on accurate assessment of not only fat distribution, but also skin tone and facial contour. Good skin and muscular tone in body liposuction patients will also help optimize surgical results. Contraindications to liposuction include, but are not limited to, clotting disorders that risk thromboembolism, bleeding diatheses, severe cardiovascular disease, pregnancy, and recent abdominal surgery. Any liver disease or current medications that might interfere with the metabolism of lidocaine should also be considered. It is imperative that all patients are informed that liposuction is not a weight loss procedure; it may remove inches from one’s waistline, but one’s weight will predominantly remain unchanged. The ideal candidate for liposuction has a lifestyle focused on maintenance of weight and muscular tone. If weight gain occurs following the procedure, it is common for weight to preferentially localize to the breast and buttocks. Similarly, it should be emphasized that liposuction is not a treatment for cellulite, striae, poor muscular tone, or inelastic skin tissue. Liposuction can be safely performed in an office or ambulatory surgical setting. The patient first undergoes intravenous conscious sedation. Next, the tumescent anesthesia is infused into the localized fatty deposits through cannulas, hydrodissecting the tissue plane. Subsequently, suction-assisted cannulas are introduced through the same incisions used for tumescent anesthesia delivery. The syringe-assisted cannulas tunnel through the fat and remove excess fat from the treatment area. Cannulas come in many different sizes, diameters, lengths, and tip shapes. Treatment location, as well as fat density and thickness or the presence of any fibrosis, dictates the choice of cannula employed (> Figs. 105.29 and > 105.30). Currently, it is recommended that not more than 4 L of supranatant fat should be removed during one operative session [24]. After the procedure, patients are required to wear special surgical garments that help support the skin over the treatment area. These binders, girdles, or elastic tapes worn for about 1–4 weeks postoperatively, deliver compression and minimize fluid shifts, bruising, and discomfort. There may be some drainage from the incision sites for several days, but overall the recovery period is remarkably rapid. Pain is minimal and is usually controlled by non-narcotic analgesic agents. Although some pain and bruising are normal, they are usually minimal and well tolerated. On occasion, there is temporary fluid or blood accumulation under the skin. However, this is easily treated and resolves with no long-term adverse effects. Skin overlying the treatment area may be numb during
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. Figure 105.29 Cannulas used for body liposuction
. Figure 105.30 Cannulas used for facial liposuction
the healing process but generally numbness resolves over 3–6 months. Many recent studies have documented the safety of tumescent liposuction [24, 26, 27]. In 2002 in a national survey of over 66,000 liposuction cases performed using the tumescent local anesthesia technique, no deaths were reported and the rate of serious adverse events was 0.68 per 1,000 cases [24]. When complications do occur secondary to liposuction they can be either systemic or localized. Systemic complications include fat and pulmonary emboli, infection, perforations, excessive blood loss, lidocaine toxicity, or death. Local complications include contour irregularities, paradoxical weight gain, hematoma or
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seroma, paresthesias, scarring, pigment irregularities localized to incision sites, and superficial skin necrosis. The first few days after liposuction, patients may be both sore and/or weak, but most are able to return to work within a week’s time. Red tinged drainage from incision sites, dependent swelling, and temporary numbness in overlying skin can all be encountered postoperatively. Pain and bruising can be controlled with oral narcotics. The incisional drainage should not persist more than 3 days. The tight-fitted binder garments help to prevent any collection of this fluid. However, regardless of garment wear after abdominal liposuction, fluid/edema will often collect in the groin and genital region. This swelling can persist for about 6–8 weeks. Any numbness in the skin overlying the treatment area usually improves by 6 months. Patients are also often frustrated by lack of immediate change in body contour following the procedure. It may take anywhere from 6 weeks to 6 months to appreciate the new, altered post-liposuction silhouette. Patients are advised to avoid alcohol, hot tubs, smoking, and any blood thinning medication such as aspirin for about 2 weeks after the procedure. Nonvigorous daily walking is also strongly encouraged, as it helps to speed recovery. Liposuction, in essence, is a tool used to sculpt the body by removing localized fat deposits. It is clearly not a form of weight loss, and is ideally suited for younger patients who maintain good skin elasticity and muscular tone, although good results can be achieved in the older population. Whether large or small volumes of fat are removed, in the appropriate candidate, the results can be drastic. Clothes fit better. A facial profile appears more youthful. Therefore, liposuction can remove not only fat, but years off one’s appearance. Perhaps, this is why it has become one of the most popular cosmetic procedures currently performed by dermatologic and plastic surgeons [27].
Blepharoplasty As Ralph Waldo Emerson once said, ‘‘The eyes indicate the antiquity of the soul.’’ With the eyes being such an age defining feature, there exists a natural tendency for patients to desire correction of periorbital signs of aging to rejuvenate facial appearance as a whole. Blepharoplasty is a pivotal procedure in ‘‘turning back the years’’ with regard to aging changes in the upper third of the face. When considering blepharoplasty in the elderly population, it is important to also note brow position. A sagging brow will significantly add to upper eyelid skin
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redundancy, as well as contribute to an overall tired appearance. It is therefore advised that significant brow ptosis be corrected prior to undergoing blepharoplasty, or concomitantly. Although contributory, brow ptosis is a distinct phenomenon from the periocular bags, sags, and folds, which develop over time secondary to loss of skin strength and supporting structures. It is specifically these unwanted periocular changes that blepharoplasty, alone, can ‘‘repair.’’ Although differences in opinion exist across cultures, the Western world has a fairly clear definition of ‘‘beautiful’’ eye architecture. For males, a flat eyebrow that rests upon the orbital rim is considered attractive. Whereas, for females, a brow that medially is positioned at or slightly above the orbital rim and ascends gently in the lateral aspect to reach a peak at the lateral limbus, is preferred. Male and female differences also exist regarding the esthetic position of the eyelid crease. Generally, the male eyelid crease is positioned at approximately 10 mm above the eyelid margin at the superior border of the tarsus. The female eyelid crease is positioned slightly higher than the male, creating a more open appearance with greater depth. It is the attachment of the levator aponeurosis to the orbicularis muscle and subdermal skin that contributes to the eyelid crease structure. In the upper eyelids, there are two fat pads: a small medial pad and a larger central one. There is no lateral fat pad. The anatomic caveat here is not to mistake a prolapsed lacrimal gland in the lateral position for a fat pad; if a prolapsed lacrimal gland is accidentally removed, chronic dry eye is likely to result. The lower eyelid has three fat pads: medial (nasal), central, and lateral (temporal). The inferior oblique muscle separates the medial and central fat collections and must not be injured, as trauma to this muscle can result in permanent diplopia on upward gaze. Additionally, the preaponeurotic fat pads are highly vascular and great care must be taken when excising them so as to control bleeding and prevent retrobulbar hematoma. Eyelid surgery can treat loose or sagging skin that creates folds or disturbs the natural contour of the upper eyelid, sometimes impairing vision; excess fatty deposits that appear as puffiness in the upper eyelids; bags under the eyes; droopiness of the lower eyelids causing scleral show; or excess skin and fine wrinkles of the lower eyelid. Unfortunately, some of these ocular findings may have hereditary associations, and neither diet nor exercise will change these unwanted periocular characteristics. As discussed previously, all cosmetic procedures require a thorough consultation outlining realistic expectations. Preoperative condition is documented with a series of photos and the eyelids are evaluated for excess skin
texture, herniated fat, level of supratarsal crease, ptosis, lacrimal gland position, and lower eyelid laxity. Special attention is directed to any evidence of contributory brow ptosis, a consequence of aging that can give the illusion of excess skin (pseudoblepharochalasis). It is critical to first surgically correct significant brow ptosis before proceeding with blepharoplasty. General medical clearance, with specific screening for a history of Graves’ disease, connective tissue diseases associated with a clinical or subclinical sicca syndrome, or a history of a bleeding disorder is recommended. Additionally, an ophthalmology consult should provide baseline visual acuity testing, visual filed mapping, fundoscopic examination, and a Shirmer test to determine tear secretion. Specific ophthalmologic history pertaining to laser-assisted in situ keratomileusis and other refractive surgery should be documented, as they predispose the patient to postblepharoplasty dry eye exposure and visual changes [28]. The generic term, blepharoplasty, can be further divided into procedures specific to the upper and lower lid. Upper blepharoplasty can be employed to correct cosmetic and functional facial imperfections like drooping eyelids with skin redundancy (> Figs. 105.31–105.36). In comparison, lower blepharoplasty, which helps correct the ‘‘bags’’ created from anteriorly, herniated retro-orbital fat, is employed primarily for cosmesis (> Figs. 105.37 and > 105.38). Depending upon the location and degree of necessary correction, patients may require excision of excess skin alone, skin and fat, or skin, fat, and muscle. Most patients seek upper lid blepharoplasty for correction of redundant upper lid skin. This can either be a cosmetic concern secondary to unfavorable signs of aging, or a functional concern in which the excess skin impedes vision or activities of daily living. The upper eyelid skin flap technique is exercised in instances where it is necessary to only excise excess skin and/or fat, as the obicularis musculature is left intact. The skin is first appropriately marked with the patient in an upright position, followed by eyelid excision, undermining, exposure
. Figure 105.31 Pre-upper blepharoplasty
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. Figure 105.32 Post-upper blepharoplasty
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. Figure 105.35 Pre-upper blepharoplasty, sideview
. Figure 105.33 Pre-upper blepharoplasty
. Figure 105.34 Post-upper blepharoplasty
of the obicularis, dissection and removal of any unwanted fat, and finally a sutured closure. The myocutaneous upper blepharoplasty is exceedingly similar, with one exception. After the orbicularis muscle is dissected and visualized, a 5–6-mm strip of orbicularis muscle is excised just above the interior wound margin, exposing the orbital septum and levator aponeurosis. Subsequently, excess fat is also removed and the area closed with suture. This myocutaneous technique is primarily reserved for patients who have not only redundant skin and fat, but also a component of hypertrophic musculature. If there also exists the need to recreate the supratarsal crease, nylon sutures are then placed with deep fixation to the underlying levator aponeurosis. In contrast to upper lid blepharoplasty, lower lid procedures are usually performed for cosmetic reasons. If the desired goal is to simply remove lower eyelid skin or fat ‘‘bags,’’ a skin flap that allows for concomitant fat
. Figure 105.36 Post-upper blepharoplasty, sideview
removal, is all that is needed. An alternative method, in which the conjunctiva, rather than the skin, is incised, is the transconjunctival approach. The transconjunctival approach places the incision line approximately 10 mm
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. Figure 105.37 Pre-lower blepharoplasty
. Figure 105.39 Pre-upper and lower blepharoplasty
. Figure 105.38 Post-lower blepharoplasty
inferior to the eyelid margin. This technique is preferred over transcutaneous incision, as it touts two major advantages. Risk of ectropion is decreased and cosmesis is superior due to lack of visible scar. Unfortunately, if there is also a need for lower lid tightening, or if there exists excess fat and minimal excess skin (common in younger patients), a transcutaneous approach is necessary to address the needed myocutaneous abnormalities. It is recommended that the blepharoplasty procedure be performed under a combination of local (injectable and topical [eyeball]) anesthetic and conscious sedation. Blepharoplasty-associated risks include corneal irritation, milia, cyst formation, allergic reactions, chemosis, dehiscence, loss of eyelashes, lagophthalmos (inability to close eyelids), overresection of fat, ectropion, ptosis, lacrimal gland injuries, diplopia, hematoma, asymmetry, persistent edema, and rarely, retrobulbar hemorrhages with associated loss of vision. Penetration of the orbital septum introduces increased risk for retrobulbar hemorrhages. The overall incidence of blindness resulting from a retrobulbar hematoma following blepharoplasty is estimated to be only 0.04% [29]. Signs of retrobulbar bleeding include pain, ecchymosis, proptosis (bulging eye), and visual loss. Postoperatively, patients should be instructed regarding aggressive corneal lubrication with eye protection, eye
drops, and ointment [28]. Most patients do very well, but many may need reassurance that any swelling or bruising will resolve by the end of 2 weeks. Most patients resume normal activities within 2–3 days and return to work within a week. Contact lens wearers may return to contact eyewear 7–10 days after surgery. And women may return to eye makeup wear 10–24 days postoperatively. The quick healing and immediate results seen in association with blepharoplasty make it quite appealing to patients. But even more attractive are the procedure’s long-lasting results. Depending on the age and inherent skin properties of an individual, the procedure’s effects may endure from 5 to 15 years. Blepharoplasty, though namely targeting aging in the periocular region, can drastically change overall appearance (> Figs. 105.39 and > 105.40). Caution is advised not to remove too much fat and underlying tissue from the periocular region, as a hollowed appearance will threaten cosmesis. In skilled hands, blepharoplasty will open up the eye and leave expressions of tiredness behind. By coupling blepharoplasty with browpexy, and other cosmetic procedures (> Figs. 105.41 and > 105.42), the look of even younger years can be achieved. Laser resurfacing is commonly combined with eyelid surgery, to further improve skin tone and pigment irregularities. It is also the standard practice to combine the blepharoplasty procedure with CO2 laser resurfacing. The tissue contraction
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. Figure 105.40 Post-upper and lower blepharoplasty
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. Figure 105.42 Post-combination upper and lower blepharoplasty, jawline tuck, neck jawline liposuction, and fractional laser to lower eye area
associated with the CO2 laser can further enhance blepharoplasty outcome, especially when treating lid laxity. . Figure 105.41 Pre-combination upper and lower blepharoplasty, jawline tuck, neck jawline liposuction, and fractional laser to lower eye area
Face Lift The ultimate technique in surgical management of facial restoration has traditionally been considered the facelift. However, with ongoing advancements in laser technology, this dogma may change in the future. Regardless, the primary means of manipulating both skin and underlying structures to restructure facial appearance in one surgical procedure, remains the facelift. The degree of necessary alteration varies with each patient and is dependent on inherent facial structure, the presence of intrinsic and extrinsic aging change, as well as the patient’s expectation for dramatic improvement. Clearly, risks and morbidity are directly proportional to the invasiveness of a facelift. Therefore, it has been the authors’ goal to employ an individualized facelift technique for each patient; the technique is minimally invasive but delivers equivalent if not superior results, in comparison to the traditional face and neck lifting. Using a limited undermining technique and superficial musculoaponeurotic system (SMAS) plication, the SMAS can be tightened and skin carefully removed,
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restoring an oval jawline and well-defined chin-neck angle. Although early intervention is not uncommon for younger patient (age 38–45 years), this technique can also be modified to treat an elderly population (65–80 years of age). In more mature patients, it is necessary to not only tighten the SMAS, but also actually resuspend it. Due to limited undermining and lack of compromise to the facial vascular supply, this method may be safely used for facelifts in certain cases where the patient’s underlying health may be partially compromised. As with most cosmetic, surgical procedures, there is a trade-off of invasiveness, risk, recuperation, and expense, with degree of improvement in appearance. Thus, there is a range of different techniques, which vary in aggressiveness and should be tailored to the patient’s present appearance and the cosmetic state they hope to achieve. Patient selection criteria include general health, age, asymmetry, dysmorphic fat, weight gain or loss, muscle tone, bony structure, skin tone, skin thickness, and patient expectations. Of note is that age is not as important as the integrity of the facial skin and its underlying structures. Early tissue sagging and mild rhytids, depending on the skin’s elasticity, can be substantially helped by limited skin procedures, whereas severe photodamage, prior trauma, scarring, and radiation changes may require more involved techniques. The optimal candidate for a sutureless facelift technique includes a patient with early mandibular angle blurring and potentially subtle neck droop with minimal fatty deposit. Conversely, a patient who presents with similar but more severe changes, as well as mild to moderate skin laxity, is a better candidate for a modified facelift. And on the most severe end of the spectrum, those patients that have a large degree of tissue laxity and sag and/or platysmal banding are better suited for the conventional facelift. After a thorough patient consultation including an exam and preoperative photographs, informed consent with appropriately set patient expectations, and medical clearance, a patient is ready to undergo the facelift procedure. All the authors’ patients undergo cervicofacial rhytidectomy in an outpatient ambulatory surgical setting. Intravenous conscious is employed to ensure a comfortable patient experience. Patients are marked while seated in an upright position, then prepped and draped for sterility. The procedure to which the authors subscribe involves incision, dissection, plication, skin removal, redraping, and closure. First, small skin incisions are placed bilaterally in the preauricular fold inferior to the tragus; at the base of the earlobe; postauricularly in the auricular sulcus to the level of the tragus; and in the midline under the
. Figure 105.43 Presurgical markings for jawline tuck procedure
. Figure 105.44 Presurgical markings for jawline tuck procedure
mentum (> Figs. 105.43–105.45). Through these minute incision, tumescent anesthesia is infused and hydrodissection, along with undermining, ensues to create a cervical neck flap. As more invasive technique is required to
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. Figure 105.45 Presurgical markings for jawline tuck procedure
. Figure 105.47 Pre-jawline tuck; female
. Figure 105.46 Intraoperative facelift flap dissection
. Figure 105.48 Post-jawline tuck; female
accomplish the aesthetic goal, the incisions can be lengthened, allowing for better access and further alteration of underlying tissue structures, and creation of a larger flap (> Fig. 105.46). The degree of undermining and extent of flap creation, as well as the degree of restructuring the SMAS, is what distinguishes a lunchtime lift, from a modified ‘‘minilift,’’ from a traditional facelift. Facial liposuction may also be employed at this time to remove any excess fat from the area. Subsequently, the SMAS
tissue is plicated by being folded over itself and pulled up with the use of nonabsorbable suture. Redundant skin is trimmed, redraped, and sutured, depending on the extent of the lift. The Webster variation lift is defined by
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. Figure 105.49 Pre-jawline tuck; male
. Figure 105.50 Post-jawline tuck; male
The postoperative course involves typical wound care with twice a day topical antibiotic ointment and any necessary suture removal in about 7–10 days. Patients with more involved procedures may be fitted with a facial elastic garment. All patients are advised to avoid bending and lifting and are instructed to sleep in a 30–45 upright position so as to minimize swelling. While any swelling and bruising usually resolves by about postoperative day 10, the face may feel numb, especially along the neck, mandibular line, and in the periauricular area, for about 6–12 weeks after the procedure. Sun exposure also should be avoided. Overall, complications are few and the major complications (large hematomas, skin loss, hair loss, and nerve loss) more commonly associated with the traditional facelift are avoided. The patients’ high level of satisfaction with their outcomes is a testament to the safety and efficacy of the Webster-style facelift. Although there are countless different approaches to rejuvenating the face within the realm of cervicofacial rhytidectomy, none has been shown to produce consistently better or longer-lasting results. Many of the more aggressive techniques extend operating time, heighten the potential morbidity of the operation, and prolong the duration of convalescence. The Webster-type lift, to which the authors subscribe, has proven time and again, that a less invasive approach often gives the best and longest-lasting result, while limiting risk and avoiding serious complications (> Figs. 105.47–105.50).
Cross-references >A
New Paradigm for the Aging Face Rejuvenation: A Chronology of Procedures
> Facial
References
sutures limited to the natural anterior and posterior cosmetic units of the ear, whereas in the traditional lift the incision/closure rises above the ear and into the temporal hair line.
1. Rabe J, et al. Photoaging: mechanisms and repair. J Am Acad Dermatol. 2006;55(1):1–19. 2. Carruthers J, et al. Botox Consensus Group. Consensus recommendations on the use of botulinum toxin type a in facial aesthetics. Plast Reconstr Surg. 2004;114(6):1S–22S. 3. Carruthers J, et al. Facial Aesthetics Consensus Group Faculty: advances in facial rejuvenation: botulinum toxin type a, hyaluronic acid dermal fillers, and combination therapies – consensus recommendations. Plast Reconstr Surg. 2008;121(5):5S–30S. 4. Carruthers A, Carruthers J. Botulinum toxin type A: history and current cosmetic use in the upper face. Semin Cutan Med Surg. 2001;20:71–84. 5. Carruthers A, Carruthers J. Dose dilution and duration of effect of botulinum toxin type A (BTX-A) for the treatment of glabellar
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rhytides. Presented at the American Academy of Dermatology Winter Meeting, 22–27 Feb 2002, New Orleans, 2002. Wise JB, Greco T. Injectable treatments for the aging face. Facial Plast Surg. 2006 May;22(2):140–146. Lupo MP. Natural look in volume restoration. J Drugs Dermatol. 2008 Sept;7(9):833–839. Thioly-Bensoussan D. Non-hyaluronic acid fillers. Clin Dermatol. 2008;26:160–176. Narins RS, Brandt FS, Lorenc ZP, Maas CS, Monheit GD, Smith SR. Twelve-month persistency of a novel ribose-cross-linked collagen dermal filler. Dermatol Surg. 2008 June;34(Suppl 1):S31–39. Romagnoli M, Belmontesi M. Hyaluronic acid–based fillers: theory and practice. Clin Dermatol. 2008;26:123–159. Brandt FS, Cazzaniga A. Hyaluronic acid gel fillers in the management of facial aging. Clin Interv Aging. 2008;3(1):153–159. Lemperle G, et al. Soft tissue augmentation with Artecoll: 10-year history, indications, techniques, and complications. Dermatol Surg. 2003;29:573–587. Ortonne JP, Marks R. Photodamaged Skin. London/England: Martin Dunitz Ltd., 1999, pp. 11–28. Clark E, Lawrence S. Superficial and medium-depth chemical peels. Clin Dermatol. 2008;26:209–218. Landau M. Chemical peels. Clin Dermatol. 2008;26:200–208. Truppman F, Ellenbery J. The major electrocardiographic changes during chemical face peeling. Plast Reconstr Surg. 1979; 63:44. Bhalla M, THAMI GP. Microdermabrasion: reappraisal and brief review of literature. Dermatol Surg. 2006 June;32(6):809–814. Sadick NS, Finn, NA. New applications for microdermabrasion technology. Int J Cosmet Surg Aesthet Dermatol. 2002 Nov; 4(63):44.
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19. Campbell RM, Harmon CB. Dermabrasion in our practice. J Drugs Dermatol. 2008 Feb;7(2):124–128. 20. Lawrence N. History of dermabrasion. Dermatol Surg. 2000;26:95–101. 21. Alexiades-Armenakas MR, et al. The spectrum of laser skin resurfacing: nonablative, fractional, and ablative laser resurfacing. J Am Acad Dermatol. 2008 May;58(5):719–737. 22. DeHoratius D. Nonablative tissue remodeling and photorejuvenation. Clin Dermatol. 2007;25:474–479. 23. Rinaldi F. Laser: a review. Clin Dermatol. 2008;26(6):590–601. 24. Coldiron B, et al. Liposuction Council bulletin: ASDS guidelines of care for tumescent liposuction. Dermatol Surg. 2006;32:709–716. 25. Butterwick KJ, Goldman MP, Sriprachya-Anunt S. Lidocaine levels during the first two hours of infiltration of dilute anesthetic solution for tumescent liposuction: rapid versus slow delivery. Dermatol Surg. 1999 Sept;25(9):681–685. 26. Coleman W, et al. American Academy of Dermatology Guidelines/ Outcomes Committee. Guidelines of care for liposuction. J Am Acad Dermatol. 2001;45(3):438–447. 27. Housman T, et al. The safety of liposuction: results of a national survey. Dermatol Surg. 2002;28(11):971–978. 28. Trussler A, Rohrich R. Blepharoplasty. Plast Reconstr Surg. 2008; 121(1):1–10. 29. DeMere M, et al. Eye complications with blepharoplasty or other eyelid surgery: a national survey. Plast Reconstr Surg. 1974;53:634–637.
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98 Cosmetics and Aging Skin Robert L. Bronaugh . Linda M. Katz
Introduction Cosmetics are a category of consumer products that is sold worldwide. Many trade and regulatory agencies now refer to cosmetics as personal care products to try and better account for some of the diversity of these products; however not all personal care products marketed in the USA are regulated as cosmetics. For the 12-month period up to December 2008, the personal care products industry worldwide sales grew by 4% to 247 billion [1]. The use of some categories of personal care products to improve the appearance of aging is discussed here. As skin ages, it loses its natural elasticity and becomes thinner, more fragile and lax, taking on a wrinkled appearance [2]. Aging of the skin has been attributed to two processes referred to as intrinsic or extrinsic processes [3]. The intrinsic process occurs through the passage of time, and appears as fine wrinkles on the skin. The extrinsic process is often referred to as the effects that the environment (such as sun) and other exposures (such as weather) have on the skin. These changes often appear as deeper or coarser wrinkles, crevices, scaling or skin dryness, and age spots. In addition, wrinkles have been classified into three different types based on morphology [4], and are influenced by environmental and genetic factors. Crinkles are defined as fine wrinkles that occur as a result of the loss of elastin, and may be accelerated by exposure to sun. These changes are particularly apparent on the face and other sun-exposed areas. Glyphic wrinkles are creases that accentuate normal skin markings and occur in prematurely aged skin, again accelerated by exposure to sun. The third type is linear furrows, which are long, straight, or curved grooves usually seen on the face. An example would be ‘‘crow’s feet,’’ which is also accelerated by sun exposure. The interventions (not including surgical) by which the appearance of wrinkles may be altered, prevented, or reduced are discussed under more than one regulatory oversight within the USA, such as cosmetics, drugs or devices. The regulatory authority that has jurisdiction over the specific product categories marketed within the USA is identified. Specific focus is given to alpha hydroxy acids (AHAs), retinoids, collagen synthesis, moisturizers
and skin hydration, sunscreens, hydroquinone, and peels or scrubs, which will embrace the bulk of the products available over-the-counter (OTC) to consumers. Some products, such as Botox, which is available only through a physician, are also briefly discussed.
Drug Versus Cosmetic Cosmetics have been used worldwide for over thousands of years to enhance appearance. In addition to improving appearance, they are also used to cover up flaws. However, as labeling claims have become more creative, consumers also have greater expectations from their cosmetic products, to the point that they are often looking for a quick fix from a bottle. The Federal Food, Drug, and Cosmetic Act (the Act) broadly defines a drug and a cosmetic, and specifies requirements that need to be followed to market these products. In the Act a cosmetic is defined as: ‘‘(i) articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance, and (ii) articles intended for use as a component of any such articles, except that such term shall not include soap’’ [5]. Soaps are generally exempt from the cosmetic provisions of the Act, and are often classified as consumer care products regulated either by the Consumer Product Commission or as a drug, depending on the nature of the product and its claims. In other words, cosmetics in the USA are intended to make the user look better, without affecting the structure or function of the body. A drug, on other hand, is defined in the Act as ‘‘(B) articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man. . .(C) articles (other than food) intended to affect the structure or any function of the body. . .and (D) articles intended for use as a component of any [such] articles’’ [6]. In other words, drugs are intended to be used to prevent, mitigate, treat, or cure a problem. Thus, in the USA, products such as sunscreens, which are used to prevent exposure to harmful rays from the sun, are regulated as drugs. In addition,
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_98, # Springer-Verlag Berlin Heidelberg 2010
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several other agents that will be discussed may also be regulated as drugs, because their intended purpose is either to affect the structure and function of the body or to treat or prevent a condition.
Sunscreens Sunscreens in the USA are regulated as drugs, because their intended purpose is to prevent the effects of harmful exposure to the sun, such as sunburn, which may ultimately lead to accelerated wrinkling, thickening of the skin, and cancer. Currently, marketed sunscreens are rated by their sun protection factor (SPF) and more recently have added UVA protection. SPF is a measure of how much solar energy (UV radiation or UVB) may be required to produce sunburn on protected skin [2]. A common misunderstanding is that the number on the product implies the amount of time a person can be in the sun and not develop sunburn. SPF, however, is a measure of exposure to the amount of solar energy, not the time. It is commonly known that there are multiple factors influencing whether or not a person will develop sunburn. These include: amount of solar exposure, which is influenced by time of day, geographic location (latitude), weather, skin type, amount of sunscreen applied and reapplication frequency, and activities being carried out. In May 1999, the US Food and Drug Administration (FDA) published a final rule that set standards for formatting, testing, and labeling of OTC sunscreen products that protect against UVB, which is responsible for causing sunburn. In August 2007, the FDA proposed new sunscreen regulations that focused on sunscreen protection from UVA exposure. In this proposal, sunscreen rating was proposed via two tests: one would be used to determine a sunscreen’s ability to reduce the amount of UVA light that passes through it; the other would determine a sunscreen’s ability to prevent tanning [7]. Further, this proposed rule would require manufacturers to print a warning statement advising consumers of risk from sun exposure and ways to limit exposure. Thus, the importance of sunscreens is their use in the prevention of harmful consequences of sun exposure, rather than being able to reduce or eliminate wrinkles and other signs once they have occurred.
Botox and Other Injectables This category consists of a wide range of products that can either be used alone or in combination with other
products, most commonly peels. The products in this category are regulated in the USA as drugs or biologics. Botox, the oldest injectable in use to decrease wrinkling, was approved by the FDA in 2002. Botox is injected into multiple sites and causes the reduction of wrinkles by the temporary paralysis of the cutaneous nerves, usually wearing off within three to six months. As the Botox wears off, patients are again able to wrinkle their foreheads and the facial lines gradually begin to reappear. Bovine collagen has also been used to improve facial scars and wrinkles. Like Botox, the effects are also temporary, with wrinkling reappearing within approximately six months. Glutaraldehyde cross-linked collagen was introduced as a way to extend the effect, but this has not been found to be the case [8]. Approximately 3% of patients react to test doses, but late allergic reactions appear to be less common. Injection with silicon, gelatin matrix implant, and polytetrafluoroethylene has gone out of favor. These substances were felt to give permanent results. However, with their permanence also came reports of unexpected and imperfect consequences. In addition, their use has been associated with the development of inflammatory reactions [9].
Hydroquinone Hydroquinone has been used in cosmetic and drug products. The latter use has usually been for higher doses and for longer duration, as a skin bleaching product, especially to remove the dark (‘‘age’’) spots that have been caused by sun exposure. The mechanism of action of hydroquinone is unknown but it has been hypothesized that it may affect the cellular processes of melanogenesis [10]. Over the past decade, much research has been done to evaluate the safety and toxicity from recurrent use. The FDA, in addition to the NDMA (Nonprescription Drug Manufacturer’s Association, now know as CHPA), has evaluated the carcinogenic risk from topically applied hydroquinone, as well as the risk for ochronosis. Because of these potential risks, the FDA has proposed [11] that the use of skin bleaching drug products should be restricted to prescription use, so that individuals can be closely monitored by a physician. Further, the FDA has concluded that the actual benefits from use of OTC skin bleaching drug products are insignificant when compared to the relative benefit. It appears from research that the benefit is directly related to the absorption of the product, which also increases the potential for harm.
Cosmetics and Aging Skin
Moisturizers Moisturizers are formulations designed to maintain the water content of the skin between 10% and 30% [12]. Creams and lotions are the most popular moisturizers for consumers, and these are emulsions of oil and watersoluble ingredients [13]. The ingredients in moisturizers that cause hydration of the skin are humectants and emollients. Humectants are ingredients that attract moisture into the stratum corneum, resulting in increased hydration of the skin [13]. Examples of humectant ingredients in cosmetics are glycerin, urea, and propylene glycol. Emollients are lipid-based ingredients that form a barrier on the surface of skin to trap moisture in the skin to prevent its evaporation [13]. Examples of emollients in cosmetics are octyl dodecanol, oleyl oleate, and isopropyl myristate. Moisturizers are designed to mimic the function of epidermal lipids in the skin barrier [12]. The lipid composing the intercellular space in the stratum corneum is composed of a mixture of ceramides, cholesterol, and fatty acids. The barrier properties of the stratum corneum are related to the organizational arrangement of these lipids into lamellar granules within the intercellular space [14]. Extraction of these lipids from the skin with solvents leads to xerosis (dry skin), directly in proportion to the amount of lipid removed [12]. This mixture of lipids also contains natural moisturizing factors consisting of amino acids, lactic acid, pyrrolidone carboxylic acids, and urea. Due to the hydroscopic properties of this mixture, water can be maintained within the stratum corneum even under very extreme conditions [15]. The water content within skin is also essential for normal desquamation and barrier integrity [15]. Normal plasticity of the tissue depends on water content [12]. Low content of water in the tissue inhibits enzyme activity of the proteinases, glycosidase, and lipases essential for shedding of the corneocytes. Skin aging occurs based on the sum of its two components – intrinsic or chronological aging and photoaging [13, 15]. Intrinsic skin aging occurs from the normal physiological changes in the body as people age. Photoaging is due to the damage caused by UV irradiation on sun-exposed areas of the body. Photoaging therefore results in premature aging of the skin. Dry skin is the result of a decreased ability of aged skin to retain water in the stratum corneum. Low moisture content in skin can result in abnormal desquamation by inhibiting the enzymatic activity of proteases responsible for corneodesmosomal degredation [15]. The corneodesmosomes are the principal cohesive linkage between the corneocytes that make up the stratum corneum.
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Relative humidity was shown to affect the water content of the stratum corneum, with a dramatic decrease in the normal process of desquamation when the relative humidity was below 80%. A glycerol containing moisturizing lotion increased desquamation of freshly obtained pig skin in vitro, demonstrating that water content of the stratum corneum was the rate-limiting factor in the final stages of desquamation [16]. Visual observations of the skin are often used to assess its dryness. However, different bioengineering methods have been developed to assess the dryness of the skin more objectively. A measurement of the capacitance of skin is most commonly used to assess water content. Trans-epidermal water loss (TEWL) can measure a compromised stratum corneum, as the passage of water from beneath the stratum corneum to outside the body is rapidly increased. The use of adhesive tape to obtain samples of the surface stratum corneum can be used to measure the extent and thickness of skin scaling [13].
Safety Studies on Alpha Hydroxy Acids Hydroxy acids are thought to act by reducing cohesion between newly formed corneocytes in the newly formed stratum corneum, thereby enhancing desquamation of skin [17, 18]. The ability of these chemicals to penetrate into the stratum corneum, and possibly deeper into the skin, likely affects the activity of hydroxy acids on skin. The pH of the formulation was determined to markedly affect the absorption of hydroxy acids into the different layers of the skin [19]. A comparison of absorption values from oil-in-water (O/W) emulsions at pH 3 and pH 7 showed that stratum corneum concentrations of glycolic acid and lactic acid were greater at the lower pH by 4.8-fold and 2.0-fold, respectively. Because the AHAs were unionized at pH 3.0 and therefore more lipophilic, they penetrated into the skin much more readily. Reducing the pH of the formulation to the pKa (or below) greatly increases the activity of AHAs in eliciting desquamation. The pKa of glycolic acid is 3.8.
Dermal Effects of AHAs and UV Light The effect of glycolic acid pretreatment of skin on UV light irradiation has been examined in several studies. In one study, a formulation containing 10% glycolic acid at pH 3.5 was applied once daily for four days to 15 volunteers with skin types suggesting increased sensitivity to
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sun (Fitzgerald skin types I and II) [20]. Other sites on the skin of the volunteers were treated daily either by rubbing with a moistened mechanical sponge for 15 s or applying 8% glycerin. The test sites were irradiated with one minimum erythema dose (MED) of primarily UVB light 15 min after the last dosing of the treatments applied each day. Biopsies were taken from the test sites to evaluate the formation of sunburn cells. The glycolic acid formulation did not statistically increase sunburn cell formation when compared to untreated skin, the 8% glycerin, or the mechanical exfoliating sponge. Another study was conducted using similar procedures, except that the skin test areas were treated for a much longer period of time – 12 weeks [20]. Two groups were examined in this study with 16 subjects each and with different glycolic acid formulations. Group A used 10% glycolic acid in a thickened aqueous vehicle at pH 4.0. Group B used a 10% formulation of glycolic acid at pH 3.5. Treatment of both groups with glycolic acid for 12 weeks, followed by UV irradiation, resulted in a significant increase in sunburn cells compared to untreated skin and control vehicles. A clinical study conducted by other investigators evaluated the effects of daily glycolic acid treatment (six days per week) on the MED, sunburn cells, and cyclobutyl pyrimidine dimers (CPDs). Treatment lasted for 4 weeks, followed by exposure to UV light [21]. Either a 10% glycolic acid formulation (pH 3.5) or a placebo (formulation without glycolic acid) was applied to the backs of 29 Caucasian volunteers. Subjects were primarily Fitzgerald skin type III and were placed in either Group 1 or Group 2. After the 4 weeks of treatment, subjects in Group 1 were irradiated with a solar simulator. One week later they were irradiated again to determine recovery. Biopsies were removed from the test sites to determine both the MED and sunburn cell formation. There was a statistically significant decrease in the MED of the treated skin after 4 weeks of treatment with the glycolic acid formulation compared to the placebo or untreated skin. The MED on the treated site returned to normal after glycolic acid treatment was discontinued for 1 week. Compared to sunburn cells formed in the sites treated with the placebo, the number of sunburn cells induced by UV light following 4 weeks of glycolic acid treatment increased 1.9-fold. However, the number of sunburn cells at the glycolic acid-treated sites and the placebo sites were not significantly different after one week of discontinued treatment. The 12 subjects in Group 2 received 4 weeks of treatment with glycolic acid and placebo, followed by 1.5 MED irradiation with UV light. The difference in CPDs measured
in the glycolic acid treated sites compared to placebo was not significant. The FDA issued a Guidance for Industry document in January 2005 entitled ‘‘Labeling for Topically Applied Cosmetic Products Containing Alpha Hydroxy Acids as Ingredients.’’ Because of evidence suggesting that topically applied cosmetic products containing AHAs might increase the sensitivity of skin to sunlight, the FDA recommends that the following statement appear on the labels of these products: "
Sunburn Alert: This product contains an alpha hydroxy acid (AHA) that may increase your skin’s sensitivity to the sun and particularly the possibility of sunburn. Use a sunscreen, wear protective clothing, and limit sun exposure while using this product and for a week afterwards.
Alpha hydroxy acids are often found in cosmetic products formulated to approve the appearance of skin. Care should be taken when using these products and spending time in the sun.
Retinoids Retinoic acid (tretinoin) seems to have some effectiveness in treating the appearance of photoaging [22]. The mechanisms responsible for this activity may include the shedding of corneocytes due to the proliferation of keratinocytes [23], and may also be associated with new collagen produced in the upper dermis [24]. The action of retinoic acid in skin may be ultimately associated with activation of retinoid receptors [25]. The application of retinol to skin has been reported to induce expression of cellular binding proteins and to cause other molecular changes that are the same as those observed after treatment with retinoic acid [26]. Retinol and its ester retinyl palmitate are frequently used in cosmetic products. In vitro skin penetration techniques that maintained skin viability demonstrated that retinyl palmitate could be absorbed into human and hairless guinea pig skin [27]. Only 0.2% of the applied dose was absorbed through human skin, but 18% of the dose was found in skin at the end of a 24-h study. Approximately half of the retinyl palmitate remaining in skin had been metabolized to retinol. No further biotransformation of retinol to retinoic acid was observed at the level of detection in this in vitro system. Retinol penetration through excised human skin has been measured following application of cosmetic formulations to skin assembled in diffusion cells [28]. Penetration through skin into the receptor fluid was 1.3% of the
Cosmetics and Aging Skin
applied dose from an emulsion vehicle and 0.3% of the dose from a gel vehicle in 24-h studies. Retinol and retinoic acid skin penetration were found in vivo in human subjects, with induction of retinoic acid 4-hydroxylase activity used as an endpoint for making the comparison [29]. Significant induction of enzyme activity was observed when retinoic acid was applied to skin (under occlusion) in concentrations as low as 0.001%. Retinol (also applied under occlusion) required a concentration of 0.025% to produce significant effects on induction of 4-hydroxylase enzyme activity. Liposomes have been reported to enhance the penetration of retinol through human skin assembled in diffusion cells [30]. The skin penetration rates following infinite dosing of retinol were compared following application of retinol in either a control vehicle without liposomes or in deformable (flexible) liposomes made with between 20. At the end of 24 h, control retinol absorption was found to be approximately 1 ug/cm2, while approximately 30 mg/cm2 skin of retinol had penetrated through skin from the flexible liposomes. Enhanced penetration of retinol was found from solid lipid nanoparticles contained in an oil-in-water cream as compared to a conventional cosmetic formulation [31]. Highest retinol levels were found in the stratum corneum and the upper viable epidermal region. The absorption of retinyl palmitate was influenced even more by delivery with the solid lipid nanoparticles. Certain retinoids (retinol, retinyl palmitate) are frequently found in cosmetic products formulated to improve the appearance of skin. Additional studies are needed to completely clarify the mechanisms of action of these retinoids. As with AHAs, irritation of the skin can be caused by higher dosage levels of these ingredients, especially with exposure to the sun.
Collagen Synthesis and Aging Skin Many of the skin changes associated with aging, including changes in pigmentation and deep wrinkling, are the result of overexposure to the sun [32]. Chronologic aging is characterized by changes in skin, such as fine wrinkling and skin laxity. Both chronologic aging and photoaging are associated with decreased collagen levels in the dermis. As collagen fibers serve as the primary structural support of the skin, it follows logically that a reduction in skin collagen levels would be associated with the formation of skin wrinkles. Photo-effects on aging include the formation of activator proteins that inhibit the production of collagen and the formation of reactive
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oxygen species (ROS), leading to increased breakdown of collagen. Similar mechanisms seem to be involved, but at a reduced level, in the chronologic aging of skin [32, 33]. Recent work has focused on the dermal collagen matrix and resultant fragmentation by matrix metalloproteinases. Fragmentation of collagen in the matrix prevents the activity of fibroblasts (where collagen is synthesized), resulting in low levels of collagen in the dermis [34]. Topical drug products such as retinoic acid appear to have anti-aging effects based on stimulation of collagen synthesis. Other topical products that affect collagen synthesis may be drug or cosmetic products, depending on the claims that are made. Several commercially available products were evaluated for their effects on extracellular matrix proteins, using a 12-day occluded patch test in nine volunteers. One product contained 2% of a lipopentapeptide, and another product contained the same peptide at a 6% concentration and retinyl palmitate in a basic moisturizer cream. A third product contained retinoic acid as a positive control for collagen increase, and a fourth product was the moisturizer lotion. Retinoic acid treatment produced a significant increase in fibrillin-1, a biomarker for connective tissue. And the 6% lipopeptide, retinyl palmitate formulation produced significant increases in fibrillin-1 and procollagen 1 levels in the dermis. It was suggested that these levels indicated partial repair of photoaged human skin [35]. A human tripeptide GHK (glycyl-L-histadyl-LIysine) complexed with copper has been reported to increase skin collagen and to reduce fine lines and depth of wrinkles in clinical studies [36].
Conclusion In conclusion, several approaches for reducing the appearance of wrinkles have been used in various cosmetic products. In some cases, such as with sunscreens and botox, the products are not cosmetics and are regulated by other FDA centers. AHAs and retinoids can be irritating to the skin, particularly when users are exposed to the sun. Moisturizers and sunscreens can be used with fewer side effects.
Cross-references > Aging
and Anti-aging Strategies Anti-aging Ingredients > Topical Growth Factors for Skin Rejuvenation > Topical Peptides and Proteins for Aging Skin > Cosmetic
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References 1. Pitman S. Personal care products buck downward retail sales trend (2009) Cosmeticsdesign.com. http://www.cosmeticsdesign.com/ financial/personal-care-bucks-downward-retail-sales-trend (accessed on January 30, 2009). 2. Kligman AM, Lavker RM. Cutaneous aging. The difference between intrinsic aging and photoaging. J Cutan Aging Cosmet Dermatol. 1988;1:5–12. 3. Watson REB, Long SP, Bowden JJ, et al. Repair of photoaged dermal matrix by topical application of cosmetic ‘‘antiageing’’ product. Br J of Dermatol. 2008;158:472–477. 4. Kligman AM, Zheng P, Lavker RM. The anatomy and pathogenesis of wrinkles. Br J Dermatol. 1985;113:37–42. 5. Federal Food, Drug, and Cosmetic Act, Section 201 (i). 6. Federal Food, Drug, and Cosmetic Act, Section 201 (g). 7. FDA – CDER web. Summary of Key Points on Over-the-Counter Sunscreen Products. August 23, 2007. 8. Walker NPJ, Lawrence CM, Barlow RJ. Physical and laser therapies. In: Burns, T, Breathnach, S, Cox, N, Griffiths, C (eds) Rook’s Textbook of Dermatology, 7th ed, Vol 77. Oxford: Blackwell Publishing, 2004, pp. 1–24. 9. Clark DP, Hanke CW, Swanson NA. Dermal implants: Safety of products injected for soft tissue augmentation. J Dermatol Surg Oncol. 1989;21:992–998. 10. Le Palumbo A, d’Ischia M, Misuraca G, et al., Mechanism of inhibition of melanogenesis by hydroquinone. Biochim Biophys Acta. 1991;1073:85–90. 11. Skin bleaching drug products for over-the-counter human use; Proposed rule. Federal Register. 71(167):51146–51155, August 29, 2006. 12. Draelos D. Therapeutic moisturizers. Dermatol Clin. 2000;18: 597–607. 13. Bikowski J. The use of therapeutic moisturizers in various dermatologic disorders. Cutis. 2001;68:3–11. 14. Wertz P. Lipids and barrier function of the skin. Acta Derm Venerol. 2000;(Suppl 208):7–11. 15. Hashizume H. Skin aging and dry skin. J Dermatol. 2004;31: 603–609. 16. Watkinson A, Harding C, Moore A, et al. Water modulation of stratum corneum chymotryptic enzyme activity and desquamation. Arch Dermatol Res. 2001;293:470–476. 17. Van Scott E, Yu R. Alpha hydroxy acids: Procedures for use in clinical practice. Cutis. 1989;43:222–228. 18. Van Scott E, Yu R. Actions of alpha hydroxy acids on skin compartments. J Geriatr Dermatol. 1995;3(Suppl A):19A–24A. 19. Kraeling M, Bronaugh R. In vitro percutaneous absorption of alpha hydroxy acids in human skin. J Soc Cosmet Chem. 1997;48:187–197. 20. Anderson F (ed). Final report on the safety assessment of glycolic acid, ammonium, calcium, potassium, and sodium glycolates, methyl, ethyl, propyl, and butyl glycolates, and lactic acid, ammonium, calcium, potassium, sodium, and TEA-lactates, methyl, ethyl, isopropyl, and butyl lactates, and lauryl, myristyl, and cetyl lactates. Int J Toxicol. 1998;17(Suppl 1):1–241. 21. Kaidbey K, Sutherland B, Bennett P, Wamer W, Barton C, Dennis D, Kornhauser A. Topical glycolic acid enhances photodamage by
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Gender, Ethnicity and Lifestyle Differences
92 Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences Miranda A. Farage . Kenneth W. Miller . Howard I. Maibach
Introduction The number of those over the age of 60 in the USA is expected to nearly double by 2025 [1], with half the US population predicted to be older than 65 by 2030 [2]. Life expectancy in industrialized countries is expected to reach 100 years by about 2025 [3]. Aging is a complex, multifactorial process characterized by a deterioration of the ability of the body to maintain homeostasis, an increase in vulnerability to environmental insults, and a progressive loss of both structural integrity and physiological function [4]. Obviously, however, the process of physiological aging does not occur in all individuals at the same rate. Aging involves both intrinsic and extrinsic processes, occurring in parallel [5], that individually affect the pace at which the integument ages [6]. Intrinsic aging, the inevitable natural deterioration of body structure and function with age, is genetically determined and proceeds at a fairly predictable rate, affected by the inherent toxicity of certain by-products of metabolism and a lack of sufficient physiological resources dedicated to somatic maintenance and repair [7]. Estrogen depletion after menopause drives gender differences at the rate at which skin ages [8]. Significant differences in skin structure and lipid content at different anatomical sites also have the potential to influence the process of intrinsic aging [9]. Extrinsic aging is the result of exogenous insults such as infectious agents, environmental toxins, and ultraviolet (UV) radiation [10] which attack skin integrity daily, causing thousands of DNA alterations per cell each day [10]. UV exposure, in particular, creates cumulative skin damage which accentuates the results of chronological aging [11]. More than 90% of deleterious age-related changes in the appearance of skin, particularly facial skin result from UV damage [12] (> Fig. 92.1). In addition, lifestyle factors like smoking or nutritional choices can modulate the effects of both genetic and environmental aging [7].
Although hormone replacement therapy can delay the effects of intrinsic aging in estrogen-deficient older women [13], intrinsic aging is inevitable. Many of the components of extrinsic aging, however, can be avoided, with substantial decreasing of both morbidity and premature mortality in older individuals [6].
Factors That Contribute to Skin Aging Intrinsic Factors Gender Estrogen and other sex steroids have a significant influence on skin biology and structure, and are known to affect epidermal keratinocytes, dermal fibroblasts, melanocytes, hair follicles, and sebaceous glands [14]. Women have more estrogen receptors than men. Skin thickness has been observed to increase over the menstrual cycle with rising levels of estrogen [15]. Estrogen increases DNA repair capacity by 25% [16]. A woman’s skin contains numerous estrogen receptors; nearly every structural and functional change which accompanies the menopause in women has been demonstrated to be at least partially reversible with estrogen replacement therapy. It is believed that threshold levels of estrogen are required to maintain skin integrity [17]. Skin aging can be significantly delayed by the administration of estrogen at menopause [14]. Estrogens may be involved in regulating the inflammatory response, as improvements in inflammatory skin disorders such as psoriasis have been reported during pregnancy [18]. It also appears to offer some protection against skin photoaging [14, 19]. Numerous differences between the skin of men and women have been observed. In young women, the skin has less collagen content than in men; collagen content decreases dramatically after menopause, which makes
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Figure 92.1 The deep wrinkles, age spots, and leathery skin indicate premature aging caused by years of unprotected exposure to the sun (Courtesy Dr. Brian Gray)
women [24]. In general, skin changes in women were correlated more strongly with the estrogen deficit of menopause than with chronological age.
Ethnicity
older women’s skin age rapidly [16]. In men, skin thins gradually, about 1% per year; the thickness of women’s skin remains relatively constant until menopause, and then thins dramatically in a fairly short time [20]. Women experience a substantial decrease in skin elasticity after menopause, a drop which does not occur to the same degree in men [16]. Skin roughness, however, increases with age at a lesser rate in women [21]. Dermal wounds heal more quickly in women than in men [22]; but mucosal wounds heal more rapidly in men than in women, probably due to relatively high levels of testosterone in saliva [22]. A direct relationship exists in men between skin thickness and collagen content, a correlation which is absent in women of reproductive age [20]. Mortality rates for all skin cancers are significantly lower in women [23]. The distinct impact that the hormonal deficiency of menopause has on the skin has recently begun to be recognized [16]. Many women report a sudden onset of skin aging – thinner skin, wrinkling, dryness, and decreasing firmness and elasticity – several months after onset of menopause, as estrogen levels plummet [13]. In arguably the clearest evidence of a genuine gender difference in the rate of skin aging, non-invasive laser imaging of collagen and elastin revealed that the ratio of collagen to elastin fibers in the dermis decreased with age at a faster rate in
Differences in skin pigmentation have the greatest effect on skin aging related to ethnicity, as this property lends the skin differing abilities to respond to UV-light insult [25]. High levels of pigmentation offer protection from the cumulative effects of photoaging, with blacks showing little cutaneous difference in signs of aging between exposed and unexposed sites [26]. In addition, pigmentation provides black skin up to a 500-fold level of protection over white skin from UV radiation (based on skin cancer rates) [27]. Basal cell carcinoma and squamous cell carcinoma occur almost exclusively on sun-exposed skin of light-skinned people [28]. Studies of ethnic differences in skin aging have been confounded by non-uniformity in various investigational parameters, particularly by varying definitions of race versus skin type. Often, small numbers of people were investigated. Most studies have focused on white, black, or Asian skin, with Hispanic or Native Americans less often included, with little attempt to delineate among the significant variety of skin types that each category may contain. For example, American blacks are now considered racially distinct from African blacks [29]. Other factors which need standardization are anatomical site, season, and hormonal status. For example, very substantial differences in desquamation have been found by anatomical site [25]. Despite methodological issues, however, some distinct racial differences in the aging of skin have been observed. Caucasian skin wrinkles and sags, with a much higher risk of skin cancers. Darker skin winkles at a much slower pace [30]. Aging Asian and black skin experiences hyperpigmentation and uneven skin tone [31, 32]. Understanding ethnic differences in skin biochemistry would provide foundation for more effective skin care products and treatments targeted to ethnically diverse populations [33]. A comparison of structural and functional differences in skin of different races is displayed in > Tables 92.1 and > 92.2.
Anatomical Variations Huge variations in some skin parameters have been observed with respect to the body site studied, underscoring
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
92
. Table 92.1 Comparison of racial differences in structural skin properties Parameter
Comparison
Reference
Stratum corneum Thickness
Equal in blacks and whites
[91, 92]
Number of cell layers
Higher in blacks than whites
[93]
Higher in whites than Asians Resistance to tape stripping
Higher in blacks than in whites
[93]
Lipid content
Higher in blacks than in whites (>two-fold)
[94]
Electrical resistance
Higher in blacks than in whites (two-fold)
[95]
Desquamation
Higher in blacks than in whites (two-fold) lower in blacks than in whites
[96, 97]
Corneocyte size
Equal in blacks, whites, and Asians
[96]
Ceramide content
Asians > Hispanic > Caucasian > blacksa
[98]
Variability in parameters
Higher in blacks than in whites
[93]
Water content
Higher in blacks than in whites
[41, 97, 98]
Higher in Asians than in blacks Higher in Hispanics than in blacks NMF levels
Lower in Asians than in blacks or Caucasians
[25]
Higher in Asians
[99]
Dermis Dermal thickness Collagen content
Higher in Asians
[99]
Melanin content
Higher in Asians
[99]
Melanosome size and distribution
Black skin: large, uniform size, single units with membrane
[100]
Asian skin: small, uniform size, clustered in groups of up to 10
[100]
White skin: Melanocyte size and shape highly variable, clustered in groups of up to 10
[100]
Black skin: keratinocytes contain mainly Stage IV melanosomes
[100]
Asian skin: keratinocytes contain Stage II, III, and IV melanosomes, Stage IV predominates
[100]
White skin: keratinocytes contain mainly Stage II and III melanocytes
[101]
Black skin; UV induces all stage melanosomes
[101]
Asian skin: UV induces primarily Stage II and III melanosomes
[101]
White skin: UV induces primarily Stage IV melanosomes
[101]
Less in Asians
[100]
Melanosome stage in keratinocytes
Melanosome induction
Ground substance Sebaceous glands Sebum production
Glands bigger, sebum production higher in blacks than in East Asians; East Asians [25] higher than Hispanics; Asians higher than Caucasians
a
50% lower in blacks compared to Caucasians or Hispanics.
a need to standardize both the study site as well as the age range of the population in order to obtain meaningful comparative results [34]. Large differences in skin SC thickness exist at different body sites: about 3 stratum
corneum cell layers on the eyelid, 5 layers in most nonexposed areas of the body, but up to 50 on the soles of feet [35]. The decrease in epidermal thickness with aging was found to be smaller at the temple than at the volar
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Table 92.2 Comparison of racial differences in functional skin properties Permeability
In vitro penetration of fluocinolone acetonide
Lower in blacks than in caucasians
[102]
In vitro penetration of water
No difference
[102, 103]
Differences
Transepidermal water loss
Topical application of anesthetic mixture
Less efficacy in blacks than in Caucasians
[104]
In vivo penetration of C-labeled dipyrithione
Lower in blacks (34% lower) than in Caucasians
[105]
Methylnicotinate-induced vasodilation
Time to peak response equal than Caucasians
[106–108]
Slower in blacks
Baseline TEWL (in vitro)
Higher in blacks
TEWL in response to SLS irritation Higher in blacks and Hispanics (in vivo) Baseline TEWL (in vivo)
Skin irritant reactivity
[108, 109]
Higher in blacks (in vitro)
Blacks > Caucasians > Asians
[110] [98]
Return to baseline TEWL after tape Blacks faster than whites stripping
[111]
Reactivity to SLS (measured by TEWL)
Higher in blacks than in Caucasians
[109]
Reactivity to dichlorethylsulfide (1%)
Lower in blacks (measured by erythema, 15% vs. 58%) [112] than in Caucasians
Reactivity to 0-chlorobenaylidene Lower, longer time to response in blacks than in malonitrile Caucasians
[113]
Reactivity to dinitrochlorobenzene
Lower in blacks, but trend toward equalization after removal of stratum corneum than in Caucasians
[93]
Reactivity to octanoic acid, 20% SLS, 100% decanol, 10% acetic acid
Asians more reactive than in Caucasians (react more [114] quickly)
Stinging response
Lower in blacks than in whites
[11, 115–117]
Equal in blacks and whites Higher in Asians than in whites Stinging response
UV protection factor of stratum corneum
Higher in blacks (about 50% higher) than in Caucasians
[87]
Skin transparency
UVB transmission in stratum corneum
Lower in blacks (about 50% lower)
[87]
Spectral emittance
Lower in blacks (above 300 nm: 2–3-fold)
[118]
Photoprotection of epidermis
UV protection factor of epidermis Higher in blacks (fourfold)
[87]
UVA transmission through epidermis
Lower in blacks (almost fourfold)
[87]
UVB transmission through epidermis
Lower in blacks (fourfold)
[87]
Contribution of malpighian layer
Black skin: twice as effective in absorbing UVB as white skin
[87]
Skin extensibility on dorsal (sun exposed) and volar (sun protected) forearms
Black skin maintains extensibility on sun-exposed [41] sites, but Hispanic skin extensibility is reduced on sun exposed sites
92
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Table 92.2 (Continued) Consequence of photoaging
Response to insult
Somatosensory function
Elastic recovery
Black skin maintains recovery on sun-exposed sites, white and Hispanic skins reduced
[41]
Drying
Higher in Caucasian and Asians than in Hispanics and [119] blacks
Hypertrophic scarring
Higher in Asians than in Caucasians
[120]
Pigmented dermatoses
Higher in Asians than in Caucasians
[120, 121]
Wrinkling
Average onset is 10 years later in Asians than in Caucasians
[121]
Wrinkling
Average onset 20 years later in blacks than in Caucasians
[100]
Thermal tolerance
Blacks have a lower threshold than whites
[122]
Elastic recovery (tested on the cheek)
1.5 times greater in black as compared with white subjects
[25, 97]
SLS = sodium lauryl sulfate; TEWL = transepidermal water loss; UV = ultra violet; UVA = UV band A; UVB = UV band B.
forearm [36, 37] – which may be the effect of cumulative photoaging. The lipid composition of human stratum corneum displays striking regional variation in both content and compositional profile [38]. There is a much higher proportion of sphingolipids and cholesterol in palmoplantar stratum corneum than on extensor surfaces of the extremities, abdominal, or facial stratum corneum [38]. There is also an inverse relationship between the lipid weight percentage of a particular body site and its permeability [38]. Subcutaneous tissue with age depletes selectively in the face as well as the dorsal aspects of hands and shins [39]. Skin rigidity is much higher at the forehead than at the cheek in postmenopausal women [40]. In areas of the body with high blood flow (e.g., lip, finger, nasal tip, forehead), blood flow decrease with age [34]; in areas with normally low blood flow, no difference was observed [34]. In all races, significant differences in conductance exist between the volar and dorsal forearms [41]. The decrease in sensory perception with aging is more pronounced in the nasolabial fold and cheek, than in the chin and forehead [38]. It is commonly assumed that aged skin is intrinsically less hydrated, less elastic, more permeable, and more susceptible to irritation [36, 42] due to an apparently impaired functional barrier [43] as measured by higher TEWL [36, 43]. The highest TEWL in all phases of life is measured on the lips, a value three times higher than that of the surrounding face, which is also higher than most other body sites [44]. Interestingly, the upper lip more hydrated than the lower [45].
A high density of estrogen receptors is present on the genitalia, as well as on the face and lower limbs. Estrogen acts on both epidermis and dermis [16]. Estrogen receptors alpha and beta are found in varying densities by anatomic site and have different affinities for specific estrogen types, so that the potency of specific estrogens varies from tissue to tissue [16]. The morphology and the physiology of the vulva and vagina undergo numerous changes associated with hormonal changes at menopause [42]. Skin in the vulvar area derives from three different embryonic layers. The cutaneous epithelia of the mons pubis, labia, and clitoris originates from the embryonic ectoderm and exhibits a keratinized, stratified structure similar to the keratinized, stratified skin at other sites [46]. The mucosa of the vulvar vestibule originates from the embryonic ectoderm and is non-keratinized. The vagina is derived from the embryonic mesoderm and is responsive to estrogen cycling [36, 42]. There are fewer estrogen receptors on the vulva than on the vagina [16]. After menopause the following changes occur: vaginal epithelium atrophies, cervico-vaginal secretions become sparse, vaginal pH rises, atrophic vaginitis becomes more common [36, 42], collagen and water content decrease, pubic hair grays and becomes sparse, the labia majora loses subcutaneous fat, and also the labia (labia minora, vestibule, and vaginal mucosa) atrophies [36, 42]. In addition, vaginal secretions decrease and the thinned tissue is more easily irritated and susceptible to infection [36, 42]. The cumulative effect of estrogen deficiency contributes to poor wound healing [47]. Skin collagen content and thickness also decrease as a consequence of
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
hormonal decline after removal of the ovaries [48]. Also, dramatic hormonal changes, particularly thyroid, testosterone, and estrogen, alter epidermal lipid synthesis [38]. In people with limited mobility, atrophied genital tissue is more susceptible to shear forces and may be more susceptible to the pH changes and enzymatic action associated with incontinence [36, 42]. Vulvar skin is more resistant to tape stripping and recovers more quickly in younger subjects than in older ones [49]. Also, vulvar skin has an increased rate of epidermal turnover [36, 49] and increased basal cutaneous blood flow [36, 49]. Although aged forearm skin has a less frequent and slower reaction to sodium lauryl sulfate (SLS) irritation younger forearm skin, while no age-related differences were observed in the vulvar area [50]. Elderly patients are susceptible to contact dermatitis in the vulvar area, principally due to urinary moisture under occlusion. Urinary ammonia elevates local pH, which alters barrier function, further compromising skin integrity thereby increasing risk of infection [36, 42]. The vulvar area is more susceptible to persistent vulval itch and irritation in old age [51, 52]. A combination of factors contributes to this, including sweating, occlusion, vaginal discharge, friction, use of hygiene products, and incontinence. There is a significant decrease in the size and number of free nerve endings in aged skin in genital mucous membranes, with a corresponding decrease in sensory perception in the genital area [36, 39].
Extrinsic Factors Lifestyle Influences The skin is at high risk of damage from reactive oxygen species because its high level of vascularity creates a high degree of exposure to oxygen and because its exterior surface is exposed to atmospheric oxygen. The skin is also exposed to reactive oxygen species generated by UV light. Damage to epidermal cells and subcutaneous tissues caused by the reactive oxygen species manifests as skin aging [53]. The skin is particularly susceptible to oxidative damage because of its high lipid content. Skin proteins and DNA are also sensitive to oxidation [54].
Nutrition Over the last decade, fruits, vegetables, legumes, herbs, and teas have been found to contain antioxidative
compounds [53, 55]. In a study evaluating 4,025 middle-aged American women, higher vitamin C intakes were associated with a decreased risk of wrinkles (odds ratio 0.89; 95% confidence interval [CI] 0.82 to 0.96) and a decreased risk of senile dryness (odds ratio 0.93; 95% CI 0.87 to 0.99) [56]. Higher linoleic acid intakes were associated with a decreased risk of senile dryness (odds ratio 0.75; 95% CI 0.64 to 0.88) and a lower likelihood of skin atrophy (odds ratio 0.78; 95% CI 0.65 to 0.95) [56]. In a study of the protective effects of nutrition among Greek, Australian, and Swedish subjects, resilience to photoaging was associated with a higher intake of vegetables (p < 0.0001), olive oil (p < 0.0001), fish (p < 0.0001), and legumes (p < 0.0001); and with a lower intake of butter (p < 0.0001), margarine (p < 0.001), sugar (p < 0.01), or dairy products (p < 0.01) Vegetables, legumes, and olive oil appeared to be particular protective, collectively explaining 20% of the variance in resistance to photoaging.. Among Australian Anglo-Celtics, prunes, apples, and tea explained 34% of protective variance [53]. In another study, higher fat intake was associated with an increased risk of wrinkles and skin atrophy. Higher carbohydrate intake was associated with increased risk of skin atrophy, with findings independent of age, race, social status, UV exposure, menopausal status, body mass index, exercise, or energy intake [56]. Vitamin A has also been observed to decrease production of matrix metalloproteinases (MMPs), associated with degradation of the extracellular matrix [57]. Fish oil consumption can confer an SPF as high as 4 [58]. Vitamin C deficiency delays healing [59]. Ambient conditions such as temperature and humidity also affect the skin. An increase in skin temperature of 7 F to 8 F doubles the evaporative water loss [9]. Low temperature stiffens skin and decreases evaporative water loss even with plenty of humidity in air, as structural proteins and lipids in the skin are critically dependent on temperature for appropriate conformation [9]. Extreme changes in ambient humidity in either direction lower skin hydration [9]. Some medications affect the skin as well, particularly hypocholesterolemic drugs, which may induce abnormal increased desquamation [60]. By far, however, the two exogenous factors that exact a heaviest toll on the skin are smoking [61] and exposure to UV light. Cigarette smoking is strongly associated with elastosis in both sexes, and with telangiectasia (red spots on skin) in men [62]. Smoke causes damage to collagen and elastin in lung tissue and may do so in skin as well [62]. Nicotine constricts the vasculature [62], causing a decrease in capillary blood flow in skin, which may
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Figure 92.2 Smoking effect and wrinkles (with permission from the Procter & Gamble Co.)
contribute to wrinkling [48]. Smoking significantly affects the ability of the skin to repair itself, most likely related to effects on the vasculature [63–65]. Smoking increases the risk of poor outcomes such as skin necrosis after facial cosmetic surgeries (including rhytidectomies and face lifts) [66]. Frequent smokers (defined as greater than 1 pack/day) were observed to have three times the risk of skin necrosis after facial skin procedures than former smokers, those who had never smoked, and those who smoked at a much less frequent level [67]. Smoking increases keratinocyte dysplasia and skin roughness [68]. A clear dose–response relationship between wrinkling and smoking has been demonstrated [61]; (> Fig. 92.2) smoking contributes more to facial wrinkling than sun exposure [62]. In fact, smoking was shown to be an independent risk factor for premature wrinkling when age, sun exposure, and pigmentation were controlled [62]. Moreover, although hormone-replacement therapy reverses wrinkling in older patients, the skin of long-time smokers did not respond [48]. In one study, the relative risk for moderate to severe wrinkling for current smokers compared to that of life-long nonsmokers was 2.57 (CI: 1.83 to 3.06; p < 0.0005) [48]. Wrinkle scores were three times greater in smokers than in nonsmokers, with a significant increase in the risk of wrinkles after 10 pack-years [69]. (Pack years are calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked; e.g. 10 pack years is the equivalent of smoking 1 pack a day for 10 years, or 2 packs a day for 5 years.) [69] Smoking is an important risk factor in cutaneous squamous cell carcinoma [62]. Specific effects of smoking on the skin are shown in > Table 92.3.
92
. Table 92.3 Effects of smoking on skin structure and function Effects
References
Increased appearance of age
[123, 124]
Decreased collagen production
[125]
Induction of MMPs
[126]
Degradation of collagen by MMPs
[125]
Degradation of elastin fibers by MMPs
[125]
Increased reduction of elastotic material
[127]
Degradation of proteoglycans by MMPs
[125]
Increase in circulating free radicals
[125]
Increased sallowness
[79, 128]
Increased wrinkling
[129]
Increased roughness
[129]
Decreased cutaneous blood flow
[130, 131]
Decreased skin temperature
[131]
Reactive hyperemia
[132]
Subcutaneous oxygen saturation
[133]
MMP = matrix metalloproteinases.
Exposure to Ultraviolet Light (Photoaging) As people age, intrinsic changes occur in the skin, such as decreases in skin cell turnover, chemical clearance, thickness and cellularity, thermoregulation, mechanical protection, immune responsiveness, sensory perception, sweat and sebum production, and vascular reactivity [36, 70]. These changes manifest as generalized skin atrophy with few structural alterations up to the age of 50, which is followed by slow deterioration in skin condition [9]. In contrast, exposure to solar UV light initiates a flurry of molecular and cellular responses that promote rapid and dynamic changes in the skin [9]. Photodamage is damage produced in tissue by single or repeated exposure to UV light (> Figs. 92.3 and > 92.4). This is believed to account for the vast majority of the skin’s age-related cosmetic changes as well as certain clinical problems [36, 70]. UV light induces photochemical changes that can lead to either acute effects (e.g., erythema or sunburn) or chronic effects (e.g., premature skin aging, neoplasms) [36, 71]. UV band B ([UVB] 290 to 320 nm) is considered the primary causative agent of UV effect on skin, causing modification gene expression as well as mutations in DNA [36]. Ultraviolet radiation is a complete carcinogen in that it both initiates cancers through DNA mutations and promotes cancer growth through the inflammatory processes inherent in cumulative UV exposure [36, 72].
989
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Figure 92.3 The spectrum of ultraviolet radiation
. Figure 92.4 The depth of penetration of UV radiation of different wavelengths into the skin: UVB mainly affects the epidermis, while UVA penetrates deeper into the dermis
Modern Western culture has promoted tanned skin as healthy, resulting in steadily increasing rates of skin cancer and prematurely aged skin [36, 51] (> Fig. 92.5). Senescence in aged cells creates an extended window of time in which DNA mutations can accumulate and eventually result in carcinogenesis. UV exposure is the largest contributor to the detrimental aging of skin [36, 51]. It is believed to account for
more than 80% of facial aging [73]. It has recently been reported that it may be UV band A (UVA) that is responsible for the bulk of epidermal skin damage (> Figs. 92.3 and > 92.4). UVA excitation of trans-urocanic acid initiates chemical processes that result in photoaging of the skin [74]. Virtually all Caucasian westerners with normal recreational practices have subclinical signs of skin damage on
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Figure 92.5 Sun tan is a defense against the sun which arises as a result of UV induced skin damage. Sunscreens protect the skin from burning rays (Reproduced with permission from the American Academy of Dermatologyß 2008. All rights reserved)
exposed skin by the time they are 15 years old [72], whereas changes become discernible in unexposed skin in the early 30s [36, 75]. In Caucasians, as little as one minimal erythema dose of UV radiation is sufficient to disrupt production of natural moisturizing factor (NMF) amino acids [76]. UV exposure which causes moderate pinkness but not sunburn in Caucasians increases MMP levels in the irradiated tissue by hundreds of times [77]. The same level of exposure also reduces collagen production by 80% [77]. The clinical signs of cutaneous photoaging include changes in visible skin color and surface texture [36, 51], including the early appearance of dyschromia and lentigines, loss of normal translucency or pink glow, sallowness, and the gradual appearance of telangiectasia, and purpura [36, 51]. Textural changes include increased roughness, frank keratoses, and the development of fine rhytides which progress to deeper folds and creases [36, 51]. Although plasma concentration of retinol increases with age, within the epidermis vitamin A is destroyed by sun exposure [78]. Although one primary effect of photodamage is skin thickening, severe UV exposure results in dramatic thinning [28, 36]. Sun damage creates a state of chronic inflammation, with ongoing release of proteolytic enzymes by inflammatory cells that disrupts the dermal matrix [28, 36]. Epidermal thickness increases, then decreases, with an eventual loss of epidermal polarity (orderly maturation) and increased atypia among individual keratinocytes [28, 36]. Another observed change is decrease in perfusion in aged skin, which is more
92
pronounced in photo-exposed areas [9]. Physical responses to UV exposure are displayed in > Table 92.4. UV radiation of the skin produces both local and distant effects [28, 36] (> Fig. 92.4). Irradiated skin was observed to have a decreased capacity for inflammatory response [36, 39]. UV light reduced the quantity of epidermal Langerhans cells and induced proliferation of suppressor T cells, facilitating tumor induction [36, 39]. Histologically, corneocytes in sun-exposed areas become pleomorphic with increasing anomalies that include retention of nuclear remnants, loss of lines of overlap, and roughening of border edges [11, 75]. However, the most dramatic histological differences between photoaging and chronological aging occur in the dermis [79]. UVA light penetrates more deeply (> Fig. 92.4). Although it does not cause pronounced erythema, it may damage dermis more than UVB light, as UVA radiation induces elafin expression in fibroblast, which inhibits proteolytic breakdown and leads to an accumulation of elastotic material in photodamaged skin [80]. The most prominent microscopic alteration in the structure of photodamaged dermis is the replacement of normal collagen fibers with large quantities of abnormal, thickened, tangled, non-functional elastic fibers, which finally degenerate into a nonfibrous, amorphous mass [81]. Damaged dermal tissue provides less support its vasculature, causing vessels to widen and become visible at the skin surface as telangiectasia [9]. At the genetic level, UV exposure modulates expression of Collagen I, III, VI genes, heat shock protein 47 (Hsp47) genes, and matrix metalloproteinase 1 (MMP 1), contributing to the general disruption of skin structure. Collagen I is time and age-dependently reduced after single UVexposure in human skin in vivo [36, 71]. Photoaging is associated with increased expression of MMP 1 and MMP 9, both involved in degradation of skin structure [36, 82]. With acute sun exposure, genes with reparative, protective, or apoptotic functions, as well as stress communication genes are rapidly activated [27, 83, 84]. Aging strikingly increases the expression of these genes when exposed to UV [85]. Skin repair processes can repair UV damage as long as chronic exposure is avoided. With chronic ongoing insult, however, acute reversible effects become chronic degenerative changes that accumulate in tissue over time [86]. One caveat to this discussion is that separating the effects of actinic damage from normal chronological degeneration of the skin over a lifetime can be difficult, as actinic effects are always superimposed on intrinsic changes [81]. The most common approach has been to compare exposed skin to unexposed skin at a different anatomic site, but constitutive anatomic differences may exist.
991
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Table 92.4 Response of human skin to UV exposure Responses
References
Acute surge in induction of MMP
[79, 134, 135]
Procollagen depleted
[79]
Procollagen synthesis upregulated, degradation of collagen by MMPs more significantly upregulated, net collagen decreased
[79]
Inflammation (acute)
[81]
Thickening of stratum corneum (acute)
[81]
Decrease in NMF and amino acid production
[76]
Induction of elafin, which binds to elastic fibers creating increase in elastotic material
[80]
Accumulation of elastic fibers
[136]
Accumulation of proteoglycans
[136]
Induction of c-fos, causing cellular proliferation
[5]
Down-regulation of c-myc, causing cellular division
[5]
Induction of GADD, response to injury
[5]
Induction of Il-1a, keratinocyte mitogen, fibroblast mitogen
[5]
Induction of Il-1b, keratinocyte mitogen
[5]
Induction of SRP2, involved in differentiation
[5]
Decreased HA levels
[73]
Increased chondroitin sulphate proteoglycans
[73]
GADD = growth arrest and DNA damage protein; HA = hyaluronic acid; Il-1a = interleukin-1 alpha; IL-1b = interleukin-1 beta; MMP = matrix Metalloproteinase; NMF = natural moisturizing factor; SRP = serine/arginine rich protein.
Pigmentation levels of black skin are photoprotective at all wavelengths of light [87], but white skin is transparent to both UV and visible wavelengths [88]. More UVA and UVB reaching the dermal layer in white skin more than in black (> Fig. 92.4). Consequently, racial differences in skin structure become more easily discerned as a result of photoaging. In whites, damage to the dermis is much more pronounced than in blacks, with focal areas of atrophy and necrosis [25]. The number of melanocytes decreases by as much as 20% per decade after the age of 30 in all races, but whites, with limited melanin-based protection, becomes increasingly susceptible with age to photodamage. What is more, melanin distribution also becomes patchy, with some areas eventually having little or no protection [81]. Characteristics of photoaging as compared with intrinsic aging are in > Table 92.5.
Pollution Chemicals in smog created by fossil fuel emissions are chemically altered in sunlight, creating ozone [7]. Ozone attacks the outer layers of the epidermis, depleting
antioxidants like vitamin E and ascorbic acid (vitamin C). With the skin’s endogenous antioxidants compromised, the skin is susceptible to oxidative damage [89]. Cutaneous exposure to common chemical components of pollution has been demonstrated to induce morphological changes to epidermal structure [90].
Conclusion In barely more than 20 years, half of the population is expected to be older than 65 [76]. Patients in this age group typically have multiple dermatological disorders that require treatment [2]. The field of gerontological dermatology, therefore, will inevitably grow in size and importance. The challenge at the present time is not only to optimize treatment of the variety of dermatological disorders associated with old age, but to gain understanding of the intrinsic and extrinsic mechanisms of skin aging in order to preserve the function and appearance of the integument into old age. Skin aging depends on a combination of intrinsic and extrinsic factors. Most intrinsic factors cannot be avoided, but extrinsic factors, such as sun exposure and smoking,
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
92
. Table 92.5 Comparing photoaging to intrinsic aging Characteristic
Photoaging
Intrinsic aging
Reference
Overall Metabolic processes
Pronounced increase
Slow down
[137]
Clinical appearance
Nodular, leathery, blotchy
Smooth, unblemished
[51]
Coarse wrinkles, furrows
Loss of elasticity, fine wrinkles
[51]
Skin color
Irregular pigmentation
Pigment diminishes to pallor
[27]
Skin surface marking
Markedly altered, often effaced
Maintains youthful geometric patterns
[28]
Onset
As early as late teens
Typically 50s–60s (women earlier than men)
[138]
Severity
Strongly associated with degree of pigmentation
Only slightly associated with degree of pigmentation
[27]
SPR-2 (differentiationassociated protein)
Decreased
Dramatically increased
[139]
Interleukin-1 antagonist receptor
Decreased
Dramatically increased
[139]
Acanthropic in early stages
Thins with aging
Gene expression
Epidermis Thickness
Atrophy in end stages
[140] [141]
Proliferative rate
Higher than normal
Lower than normal
[141]
Keratinocytes
Atopic, with polarity loss and numerous dyskeratoses
Modest cellular irregularity
[142]
Dermo-epidermal junction
Extensive reduplication of lamina dense
Modest reduplication of lamina dense
[28]
Vitamin A content
Destroyed by sun exposure
Retinol content of plasma increases
[84]
Elastin
Marked elastogenesis followed by massive degeneration, dense accumulations on fibers
Elastogenesis followed by elastolysis-‘‘moth eaten fibers’’
[142]
Elastin matrix
Massive increase in elastic fibers, replacing the collagenated dermal matrix
Gradual decline in production of dermal matrix, only modest increase in the number and thickness of elastic fibers in the reticular dermis
[78]
Lysozyme deposition on elastic fibers
Increased
Modest
[28]
Collagen production
Decrease in amounts of mature collagen
Mature collagen more stable to degradation
[141]
Dermis
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Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
. Table 92.5 (Continued) Characteristic
Photoaging
Intrinsic aging
Reference
Glucosaminoglycan Production
Great increase
No change
[141]
Grenz zone
Prominent
Absent
[141]
Microvasculature
Abnormal deposition of basement membrane-like material
Normal
[28]
Microcirculation
Vessels become dilated, deranged
Microvessels decrease, remaining vessels do not change
[28]
Horizontal superficial plexus virtually destroyed
Horizontal superficial plexus largely undisturbed
[28]
Pronounced inflammation, perivenular, histocytic-lymphocytic infiltrate
No inflammatory response observed
[28]
Inflammatory response SRP = serine/arginine rich protein.
can be reduced or eliminated, with substantial impact on the visible appearance of the skin. Greater understanding of the molecular processes which contribute to skin aging, as well as how lifestyle choices accelerate or impede those changes, may enable physicians in the future to prevent the underlying biological processes, improving both physical and psychological health for the increasing percentage of the population in the latter years of their lives. In addition, because of the association between solar UV exposure and skin cancer, encouraging people to avoid detrimental behaviors such as sun tanning would promote a youthful appearance and reduce cancer related mortality. This would be an important goal of patient education.
Cross-references > Aging
in Asian Skin Differences in Skin
> Gender
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46. Farage MA, Miller KW, Berardesca E, et al. Incontinence in the aged: contact dermatitis and other cutaneous consequences. Contact Dermatitis. 2007;57:211–217. 47. Brincat MP, Baron YM, Galea R. Estrogens and the skin. Climacteric. 2005;8:110–123. 48. Castelo-Branco C, Figueras F, Martı´nez de Osaba MJ, et al. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas. 1998;29:75–86. 49. Wilhelm D, Elsner P, Maibach HI. Standardized trauma (tape stripping) in human vulvar and forearm skin. Effects on transepidermal water loss, capacitance and pH. Acta Derm Venereol. 1991; 71:123–126. 50. Elsner P, Wilhelm D, Maibach HI. Sodium lauryl sulfate-induced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal women. J Am Acad Dermatol. 1990;23:648–652. 51. Glogau RG. Physiologic and structural changes associated with aging skin. Dermatol Clin. 1997;15:555–559. 52. Marren P, Wojnarowska F, Powell S. Allergic contact dermatitis and vulvar dermatoses. Br J Dermatol. 1992;126:52–56. 53. Purba MB, Kouris-Blazos A, Wattanapenpaiboon N, et al. Skin wrinkling: can food make a difference? J Am Coll Nutr. 2001;20: 71–80. 54. Kohen R. Skin antioxidants: their role in aging and in oxidative stress – new approaches for their evaluation. Biomed Pharmacother. 1999;53:181–192. 55. Cimino F, Cristani M, Saija A, et al. Protective effects of a red orange extract on uvb-induced damage in human keratinocytes. Biofactors. 2007;30:129–138. 56. Cosgrove MC, Franco OH, Granger SP, et al. Dietary nutrient intakes and skin-aging appearance among middle-aged American women. Am J Clin Nutr. 2007;86:1225–1231. 57. Varani J, Warner RL, Gharaee-Kermani M, et al. Vitamin a antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 2000;114:480–486. 58. Rhodes LE, Durham BH, Fraser WD, et al. Dietary fish oil reduces basal and ultraviolet B-generated PGE2 levels in skin and increases the threshold to provocation of polymorphic light eruption. J Invest Dermatol. 1995;105:532–535. 59. Goode HF, Burns E, Walker BE. Vitamin C depletion and pressure sores in elderly patients with femoral neck fracture. BMJ. 1992; 305:925–927. 60. Jackson SM, Williams ML, Feingold KR, et al. Pathobiology of the stratum corneum. West J Med. 1993;158:279–285. 61. Kennedy C, Bastiaens MT, Bajdik CD, et al. Effect of smoking and sun on the aging skin. J Invest Dermatol. 2003;120:548–554. 62. Leow YH, Maibach HI. Cigarette smoking, cutaneous vasculature, and tissue oxygen. Clin Dermatol. 1998;16:579–584. 63. Mosely LH, Finseth F. Cigarette smoking: impairment of digital blood flow and wound healing in the hand. Hand. 1977;9: 97–101. 64. Wilson GR, Jones BM. The damaging effect of smoking on digital revascularisation: two further case reports. Br J Plast Surg. 1984; 37:613–614. 65. Harris GD, Finseth F, Buncke HJ. The hazard of cigarette smoking following digital replantation. J Microsurg. 1980;1:403–404. 66. Riefkohl R, Wolfe JA, Cox EB, et al. Association between cutaneous occlusive vascular disease, cigarette smoking, and skin slough after rhytidectomy. Plast Reconstr Surg. 1986;77:592–595.
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67. Goldminz D, Bennett RG. Cigarette smoking and flap and fullthickness graft necrosis. Arch Dermatol. 1991;127:1012–1015. 68. Friedman O. Changes associated with the aging face. Facial Plast Surg Clin North Am. 2005;13:371–380. 69. WebRef.org. http://www.webref.org/cancer/p/pack_year.htm. cited 18Feb2009. 70. Barland CO, Zettersten E, Brown BS, et al. Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis. J Invest Dermatol. 2004;122:330–336. 71. Su¨del KM, Venzke K, Mielke H, et al. Novel aspects of intrinsic and extrinsic aging of human skin: beneficial effects of soy extract. Photochem Photobiol. 2005;81:581–587. 72. Green B, Bluth J. Measuring the chemosensory irritability of human skin. J Toxicol-Cutan Ocul Toxicol. 1995;14:23–48. 73. Baumann L. Skin ageing and its treatment. J Pathol. 2007; 211:241–251. 74. Reed JT, Elias PM, Ghadially R. Integrity and permeability barrier function of photoaged human epidermis. Arch Dermatol. 1997;133:395–396. 75. Bergfeld WF. The aging skin. Int J Fertil Womens Med. 1997; 42:57–66. 76. Verdier-Se´vrain S, Bonte´ F. Skin hydration: a review on its molecular mechanisms. J Cosmet Dermatol. 2007;6:75–82. 77. Fisher GJ, Varani J, Voorhees JJ. Looking older: fibroblast collapse and therapeutic implications. Arch Dermatol. 2008;144: 666–672. 78. Hanson KM, Simon JD. Epidermal trans-urocanic acid and the uv-ainduced photoaging of the skin. Proc Natl Acad Sci USA. 1998; 95:10576–10578. 79. Chung JH, Lee SH, Youn CS, et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch Dermatol. 2001;137:1043–1051. 80. Schalkwijk J. Cross-linking of elafin/skalp to elastic fibers in photodamaged skin: too much of a good thing? J Invest Dermatol. 2007;127:1286–1287. 81. Wulf HC, Sandby-Møller J, Kobayasi T, et al. Skin aging and natural photoprotection. Micron. 2004;35:185–191. 82. Bernstein IA, Vaughan FL. Cultured keratinocytes in in vitro dermatotoxicological investigation: a review. J Toxicol Environ Health B Crit Rev. 1999;2:1–30. 83. Sauermann K, Jaspers S, Koop U, et al. Topically applied vitamin c increases the density of dermal papillae in aged human skin. BMC Dermatol. 2004;4:13. 84. Seite´ S, Bredoux C, Compan D, et al. Histological evaluation of a topically applied retinol-vitamin C combination. Skin Pharmacol Physiol. 2005;18:81–87. 85. Sesto A, Navarro M, Burslem F, et al. Analysis of the ultraviolet b response in primary human keratinocytes using oligonucleotide microarrays. Proc Natl Acad Sci USA. 2002;99:2965–2970. 86. Ortonne J, Marks R. Photodamaged skin: Clinical Signs, Causes and Management. London: Martin Dunitz, Ltd., 1999. 87. Kaidbey KH, Agin PP, Sayre RM, et al. Photoprotection by melanin – a comparison of black and Caucasian skin. J Am Acad Dermatol. 1979;1:249–260. 88. Pathak M, Fitzpatrick T. The role of natural photoprotective agent in human skin. In: Fitzpatrick T, Pathak MA, Harver LC, Seiji M, Kukita A (eds) Sunlight and Man. Tokyo: University of Tokyo Press, 1974. 89. Romieu I, Castro-Giner F, Kunzli N, et al. Air pollution, oxidative stress and dietary supplementation: a review. Eur Respir J. 2008;31:179–197.
90. Giacomoni PU, Rein G. Factors of skin ageing share common mechanisms. Biogerontology. 2001;2:219–229. 91. Freeman R, Cockerell E, Armstrong J, et al. Sunlight as a factor influencing the thickness of epidermis. J Invest Dermatol. 1962; 39:295–298. 92. Thomson ML. Relative efficiency of pigment and horny layer thickness in protecting the skin of Europeans and Africans against solar ultraviolet radiation. J Physiol (Lond). 1955;127:236–246. 93. Weigand DA, Gaylor JR. Irritant reaction in Negro and Caucasian skin. South Med J. 1974;67:548–551. 94. Reinertson RP, Wheatley VR. Studies on the chemical composition of human epidermal lipids. J Invest Dermatol. 1959;32:49–59. 95. Johnson LC, Corah NL. Racial differences in skin resistance. Science. 1963;139:766–767. 96. Corcuff P, Lotte C, Rougier A, et al. Racial differences in corneocytes. a comparison between black, white and oriental skin. Acta Derm Venereol. 1991;71:146–148. 97. Warrier A, Kligman AM, Harpert R, et al. A comparison of black and white skin using noninvasive methods. J Soc Cosmet Chem. 1996;47:229–240. 98. Sugino K, Imokawa G, Maibach H. Ethnic difference of stratum corneum lipid in prelation to stratum corneum function. J Invest Dermatol. 1993;100:587 [Abstract 594]. 99. Kim MM, Byrne PJ. Facial skin rejuvenation in the Asian patient. Facial Plast Surg Clin North Am. 2007;15:381–386, vii. 100. Kelly AP. Aesthetic considerations in patients of color. Dermatol Clin. 1997;15:687–693. 101. Szabo G, Gerald A. The ultrastructure of facial color difference in man. In: Riley V (ed) Pigmentation: its genesis and biological control. New York: Appleton-Century Crofts, 1972. 102. Berardesca E, Maibach H. Racial differences in skin pathophysiology. J Am Acad Dermatol. 1996;34:667–672. 103. Bronaugh RL, Stewart RF, Simon M. Methods for in vitro percutaneous absorption studies. vii: use of excised human skin. J Pharm Sci. 1986;75:1094–1097. 104. Hymes J, Spraker M. Racial differences in the effectiveness of a topically applied mixture of local anesthetics. Reg Anesth. 1986; 11:11–13. 105. Wedig JH, Maibach HI. Percutaneous penetration of dipyrithione in man: effect of skin color (race). J Am Acad Dermatol. 1981;5: 433–438. 106. Guy RH, Tur E, Bjerke S, et al. Are there age and racial differences to methyl nicotinate-induced vasodilatation in human skin? J Am Acad Dermatol. 1985;12:1001–1006. 107. Berardesca E, Maibach HI. Racial differences in pharmacodynamic response to nicotinates in vivo in human skin: black and white. Acta Derm Venereol. 1990;70:63–66. 108. Kompaore F, Marty JP, Dupont C. In vivo evaluation of the stratum corneum barrier function in blacks, caucasians and asians with two noninvasive methods. Skin Pharmacol. 1993;6:200–207. 109. Wilson D, Berardesca E, Maibach HI. In vitro transepidermal water loss: differences between black and white human skin. Br J Dermatol. 1988;119:647–652. 110. Berardesca E, Maibach HI. Racial differences in sodium lauryl sulphate induced cutaneous irritation: black and white. Contact Dermatitis. 1988;18:65–70. 111. Reed JT, Ghadially R, Elias PM. Skin type, but neither race nor gender, influence epidermal permeability barrier function. Arch Dermatol. 1995;131:1134–1138. 112. Marshall E, Lynch V, Smith H. Variation in susceptibility of the skin to dichlorethylsulfide. J Pharmacol Exp Ther. 1919;12:291–301.
Determinants in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences 113. Weigand D, Mershon M. The cutaneous irritant reaction to agent O-chlorobenzylidene malonitrile (cs); quantitation and racial influence in human subjects. Edgewood Arsenal Technical Report No.4332, 1970. 114. Robinson MK. Population differences in acute skin irritation responses. race, sex, age, sensitive skin and repeat subject comparisons. Contact Dermatitis. 2002;46:86–93. 115. Frosch P, Kligman A. A method for appraising the stinging capacity of topically applied substances. J Soc Cosmet Chem. 1981; 28:197–209. 116. Aramaki J, Kawana S, Effendy I, et al. Differences of skin irritation between Japanese and European women. Br J Dermatol. 2002; 146:1052–1056. 117. Foy V, Weinkauf R, Whittle E, et al. Ethnic variation in the skin irritation response. Contact Dermatitis. 2001;45:346–349. 118. Anderson RR, Parrish JA. The optics of human skin. J Invest Dermatol. 1981;77:13–19. 119. Diridollou S, de Rigal J, Querleux B, et al. Comparative study of the hydration of the stratum corneum between four ethnic groups: influence of age. Int J Dermatol. 2007;46(Suppl 1):11–14. 120. McCurdy J. Cosmetic surgery of the asian face. In: Paper I (ed) Facial plastic and reconstructive surgery. New York: Thieme, 2002. 121. Nouveau-Richard S, Yang Z, Mac-Mary S, et al. Skin ageing: a comparison between Chinese and European populations. a pilot study. J Dermatol Sci. 2005;40:187–193. 122. Edwards RR, Fillingim RB. Ethnic differences in thermal pain responses. Psychosom Med. 1999;61:346–354. 123. Doshi DN, Hanneman KK, Cooper KD. Smoking and skin aging in identical twins. Arch Dermatol. 2007;143:1543–1546. 124. Rexbye H, Petersen I, Johansens M, et al. Influence of environmental factors on facial ageing. Age Ageing. 2006;35:110–115. 125. Morita A. Tobacco smoke causes premature skin aging. J Dermatol Sci. 2007;48:169–175. 126. Lahmann C, Bergemann J, Harrison G, et al. Matrix metalloproteinase-1 and skin ageing in smokers. Lancet. 2001;357:935–936. 127. Boyd AS, Stasko T, King LEJ, et al. Cigarette smoking-associated elastotic changes in the skin. J Am Acad Dermatol. 1999;41:23–26. 128. Smith JB, Fenske NA. Cutaneous manifestations and consequences of smoking. J Am Acad Dermatol. 1996;34:717–732, quiz 733–734. 129. Koh JS, Kang H, Choi SW, et al. Cigarette smoking associated with premature facial wrinkling: image analysis of facial skin replicas. Int J Dermatol. 2002;41:21–27.
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130. van Adrichem LN, Hovius SE, van Strik R, et al. Acute effects of cigarette smoking on microcirculation of the thumb. Br J Plast Surg. 1992;45:9–11. 131. Bornmyr S, Svensson H. Thermography and laser-Doppler flowmetry for monitoring changes in finger skin blood flow upon cigarette smoking. Clin Physiol. 1991;11:135–141. 132. Richardson DR. Effects of habitual tobacco smoking on reactive hyperemia in the human hand. Arch Environ Health. 1985; 40:114–119. 133. Jensen JA, Goodson WH, Hopf HW, et al. Cigarette smoking decreases tissue oxygen. Arch Surg. 1991;126:1131–1134. 134. Fiers SA. Breaking the cycle: the etiology of incontinence dermatitis and evaluating and using skin care products. Ostomy Wound Manage. 1996;42:32–34, 36, 38–40, passim. 135. Fisher GJ, Wang ZQ, Datta SC, et al. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997; 337:1419–1428. 136. Yin L, Morita A, Tsuji T. Skin aging induced by ultraviolet exposure and tobacco smoking: evidence from epidemiological and molecular studies. Photodermatol Photoimmunol Photomed. 2001; 17:178–183. 137. Soter NA. Acute effects of ultraviolet radiation on the skin. Semin Dermatol. 1990;9:11–15. 138. Kligman AM. The treatment of photoaged human skin by topical tretinoin. Drugs. 1989;38:1–8. 139. Garmyn M, Yaar M, Boileau N, et al. Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation. J Invest Dermatol. 1992;99: 743–748. 140. Takema Y, Yorimoto Y, Kawai M, et al. Age-related changes in the elastic properties and thickness of human facial skin. Br J Dermatol. 1994;131:641–648. 141. Lavker RM. Cutaneous aging: chronologic versus photoaging. In: Gilchrest B (ed) Photodamage. Cambridge: Blackwell Science, 1995. 142. Kligman A, Lavker R. Cutaneous aging: the differences between intrinsic aging and photoaging. J Cutan Aging Cosmet Dermatol. 1988;1:5–12.
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106 Facial Rejuvenation: A Chronology of Procedures Alexander S. Donath
Introduction The past decade has borne witness to a remarkable increase in the demand for facial rejuvenation [1]. Fueled in part by reality television programming and the associated increase in public familiarity with, and acceptance of, the available procedures, this trend is also a reflection of increasing life expectancies and the entrance of baby boomers into their fifth through seventh decades (40s through 60s) – a period of heightened manifestation of aging face stigmata. Indeed, this cohort, born between 1946 and 1964, [2] is characterized as being the healthiest and wealthiest generation to that time, and among the first to grow up genuinely expecting the world to improve with time [3]. It logically follows that with such optimism comes the desire to maintain a youthful countenance during the good times to come. While the estimated 80 million boomers are certainly a major driving force in the economy, the marketing dollars spent in pursuit of their spending power have also piqued the interest of other generations. This chapter aims to outline the procedures which are appropriate for the various decades of adult life, beginning when the earliest signs of aging present themselves – in the thirties. It should be emphasized that the following are general guidelines for the respective decades and that major factors such as ultraviolet light exposure and tobacco smoking, along with lesser factors such as diet and exercise, can accelerate the natural aging process. Thus, a heavy smoker who also sunbathes excessively and without UV protection will perhaps be a candidate for all procedures given below in his/her early forties, in contrast to his/her healthier counterparts in the expected decades, addressed below. Additionally, one’s genetic construct also certainly influences the period in which the stigmata outlined below become evident.
Thirties Most adults begin to show mild rhytides at rest (without animation) in the glabellar, forehead, and periorbital
regions in the early to mid-thirties. Some patients wish to prevent formation of these dynamic lines prior to their presentation at rest, as they note their appearance in animation in their late twenties and are frequently fearful of permanence, but the majority of patients tend to delay intervention until the persistence of rhytides at rest. The primary treatment modality for these rhytides is neurotoxin, specifically botulinum toxin type A (e.g., Botox Cosmetic1; Allergan, Inc., Irvine, CA). Botulinum toxin causes a temporary paralysis of the targeted muscle(s) by preventing acetylcholine release from motor nerve endings. The paralysis typically lasts for 3–4 months, with increased durability of effect realized by patients after multiple treatments at regular intervals; some patients are able to extend their treatment intervals to 6 months or more. Muscles most frequently treated in this fashion include the procerus and corrugator supercilii muscles, for horizontal and vertical glabellar rhytides, respectively; the frontalis muscle for horizontal forehead rhytides; and the orbicularis oculi muscles for lateral periorbital rhytides, commonly called ‘‘crow’s feet.’’ Additional sites for the use of neurotoxins in the later decades are outlined below. The specific US FDA approval for botulinum toxin A for cosmetic purposes is for glabellar lines alone, and Botox Cosmetic has shown an excellent safety profile since its approval in 2002 [4]. For patients who do not wish to receive neurotoxin injections or for whom they are contraindicated, facial resurfacing procedures are also effective in reducing fine rhytides. A variety of modalities are available for this aim, including medium strength chemical peels (e.g., 35% trichloroacetic acid [TCA]) and light-based therapies such as the erbium laser. In addition to the role of rhytides in portending age, there has been a dramatic increase in appreciation for the role of volume loss in the aging face [5]. This volume loss is exhibited in diminishment of skin thickness, subcutaneous adipose volume, and bony volume, as well as in muscular atrophy. The earliest site to yield evidence of this volume loss is the infraorbital region, beginning in the late twenties or early thirties. Here, patients begin to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_106, # Springer-Verlag Berlin Heidelberg 2010
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show the ‘‘tear trough deformity’’ or ‘‘dark circles’’ beneath the eyes. While commonly attributed to allergic, fatigue, or vascular etiologies, the elimination of these ‘‘circles’’ is most effectively achieved by volume augmentation of the tear trough region, rather than by reversal of the other proposed etiologies. Indeed, many patients who have previously used numerous under-eye ‘‘concealers’’ unsuccessfully in hopes of camouflaging what is perceived as darkly pigmented skin are relieved when volume augmentation is quickly effective. This is because volume loss results in a depression, or concavity, between the convexities of the bony inferior orbital rim and/or lower lid fat pads above and the cheek mass below. The appropriateness of a patient for volume augmentation here may be determined by shining light directly into the ‘‘dark circle,’’ perpendicular to the skin – this will fill the shadow in the tear trough created by overhead light, such as that in a typical examination room. If the resulting appearance is desired by the patient, they are excellent candidates for this procedure; any pigmentary issues are thus excluded as causative. Volume replacement can be performed
with either synthetic materials, such as hyaluronic acid (HA) fillers, or the patient’s own fat cells (> Fig. 106.1). The specific FDA approval for HA fillers is for ‘‘correction of moderate to severe facial wrinkles and folds, such as the nasolabial folds,’’ and volume restoration in the infraorbital area and others is therefore categorized as ‘‘off-label,’’ though well-accepted in the medical community [6–10]. The next most common site of use for filling materials in the practice in this decade is the lips. Restoration or augmentation of genetically suboptimal fullness in the vermillion of the lips is easily accomplished with synthetic fillers, such as the HA fillers. Certain anatomic norms must be respected, however, if the physician is to achieve a natural result: the upper lip vermillion show should not exceed one half of the lower in vertical height; the upper lip vermillion should be viewed as three separate aesthetic units: central, left, and right, with the lateral units comprising the majority of the horizontal length; and the lower lip vermillion is comprised of two aesthetic units – left and right – with a central sulcus between them.
. Figure 106.1 A 56-year-old patient who requested improvement in the infraorbital region. Patient shown before (left) and after (right) hyaluronic acid filler augmentation of the tear trough region, using Fig. 106.2). This reinflation of the infrabrow area can create an appearance of brow elevation, as elegantly demonstrated by Coleman [17]. As described below, when infrabrow skin excess exceeds the capacity for elevation by soft tissue augmentation, upper lid blepharoplasty, with excision of redundant skin, may be entertained as an adjunctive procedure or in isolation.
Fifties One of the most important endocrinologic events contributing to the aging face stigmata in women is menopause, for which the average age is fifty-one. The associated decline in circulating body estrogen has been linked to multiple facial cutaneous aging phenomena, including wrinkling, dryness, laxity, and atrophy; it has been demonstrated that some 30% of skin collagen is lost in the first 5 years after menopause, and that the average annual decline over 20 years following menopause is 2.1% [18, 19]. Estrogen replacement, particularly in the form of topical application of estriol, a weak estrogen, may ameliorate some of these effects and should be considered as part of a comprehensive skin care regimen in postmenopausal women [20, 21]. In the periocular region, the infrabrow shadow noted above and resulting from volume loss, progresses laterally across the infrabrow region creating an appearance of darkness across the upper eyelid region. In this sixth decade, the deflation of the infrabrow area also results in
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. Figure 106.2 Patient at 50 years of age before (above) and after (below) soft tissue augmentation in the superior orbital rim/infrabrow area using an HA filler. Note the age-associated atrophy in medial infrabrow soft tissue creating concavity with resultant shadowing as well as skin redundancy prior to treatment (above), the latter seen best on patient’s left side. Note improvement of atrophy and skin redundancy in posttreatment image (below) resulting in reduction of upper eyelid show to a more youthful height
greater accumulation of folds of skin near the eyelashes, which prompt visits to the facial cosmetic surgeon for blepharoplasty consultation to a larger extent than during the fifth decade. At this relatively early age, however, the degree of skin laxity is often best treated not by skin excision but by volume restoration in the infrabrow area; refilling the subcutaneous volume in this area results in radial expansion and superior elevation of the skin (> Fig. 106.2) and, frequently, the overlying brow hair. Along with the infraorbital filling, which is begun in the thirties and forties, such infrabrow filling aids in restoring highlights around the eyes, a technique frequently termed ‘‘framing the eye’’ [22]. The reader is encouraged to review covers of beauty and fashion magazines for depictions of society’s ‘‘ideal’’ periorbital constructs: a relatively low eyebrow, full infrabrow soft tissue with associated highlight, and a full lower eyelid/cheek interface. It is this anatomy that draws the viewer’s eye toward the iris and pupil. Careful of study of these anatomic relationships allows more natural restoration of the youthful visage, as eloquently demonstrated by Coleman [23]. Indeed
without such examination one may be beguiled by the optical illusion that a hollowed infrabrow region represents a descended brow and thereafter undertake to surgically elevate the brow, all too often creating an unnatural, surprised look [22, 24]. Additionally, the lateral brow descends earlier and to a greater extent than the central or medial brow, and therefore in practice greater emphasis should be placed on lateral browlifting than the more traditional lift, which incorporates all brow components. In the lower periorbital region, the tear trough deformity described above becomes more prominent, while the lower lid fat pads become more protuberant as well. Although the latter is partially due to weakening of the lower eyelid retaining structures, it is the same atrophy that causes the tear trough deformity to manifest that also results in uncovering of the infraorbital fat pads. Pseudoherniation of the lower eyelid fat pads may necessitate surgical removal, via lower eyelid blepharoplasty, particularly if they cannot be recovered by volume restoration in the tear trough and inferior orbital rim regions. Techniques for addressing lower eyelid fat pseudoherniation
Facial Rejuvenation: A Chronology of Procedures
include transconjunctival and transcutaneous blepharoplasty. The practice employs predominantly the transconjunctival approach, so as to limit the possibility of weakening the lower eyelid support mechanisms, which may result in lid retraction and scleral show [25]. Regardless of the chosen approach, it is recommended that blepharoplasty be done in conjunction with infraorbital volume restoration, either by formal lipotransfer or simply by lower lid fat transposition [26], to both increase the longevity of the result and to create a more youthful eyelid–cheek interface, which is one of convexity. Another anatomic site of volume loss is the lips. Frequently overtreated at the patient’s unwitting request, a conservative restoration of more youthful volume in the lip vermillion is an important aspect of facial rejuvenation. Here again, both synthetic fillers and autologous fat are excellent options. As noted above for patients in their thirties, the aesthetic norms should be respected in order to prevent an unnatural appearance. In addition to restoration of vermillion volume, however, patients in their fifties frequently begin to benefit from restoration of the philtral ridges and the white rolls, including the central ‘‘cupid’s bow’’ of the upper lip.
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Many patients begin to note an interruption of their mandibular jawline with ‘‘jowls’’ in their late forties or early fifties. While frequently attributed entirely to gravityinduced descent of the cheek and loss of cutaneous elasticity, jowling is also partially a function of subcutaneous atrophy in the cheek, with resultant hanging of the deflated tissue inferiorly under the influence of gravity; inverting the head readily demonstrates the effect of gravitational pull, with redirection of the deflated soft tissue superiorly toward the infraorbital area. Thus, gravity is not primarily causative, but rather simply determines the direction in which the deflated, atrophic cheek tissue hangs. Reinflation of the anterior cheek with fat or synthetic fillers aids in the reduction of jowling along the mandibular border. However, the primary benefit of volume augmentation in the anterior and lateral cheek is in restoring the convexities of youth, which have typically become replaced by concavities in this decade (> Fig. 106.3). Bony loss is also thought to contribute to jowling: atrophy of the mandible anterior to the jowl leads to the formation of the prejowl sulcus, creating a superomedial indentation in the overlying soft tissue, which exacerbates
. Figure 106.3 A 51-year-old woman who underwent fat transfer to the midface to complement facelifting, shown before (left) and 2 months postoperatively (right). Notable is the improvement in the cheek fullness seen in both the near and far cheek, and the associated improvement in continuity of the lid–cheek interface; neither benefit is likely with facelifting alone
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the appearance of jowling [27, 28]. Bony loss also occurs in the lateral mandibular region and thus, when combined with augmentation of the prejowl sulcus, volume restoration of this postjowl region may effectively postpone surgical elevation of the jowl (> Fig. 106.4). Once accumulation of jowls has progressed to a point unwelcome to the patient and if deemed too pronounced to be addressed by volume restoration alone, or if lipotransfer is otherwise not an option, facelifting becomes a viable remedy. There are many variations of this procedure, and a complete review of the techniques is beyond the scope of this chapter [29]. Most methods, however, rely on elevation of a skin flap, suture suspension of the underlying superficial musculoaponeurotic system (SMAS) in a superoposterior direction, redraping and trimming of excess skin, and suturing the cut skin edges. Facelifting addresses primarily the lower third of the face, from the nasal ala to the jawline and jowl; improvement is expected in the labiomandibular fold and jowl, as well as platysmal banding. Importantly, however, facelifting does not appreciably efface the nasolabial fold. Liposuction of the jowls, while unlikely to independently give a satisfactory result, is a useful adjunct to the above mentioned
techniques and is frequently performed in conjunction with cervical liposuction. For patients with more advanced platysmal banding and interplatysmal (midline neck) fat accumulation, directly addressing the neck through a submental incision – submentoplasty – is beneficial. In this procedure the neck skin is elevated in the subcutaneous plane, the interplatysmal neck fat – which is poorly responsive to liposuction – is excised sharply with scissors, and the medial platysmal edges are sutured together at the midline, from the immediate submental region to the level of the hyoid bone. A notch is then frequently created in the platysmal edges at this level to further define the cervicomental angle. Excess skin may also be removed by advancing the skin flap anteriorly and excising the redundant skin prior to closure of the submental incision. Finally, an often overlooked feature of the aging face is that of ptosis of the nasal tip. Typically manifesting in the late fifties and sixties, this presentation results from weakening of the fibrocartilaginous support of the nasal tip, allowing the lower lateral nasal cartilages to become more inferiorly directed. This is effectively corrected by placement of a cartilaginous graft, usually harvested from the nasal septum and placed in the columella, and is termed
. Figure 106.4 A 64-year-old woman who requested fat transfer for facial rejuvenation, which was performed in the pre and postjowl regions, as well as the midface and superior orbital rim/infrabrow area; lower lid transconjunctival blepharoplasty was also performed. Note the atrophic pre and postjowl regions prior to treatment (left), as well as upper eyelid dermatochalasis and lower eyelid pseudoherniation. Significant improvement was evident along the jawline at the 1-month postoperative visit (right), with a straightening effect without the employment of facelifting. Also notable is improvement in upper eyelid skin redundancy via volume restoration alone, and improvement in midfacial contour achieved with combination of blepharoplasty and lipoaugmentation; the mentum was augmented as well
Facial Rejuvenation: A Chronology of Procedures
a columellar strut graft. Rhinoplasty surgeons often also employ this grafting technique in the younger rhinoplasty patient as a preventative measure.
Sixties In addition to the regions of volume loss noted above in earlier decades, patients in their seventh decade show more advanced volume loss in the temporal regions, with a resultant stepoff lateral to the lateral orbital rim. Likewise, atrophy on the inferior aspect of the zygomatic arch, in the submalar region, becomes more evident, along with that of buccal region. Left uncorrected, these areas represent subtle cues of aging, which are often overlooked by cosmetic specialists unfamiliar with volume restoration. Periorbital, labial/perioral, and mandibular atrophy also become more pronounced in this decade and can be restored in conjunction with the perizygomatic and buccal areas. Facelifting is beneficial for most patients in their seventh decade, and progression of neck laxity is such that necklifting (submentoplasty) is usually performed in conjunction with nearly all facelifts in this age group, or can be performed in isolation for those patients who have previously undergone facelifting alone and have maintained their mandibular line. If the patient has not sought a facial resurfacing procedure in earlier decades, they will usually be candidates by their sixties, with most patients having progressed to a Glogau classification of III–IV (advanced to severe) [30]. Resurfacing at these stages frequently requires a deeper chemical peel, such as a croton oil/phenol peel, or a deeper laser resurfacing procedure [31–34]. Blepharoptosis, or descent of the upper eyelid margin over the iris and even the pupil of the eye, may become apparent in this decade and is addressed by a variety of suspensory procedures [35]. Dermatochalasis, or laxity of the upper eyelid skin, may begin as early as the fourth decade, but may progress to the point of obscuring the patient’s visual fields by the sixties, thus becoming of greater functional concern than of cosmetic significance alone, and is addressed by upper lid blepharoplasty. As noted above, volume loss in the infrabrow region, as well as loss of bony orbital rim, contributes to the deflation of the infrabrow skin, exacerbating the accumulation of skin resulting from loss of elasticity, or elastosis; volume restoration to the infrabrow region to complement upper eyelid blepharoplasty is therefore routinely recommended. Correction of lower eyelid skin redundancy may also become warranted during this decade and can
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be addressed with either a resurfacing procedure such as a chemical peel or laser resurfacing, or with surgical excision via lower eyelid transcutaneous blepharoplasty or skin pinch; caution must be exercised in lower lid skin excision, however, so as not to cause lower lid retraction and scleral show, or even lagophthalmos [36]. The brow of the seventh decade is more likely to show modest descent in the middle and medial portions in addition to the lateral descent noted above during the fifties. Thus, while many patients at this point benefit from lateral browlifting in conjunction with lipotransfer to the infrabrow region, patients in their later sixties may also require conservative elevation of the more medial components, as in endoscopic, coronal, or trichophytic browlifting; the emphasis nonetheless remains on lateral brow elevation.
Seventies and Beyond The eighth decade and beyond are manifested by a continuation of the processes noted in the above sections: gradual progression of subcutaneous volume loss, depletion of skin thickness with associated progression of facial rhytides, and elastosis with resultant skin laxity. Patients who have undergone facelifting or browlifting in their fifties or sixties may wish to undergo a repeat procedure to restraighten the jawline and resuspend their brows, respectively; blepharoplasty may be successfully repeated as well. The typical facelift should be expected to provide a youthful jawline and neck for approximately 7–10 years, although the associated skin removal is permanent, the fibrosis that occurs between the elevated SMAS and the redraped skin flap is permanent, and some surgeons use permanent sutures for elevation of the SMAS. Patients are informed that the aging process continues following surgical rejuvenation, with associated continued decline in skin thickness and elasticity, but that they will always display a more youthful countenance following such endeavors than if no such procedure had been undergone. Similar longevity is to be generally expected with other surgical results, including those of volume replacement with lipotransfer.
Future Directions Much excitement surrounds the seemingly limitless potential for stem cells in the rejuvenative efforts for the aging face. Adipose tissue has been shown to provide the richest source of stem cells in the body, by mass, and such fat-derived stem cells have been successfully encouraged
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in vitro to develop into nerves and blood vessels. It is hoped that, provided the proper biochemical milieu, adiposederived stem cells will develop into these and other tissues in vivo. With associated improvement in nutrient supply to the skin, fat, muscle, and bone of the face, it may be possible to achieve more durable maintenance of the youthful facial glow. Anecdotally, this has been noted in the practice and others in the months and years following autologous fat transfer to the face as a general improvement in the texture of the facial skin [37, 38]. A novel skin analysis system (Visia1, Canfield Imaging Systems, Fairfield, NJ) may provide important documentation of this effect, which might ultimately allow physicians to offer volume restoration and modest cutaneous resurfacing with a single procedure.
Conclusion Appreciation for the causative factors in the phenotype of the aging face is rapidly evolving. Along with this enhanced understanding come novel techniques for rejuvenation in each decade of age, each more natural and durable than its predecessors, but balanced by the public’s yearning for limited downtime and maximum safety. Such procedures are unlikely to wane in widespread societal acceptance, though modest fluctuations in adoption may occur with cyclical economic phenomena; it will be interesting indeed to evaluate the effects of the recession of 2008–2009 on the various cosmetic procedures. While the specific procedures sought will undoubtedly also undergo transformation, what seems constant is the quest for improved self-esteem that accompanies what the public regards as quick and effortless solutions provided by cosmetic procedures [39]. Also constant is the duty of the physician to ensure that motivating factors are healthy and recommended remedies are appropriate for the patient’s state of facial aging, whatever the chronologic age.
Cross-references >A
New Paradigm for the Aging Face Surgery in the Elderly
> Cosmetic
References 1. Liu TS, Miller TA. Economic analysis of the future growth of cosmetic surgery procedures. Plast Reconstr Surg. 2008;121:404e–412e. 2. http://www.census.gov/Press-Release/www/releases/archives/facts_ for_features_special_editions/006105.html. Entry dated 1/3/2006.
3. Jones, L. Great expectations: America and the Baby Boom Generation. New York: Coward, McCann and Geoghegan, 1980. 4. US FDA. http://www.fda.gov/bbs/topics/ANSWERS/2002/ANS01147. html 5. Donath AS, Glasgold RA, Glasgold MJ. Volume loss versus gravity: new concepts in facial aging. Curr Opin Otolaryngol Head Neck Surg. 2007;15(4):238–243. 6. US FDA. http://www.fda.gov/cdrh/pdf2/p020023a.pdf 7. Kane MA. Treatment of tear trough deformity and lower lid bowing with injectable hyaluronic acid. Aesthetic Plast Surg. 2005;29: 363–367. 8. Airan LE, Born TM. Nonsurgical lower eyelid lift. Plast Reconstr Surg. 2005;116(6):1785–1792. 9. Goldberg RA, Fiaschetti D. Filling the periorbital hollows with hyaluronic acid gel: initial experience with 244 injections. Ophthalmol Plast Reconstr Surg. 2006;22(5):335–341. 10. Carruthers JDA, Glogau RG, Blitzer A, et al. Advances in facial rejuvenation: Botulinum toxin type A, hyaluronic acid dermal fillers, and combination therapies – consensus recommendations. Plast Reconstr Surg. 2008;121(Suppl):5S–30S. 11. Carruthers J, Fagien S, Matarasso SL, et al. Consensus recommendations on the use of botulinum toxin type A in facial aesthetics. Plast Reconstr Surg. 2004;114(Suppl):1S–22S. 12. Monheit GD. Medium-depth chemical peels. Dermatol Clin. 2001;19(3):413–425, vii. 13. Hetter GP. An examination of the phenol-croton oil peel: part IV. Face peel results with different concentrations of phenol and croton oil. Plast Reconstr Surg. 2000;105(3):1061–1083. 14. Alster TS, Lupton JR. Erbium: YAG cutaneous laser resurfacing. Dermatol Clin. 2001;19(3):453–466. 15. Berlin AL, Hussain M, Phelps R, Goldberg DJ. A prospective study of fractional scanned nonsequential carbon dioxide laser resurfacing: a clinical and histopathologic evaluation. Dermatol Surg. 2009;35:222–228. 16. Fife DJ, Fitzpatrick RE, Zachary CB. Complications of fractional CO2 laser resurfacing: four cases. Laser Surg Med. 2009;41:179–184. 17. Coleman SR. Facial augmentation with structural fat grafting. Clin Plast Surg. 2006;33:567–577. 18. Brincat M, Moniz CF, Studd JWW, et al. Sex hormones and skin collagen content in postmenopausal women. Br Med J. 1983;287:1337. 19. Schuster S, Black MM, McVitie E. The influence of age and sex on skin thickness, skin collagen and density. Br J Dermatol. 1975;93:639–643. 20. Hall G, Phillips TJ. Estrogen and skin: the effects of estrogen, menopause, and hormone replacement therapy on the skin. J Am Acad Dermatol. 2005;53:555–568. 21. Baumann L. Hormones and aging skin. In: Bauman L, Weisberg E (eds). Cosmetic Dermatology: Principles and Practice. New York: McGraw-Hill, 2002, pp. 25–28. 22. Lam SM, Glasgold MJ, Glasgold RA. Complementary Fat Grafting. Philadelphia: Lippincott Williams & Wilkins, 2006, pp. 10–11. 23. Coleman SR. Facial augmentation with structural fat grafting. Clin Plast Surg. 2006;33:567–577. 24. Chiu ES, Baker DC. Endoscopic brow lift: a retrospective review of 628 consecutive cases over 5 years. Plast Reconstr Surg. 2003;112:628–633. 25. Palmer FR, Rice DH, Churukia MM. Transconjunctival blepharoplasty. Complications and their avoidance: a retrospective analysis and review of the literature. Arch Otolaryngol Head Neck Surg. 1993;119:993–999.
Facial Rejuvenation: A Chronology of Procedures 26. Goldberg RA, Edelstein C, Shorr N. Fat repositioning in lower blepharoplasty to maintain infraorbital rim contour. Facial Plast Surg. 1999;15(3):225–229. 27. Mittleman H. The anatomy of the aging mandible and its importance to facelift surgery. Facial Plast Surg Clin North Am. 1994;2:301–309. 28. Romo T, Yalamanchili H, Sclafani A. Chin and prejowl augmentation in the management of the aging jawline. Facial Plast Surg. 2005;21(1):38–46. 29. Perkins SW, Naderi S. Rhytidectomy. In: Papel ID, et al. (eds) Facial Plastic and Reconstructive Surgery, 3rd ed. New York: Thieme, 2009, pp. 207–226. 30. Glogau RG. Aesthetic and anatomic analysis of the aging skin. Semin Cutan Med Surg. 1996;15(3):134–138. 31. Rinaldi F. Laser: a review. Clin Dermatol. 2008;26:590–601. 32. Hetter GP. An examination of the phenol-croton oil peel: part IV. Face peel results with different concentrations of phenol and croton oil. Plast Reconstr Surg. 2000;105(3):1061–1083.
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33. Fulton JE, Porumb S. Chemical peels – their place within the range of resurfacing techniques. Am J Clin Dermatol. 2004;5(3): 179–187. 34. Carniol PJ, Harmon CB, Hamilton MM. Ablative laser facial skin rejuvenation. In: Papel ID, et al. (eds) Facial Plastic and Reconstructive Surgery, 3rd ed. New York: Thieme, 2009, pp. 321–330. 35. Baroody M, Holds JB, Vick VL. Advances in the diagnosis and treatment of ptosis. Curr Opin Ophthalmol. 2005;16:351–355. 36. Morax S, Touitou V. Complications of blepharoplasty. Orbit. 2006; 25(4):303–318. 37. Coleman SR. Structural fat grafting: more than a permanent filler. Plast Reconstr Surg. 2006;118(3 Suppl):108–120S. 38. Lam SM. A new paradigm for the aging face. In: Farage MA, Miller KW, Maibach HI (eds) Textbook of Aging Skin. New York: SpringerVerlag, 2009. 39. Haas CF, Champion A, Secor D. Motivating factors for seeking cosmetic surgery. A synthesis of the literature. Plast Surg Nurs. 2008;28(4):177–182.
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91 Facial Skin Attributes and Age Perception Alex Nkengne . Georgios Stamatas . Christiane Bertin
Introduction The age of a person is an important factor of social interactions. The way one acts, the verbal and body language one chooses to address someone else depends on age. Therefore, people since childhood develop a capacity to estimate age based on physical attributes. These attributes can be related to dress code or body gesture, but they are primarily linked to facial appearance. It is obvious that anyone can distinguish the face of a baby from a young adult and from a senior one since some evident characteristics such as the size of the head and the number of wrinkles are affected dramatically by age. As people get older the entire body is altered by chronological and environmental aging factors, with facial appearance often showing the most pronounced changes due to increased exposure to the outside environment. Chronological aging or intrinsic aging refers to the ongoing natural physiological changes in tissues. Environmental factors, such as sun exposure or life-related stress, affect the apparent or perceived age of an individual by modifying the aspect and the properties of his skin. In the case of excessive sun exposure, photoaging can lead to an increased gap between chronological and apparent age. Consequently, facial appearance of a person does not always reflect his/her chronological age. Some people look younger or older than their real age, depending on several factors including genetic disposition, sun exposure, smoking habits, lifestyle choices and mood. Some events in life like the death of a loved one or a long lasting disease can accelerate the process of apparent aging. Make-up, anti-aging creams, exercise, diet and surgery are a few of the methods people use to reduce their apparent age. In fact, the perceived idea of a person’s age is a subjective judgment that is influenced by the skin aspect. To better understand the factors affecting a person’s apparent age, the following questions need to be addressed:
Since apparent age is subjectively perceived by observers, is there a consensus on the apparent age that would be given to an individual? If not, will the observer be influenced by their own experience? What are the main facial skin attributes that drive age perception? Many studies have described the changes in facial attributes (skin color, wrinkles, sagging, micro relief, etc.) with age, but few have analyzed their influence on the perceived age. The primary objective of this chapter is to analyze the contribution of individual skin attributes of the face on the perceived age. Secondary objective is to assess the influence of age and gender of observers with regard to the age perception. Firstly, the different biases that affect the estimation of age are presented. Secondly, how the facial skin attributes have an effect on the perception of age has been evaluated.
What is Perceived Age? The human capacity to estimate age has probably been developed through the evolutionary process since age is a key point for social interaction. It is generally claimed that ‘‘Humans can easily categorize a person’s age group and are often be precise in this estimation’’ [1]. However, it can also be noticed that some people are easier to categorize than others. In fact, there is no strict consensus among observers when guessing someone else’s age. Thus, the factors of variability need to be explored that lead to the differences among observers and to build a consensus on how the ‘‘perceived age’’ will be defined.
Accuracy in Age Perception An experiment was conducted, asking 48 ‘‘graders’’ (20 men and 28 women, from 20 to 64 years of age) recruited from a Caucasian population living in Paris and its suburbs to
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_91, # Springer-Verlag Berlin Heidelberg 2010
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assess the age of 173 female subjects based on their ‘‘en face’’ facial images [2]. The age of these women was uniformly distributed between 20 and 74 years. Photographs were randomly presented to the graders on a computer screen. They were requested to estimate the subject’s age using a computer interface that featured a slider bar spanning from 0 to 100 for age estimation. There was no time limit for the grading. For 77.6% of the subjects, the given age follows a normal distribution (Jarque-Bera test of normality; significance = 95%) with no outlier. Therefore for each subject, the apparent age could be defined as the mean value from the age given by all graders. > Fig. 91.1 displays the correlation between perceived age and real age. They are highly correlated (R = 0.95, p < 0.001) and the residual follows a Gaussian distribution with a mean of 0.9 years and a standard deviation of 4.49 years. Even if the difference between real age and perceived one is not high (0.9), a pair-wise t-test shows that the perceived age is significantly lower than the real age (p < 0.01). This would mean that overall, the women of the dataset were perceived as 1 year younger than they are in reality.
. Figure 91.1 Mean age versus perceived age
Biases Affecting the Perception The ability to recognize and therefore to classify faces has been demonstrated to be plagued by several ‘‘own-group’’ biases. People are generally better at recognizing, remembering and classifying individuals belonging to their own group. The own-group bias effect has been found for such characteristics as age, race and gender; but the reasons underlying these biases are not clear yet [3]. One possible explanation is that people tend to interact more with others in their own racial, age and gender group and thus, are more trained (and efficient) with these categories of faces.
Own-Race Bias The own-race bias is known as the capacity for people to better recognize faces from their own race [4]. A metaanalysis [5] involving 35 articles with around 5,000 participants clearly highlights this bias, which is now taken into account for testimonies in New Jersey Courts. In fact it seems that face recognition in general, and age estimation in particular, involves learning processes through which people are trained by their environment. Dehon et al. [4] specifically studied the own-race bias in age estimation. They asked Caucasian and African participants to estimate the age of Caucasians and Africans from their facial pictures. Caucasian participants were better at estimating Caucasian faces than Africans’. However, Africans had the same performance for the two groups. Since the study was run in Belgium, the authors suggest that Africans had been trained during their daily life to recognize and to classify Caucasian faces. This hypothesis is in line with Wright et al. [6] who stated that the ability to recognize other-group faces depends on the degree of exposure and contact with people from these groups.
Own-Gender Bias Rehnman [3] has recently published a state-of-the-art review about the own-gender bias in face recognition. He concluded that women have greater accuracy in face recognition and could more easily recognize female faces than men. On the contrary, men did not show any difference in recognizing women’s or men’s faces. Looking at gender bias in age prediction, it has also been demonstrated that women and men do not estimate age the same way [6]. A hypothesis to explain these differences is that, depending on one’s age or gender, different facial attributes are focused on when estimating someone’s age.
Facial Skin Attributes and Age Perception
In evaluating the own-gender bias from the experiment mentioned above the population of imaged volunteers included only females, while the graders were males (N = 20) and females (N = 28). If an own-gender bias existed, the female graders should be more accurate in age prediction than men. To test this hypothesis, the perceived age given by women and men was compared; the perceived age given by women versus the real age and the perceived age given by men versus the real age using a pair wise t-test. The mean perceived age within male graders (mean = 44.93) was significantly different from real age (mean = 45.85, p < 0.010) and from perceived age within female graders (mean = 45.21, p < 0.05). The difference between perceived age by female graders and real age was not significant (p = 0.06). Male graders perceived that the imaged subjects look younger than they really were and female graders were more accurate in age prediction than male graders (> Fig. 91.2).
Own-Age Bias Own-age bias has been documented by several authors [7–9], with most of them evaluating the confidence that could be given to eyewitness testimony. A literature review by Anastasi and Rhodes [10] showed mixed and inconclusive results for own-age bias.
. Figure 91.2 Women are more accurate than men when predicting women’s age. The mean perceived age within male graders (mean = 44.93) is significantly different from the real age (mean = 45.85, p < 0.01) and from perceived age within female graders (mean = 45.21, p < 0.05). (a) Correlation curve between (b) Histogram of the real age and perceived age differences between real age and perceived age (residual)
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The underlying assumption in own-age bias is that people are judging another person’s age relative to themselves and should therefore be more accurate in judging their age range. An own-age bias has been reported by George and Hole [8]. They asked two groups (25 young adults and 25 old adults) to assess the age of faces aged between 5 and 70 years. In addition, the volunteers were asked to judge three images per age group. It appears that young people overestimate the age of older people, older people overestimate the age of younger people, and finally people are more accurate when estimating age within their own age group. Based on these results, So¨rqvist [11] tried to evaluate the own-age bias and improved age estimation accuracy by training. They asked two groups of people: young (15–24 years) and middle age (34–46 years) to evaluate 78 facial images from people in the three groups of age (young; middle age; old). They found that young participants were better with their age groups than with older people but the middle age group was not significantly better when judging people from their group of age. Thus, the own-age bias hypothesis was not totally confirmed. In exploring own age bias using the experiment described earlier, the populations of graders and evaluated subjects were divided into three groups: young (under 35 years), middle age (35–50 years) and senior (over 50 years). This segmentation is relevant to what is reported in the literature. > Table 91.1 shows the mean absolute error and the standard deviation for each age group of subjects and graders. The highest error and standard deviation is
. Table 91.1 Means and standard deviations of absolute values of errors in age prediction Subjects age Graders age
Under 35
35–50
Over 50
Under 35
4.55 (3.50)
5.98 (4.82)
6.21 (4.78)
35–50
4.98 (3.78)
6.43 (4.71)
7.52 (5.75)
Over 50
5.40 (4.70)
6.65 (5.02)
6.42 (4.92)
All graders
5.01 (4.06)
6.57 (4.95)
6.78 (5.33)
The means and standard deviations are calculated from the absolute values of the differences between real age and perceived age. Each cell corresponds to the mean and standard deviation of the errors from all the graders of an age group on subjects of an age group
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observed for the senior group of subjects. The youngest group of graders (less than 35 years) has smaller errors with a narrow distribution, being more accurate and having a better agreement than the other age groups. The smallest error and standard deviation are obtained when young graders judge young subjects. Thus the results confirmed in part, the own-age bias.
Facial Skin Attributes Influencing Age Perception The estimation of age is not only based on facial attributes, but also on hair, voice, body movement and posture [12]. When looking at a face to predict someone’s age, a 3D stimulus is being processed. This stimulus includes information related to shape, texture and color [13]. The aging process affects the following groups of attributes:
– Changes in shape: They occur in the spatial dimensions with a centimetric range. They may be caused by the growth of bones or loss of bones due to osteoporosis, changes in muscle and fat distributions, and skin sagging caused by the loss of elasticity and gravity. – Changes in texture: They happen in the spatial dimensions at a millimetric range. They are linked with skin aging and can affect wrinkles, pores and microrelief. – Changes in color: They are also linked with skin aging. They involve a more yellowish tone and color unevenness with the appearance of brown spots, freckles and scars. Consequently, skin transformation will have an important influence in the perceived age. Two kinds of approaches have been used to understand the influence of facial attributes on perceived age. Mono-dimensional approaches focus on a specific group of attributes such as wrinkles or color, and evaluate its impact on global perception. Multi-dimensional methods have recently been proposed. They employ a multidimensional statistical model to depict the relative importance of each facial attribute (including shape, texture and color attributes) in the overall perception.
Mono-Dimensional Approaches The principle of mono-dimensional approaches is to present stimuli of faces on which one specific attribute has been enhanced or reduced by image processing manipulations. These manipulations may include stretching the images to simulate the growth of bones, blurring to remove wrinkles,
applying a round mask to remove information about jaw line drawing, or change the pigmentation of the skin. Using image processing manipulations, one can separately investigate the influence of the shape, the texture and the color of the face/skin, on the perceived age.
Influence of Shape One way to explore the impact of shape transformations is to distort the facial images to mimic the cranium growth. However, the effect of the cranium on age perception only enables to distinguish children from adults [14]. The second way to study the impact of shape information on perception is to present inverted faces to observers (The mouth on the top and the eyes down). These kinds of stimuli enable to capture the importance of spatial interrelationship of facial features, such as the relative position of the nose, eyes and mouth. One can also use negative pictures, with inverted bright and dark areas. These stimuli distort 3D perception of volumes, making the faces look flatter. George and Hole [14] have presented original, inverted, negative and negativeinverted images of 27 faces from people aged between 0 and 80 years to 80 observers aged between 18 and 40 years. They found that negation and inversion do not affect the estimation of age while their combination does it for subjects under 50. Negative and inverted images were considerably over-estimated, with a poor agreement among the graders. George and Hole (The role of spatial Cues) suggested that observers were able to pick up the spatial cues left by the inversion or the negation. The combination of these two transformations disrupted too many cues, making it hard to guess the real age. Probably, none has explicitly focused on the influence of the sagging of the face (jaw line and chin). The stimuli of faces [8] with the shape masked by a rounded mask, meaning without jaw line information have also been presented. They notice that the spatial configuration of facial features, the color and the textural information were used successfully in that case.
Influence of Texture Textural information is related to the skin wrinkling aspect. Wrinkles are generally considered as the most visible signs of facial aging. Their importance on the perception of age was confirmed by several authors [8, 14–16]. Burt and Perret [15] presented 28 blinded faces and 40 normal faces as stimuli to 40 observers (20 young persons
Facial Skin Attributes and Age Perception
and 20 older persons, half male and female). The blinded faces did not capture textural information. They found that blinded faces were rated younger than they were and the error increased with the age. This experiment suggested that textural information is important for the perception of age. George and Scaife [14] focused on children’s ability to predict age on unfamiliar faces. They presented four different versions of facial photographs to 134 children between 4 and 6 years old. The photographs were taken from volunteers ranging in age from 1 to 80 years. The different versions of the face presented were: original image; internal facial features only (eyes-nose-mouth), skin-blur only, and overall blur (skin + features). Performances with the four sets of images were comparable, meaning that no conclusion could be drawn about the relative contribution of each facial feature. George concluded that the facial information related to the facial features and the skin texture were alternatively used for age prediction. However, his blur image also contained color information, meaning that one cannot really conclude on the importance of the texture only.
Influence of Color Skin color is affected by chronological and photo-aging, resulting in some changes in its hue, brightness and homogeneity. The impact of color changes has been explored by Burt and Perret [15]. They captured 147 Caucasian male faces, from volunteers aged between 20 and 62 years. They defined an algorithm to modify the shape or the color of the face to simulate the aging process. This algorithm caricatures differences between older faces and younger ones. Their methods modified the red, blue and green intensity distribution of the images, thus encompassing hue, saturation and lightness. The pictures were then presented to 40 observers for age estimation and the impact of color and shape transformation was analyzed. Burt and Perret found that each transformation increased the age significantly; their combination was even more effective. Fink et al. [17] studied the influence of the color homogeneity on the perceived age. They collected front and side pictures from 169 Caucasian women aged between 11 and 79 years of age. Facial features (mouth, eyes) and textural details such as wrinkles were removed to only keep information related to the skin color, leading to 2D skin color maps. The 2D skin color maps were then applied on a 3D standardized model of face. As a result, they obtained similar faces in terms of shape and texture,
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the only difference being the color hue and homogeneity. Four hundred and thirty observers were asked to give an age to the generated facial stimuli. The perceived age spanned from 17.8 to 36.7 years and was correlated with the chronological age (r = 0.708, p < 0.01). Fink et al. consequently concluded that the perceived age is influenced by the skin color distribution.
Multi-Dimensional Approaches Few attempts have been made to comparatively assess the facial signs of aging and to rank them according to their influence on age perception [2, 12, 18]. The multi-dimensional approaches have been proposed to study the influence of a large number of facial attributes on the perceived age. They differ from the mono-dimensional approaches because the facial stimuli are not altered as in that method. In general, facial images are presented to observers who are asked to grade for age. Then, an assessment is done to link the visual age to the facial characteristics of each face. This step can be done by describing the facial attributes qualitatively (open questionnaire) or quantitatively (clinical grading).
Qualitative Analysis Resbye and Povlsen [12] asked 40 graders to evaluate the age of 74 subjects older than 70 years old from their facial photographs. Then the graders were interviewed to report the main features that have driven their perception. Resbye and Powlsen found that age was assessed stepwise. First, the given picture would be classified over or under 80. After that, age would be refined first by decades, then by 5 years, and finally by single year. Almost all informants used information related to biological attributes. The main biological markers were eyes and skin. In the eyes area, the graders would have focused on wrinkles, bags under the eyes, sunken and ‘‘watery’’ eyes and finally the vitality of the gaze. Concerning the skin, the graders would have focused on wrinkles on the face and the neck, and second on pigmentation, color and sagging. Finally, the authors suggested that age estimation is more difficult on subjects with contradictory signs of aging such as a healthy skin but with an old-fashioned hair style. However, their method does not allow precisely evaluation of the weight of each attribute to the overall perception.
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Quantitative Analysis A statistical approach was proposed [2] to link the facial attributes and the perceived age. This approach enables to compare the influence of the different attributes and to rank them using a linear regression model. Regression methods can be used to study the dependence of the perceived age on several facial attributes. When the attributes are highly correlated, Partial Least Square (PLS) regression is the appropriate tool to understand the role of each attribute in the regression [19]. This regression model handles both highly correlated variables (facial attributes) and relatively small sample size [20] (173 subjects).The more important is an attribute, the highest is its weight in the model [21]. A trained grader was asked to evaluate 20 facial skin attributes from the 173 women whose pictures were taken (section Accuracy in Age Perception). The attributes related to the shape of the face were evaluated (Nasolabial fold, jaw line, lip volume, bags under the eyes, eye opening, slopping upper eyelid), its color (overall color, brown
spots, border lip definition, dark circles under the eyes, color uniformity) and its texture (Wrinkles: crow’s feet [wrinkles and fine lines], frown line, upper lip, cheek, forehead, under the eyes, microtexture). Seven PLS models were built to predict age from these facial attributes. The first two models allowed to predict the chronological and the perceived age. The three other models were used to predict the perceived age as given by the three groups of graders: young – middle age – seniors. The last two models were built to predict perceived age as estimated by men only and by women only. The weight of the facial attributes for each model was expressed as a percentage (the sum of the weight being set to 1), making it possible to compare the relative contribution of each attribute for different models. Perceived Versus Chronological Age
The model built when predicting the chronological age and the one built for the perceived age were put side by side. Some statistically significant differences were detected with a certain number of attributes (> Fig. 91.3). ‘‘Eye opening’’
. Figure 91.3 Comparison between PLS model of real age and perceived age Each bar chart represents the relative contribution of the facial attributes while building the PLS model for age prediction. The highest the value, the more important is the attribute for the given model. The attributes are ranked from the most important to the less important for chronological age. The parameters of which the contribution differs the most between the two models are skin color uniformity (p = 1.14E-59), lips border definition (p = 6.46E-69), under eye wrinkles (p = 7.43E-68), eye opening and bags under the eyes (p = 2.12E-42)
Facial Skin Attributes and Age Perception
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. Figure 91.4 Comparison between PLS models of perceived ages for different age groups of graders Each bar chart represents the relative contribution (in %) of the facial attributes while building the PLS model for age prediction. The higher is the value, the more important is the attribute for a given model. Attributes are ranked from the most important to the least important to predict perceived age by the youngest group of graders
and ‘‘Lip border definition’’ play an important role for chronological age while ‘‘Under Eye wrinkles’’, ‘‘Dark circle’’, ‘‘Bags under the eyes’’, ‘‘Skin Color uniformity’’ and ‘‘Brown spots’’ play a more significant role on the perceived age. These results suggest that the eye area and the skin color uniformity are overused when looking at a face for age assessment. Influence of Age and Gender
The models of the three age groups also presented some differences as shown in > Fig. 91.4. Particularly, the oldest group overused the attributes ‘‘eye opening’’, ‘‘lip border definition’’ and ‘‘lip volume’’. In contrast, they disregarded the attributes ‘‘nasolabial fold’’, ‘‘dark circles’’ and ‘‘brown spots’’. The comparison between the PLS models of perceived ages by men and women did not show any statistical significant difference. The study did not highlight any difference in terms of interpretation of attributes between men and women.
Conclusion In this chapter, the influence of different facial attributes on the perceived age has been discussed. The studies
reviewed and the results presented are limited to the Caucasian population. All the studies reported were done on facial pictures which are static stimuli. In reality, faces are dynamic and expressions and emotions may also contribute to the perceived age. In addition, people also use hair, clothes and body posture information when available [12]. While focusing on skin facial attributes, an attempt has been made to list all types of clinical changes with age and to review their incidence on age perception. The attributes were divided into three categories (shape, texture, color) for easier understanding. Two different methodologies were also described. The mono-dimensional approach enables to focus on a specific attribute and to evaluate its influence. The related works give interesting insights about the importance of the attributes. However, the method has two main drawbacks. First, it can be agreed with George et al. [14] who concluded that facial attributes are always used in combination. Therefore, a missing attribute from a modified stimulus may be replaced by another when guessing the age. For subjects with contradictory signs of aging [12], the results may be meaningless. Second, a facial stimulus which has been modified is no more a face and thus does not correspond to the regular experience of observers.
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The multi-dimensional approach uses facial pictures, which have not been transformed to avoid the bias. Multiple regression models enable to rank the facial attributes according to their weight in the perception. It is found that the importance of the nasolabial fold, dark circles, bags under the eyes, skin color and uniformity as well as brown spots decreases with graders’ age. In contrast, the importance of the lip area increases with graders’ age. Taken as a whole, the results suggest that the perception of aging is highly influenced by the appearance of the lip area (volume and upper lips wrinkles), the eye area (crow’s feet and under-eye wrinkles, dark circles, bags under the eyes) and the skin tone uniformity (brown spots, skin color uniformity). The importance of the eye area confirms the finding of Lanitis [22] and Rexbye [12] who also found skin wrinkling and color as important factors in perception. Compared with the two previous authors, the results also draw attention to the lip area and this finding might justify the recent popularity of fillers for lips.
Cross-references > Assessing
Quality of Ordinal Scales Depicting Skin Aging Severity
References 1. Gandhi M. A Method for Automatic Synthesis of Aged Human Facial Images. Montreal: McGill University, 2004. 2. Nkengne A, Bertin C, Stamatas G, et al. Influence of facial skin attributes on the perceived age of Caucasian women. J Eur Acad Dermatol Venereol. 2008;22:982–991. 3. Rehnman J, Herlitz A. Women remember more faces than men do. Acta Psychol. 2007;124:344–355. 4. Dehon HBS. An ‘‘other-race’’ effect in age estimation from faces. Perception. 2001;30:1107–1113.
5. Meissner C, Brigham J. Thirty years of investigating the own-race bias in memory for faces. Psychol Public Policy Law. 2001;7:3–35. 6. Daniel B, Wright BS. An own gender bias and the importance of hair in face recognition. Acta Psychologica. 2003;114:101–114. 7. Daniel B, Wright JNS. Age differences in lineup identification accuracy: people are better with their own age. Law Human Behavior. 2002;26:614–654. 8. George PA, Hole G. Factors influencing the accuracy of age estimates of unfamiliar faces. Perception. 1995;24:1059–1073. 9. Paul Willner GR. Alcohol servers’ estimates of young people’s ages. Drugs Educ Prev Pol. 2001;8(4):375–383. 10. Anastasi J, Rhodes M. Evidence for an own-age bias in face recognition. North Am J Psychol. 2006;8:237–252. 11. Sorqvist P, Eriksson M. Effects of training on age estimation. Appl Cogn Psychol. 2007;21:131. 12. Rexbye H, Povlsen J. Visual signs of ageing: what are we looking at? Int J Ageing Later Life. 2007;2:61–83. 13. Nkengne A. Predicting people’s age from their facial image: a study based on the characterization and the analysis of the signs of aging. ED 393 - SANTE PUBLIQUE: Epide´miologie et sciences de l’Information Biome´dicale University Pierre et Marie Curie. Paris: Paris VI, 2008, p. 137. 14. George PA, Hole GJ. The role of spatial and surface cues in the ageprocessing of unfamiliar faces. Visual Cognition. 2000;7:485–509. 15. Burt D, Perrett D. Perception of age in adult caucasian male faces: computer graphic manipulation of shape and colour information. Proc Roy Soc Lond B. 1995;259:137–143. 16. Montepare JM, McArthur LZ. The influence of facial characteristics on children’s age perceptions. J Exp Child Psychol. 1986;42:303–314. 17. Fink B, Grammer K, Matts P. Visible skin color distribution plays a role in the perception of age, attractiveness, and health in female faces. Evol Hum Behav. 2006;27:433–442. 18. Guinot C, Malvy D, Ambroisine L, et al. Relative contribution of intrinsic vs extrinsic factors to skin aging as determined by a validated skin age score. Arch Dermatol. 2002;138:1454–1460. 19. Tenenhaus M, La Re´gression P. The´orie et Pratique. Paris: Technip, 1998. 20. Wold S, Sjo¨stro¨m M, Eriksson L. PLS-regression: a basic tool of chemometrics. Chemom Intell Lab Syst. 2001;58:109–130. 21. Burnham A, MacGregor J, Viveros R. Interpretation of regression coefficients under a latent variable regression model. J Chemom. 2001;15:265–284. 22. Lanitis A. Digital Signal Processing, 2002. DSP 2002. 2002 14th International Conference on the significance of different facial parts for automatic age estimation. Department of Computer Science and Engineering, Nicosia, 2002.
93 Gender Differences in Skin Sarah Fitzmaurice . Howard I. Maibach
Introduction In order to intervene in skin aging, one must first have knowledge of the intricacies of gender-related skin differences. That men and women are genetically different goes without saying. How this affects phenotypic and functional differences between the sexes has been a topic much researched. In studying the skin, consideration is warranted as to the role that genetic and sex-specific environmental exposures may play in cutaneous appearance and structure. Environmental and occupational exposures are likely dictated by the social culture and biases therein that characterize each sex’s experience. These differences are biologically important as they can affect the interpretation of various skin examinations and the therapeutic decisionmaking process.
Structural and Anatomical Characteristics Skin thickness has been measured using echographic evaluation, ultrasonic echography, optical coherence tomography, x-ray, and histological analysis. Men have thicker skin than women across all age ranges [1–4], but no difference exists between men and women regarding the thickness of the stratum corneum [5, 3] or of the epidermis [6, 7]. Both men and women experience thinning of the epidermis and dermis with increasing age [4, 7, 8]. Hormone treatment in postmenopausal women increases dermal thickness [9, 10] as well as the number of collagen fibers [11, 10]. Both sexes show decreasing collagen content of the skin with age with the same rate of collagen loss, but total skin collagen content is less in women than men at all ages [8]. The evolutionary significance of these differences requires explanation. The distribution of body fat differs between sexes for both obese and non-obese individuals [12]. Men tend to accumulate fat in the upper body and abdomen whereas women tend to accumulate fat in the lower body, particularly in the gluteals and upper portions of the legs [13]. In a cohort of more than 2,000 subjects aged 6–18, there was no difference between the sexes up to age 12 in
fat distribution. After age 12, in females only, the relative mass of the subcutaneous fat continued to increase whereas in males it did not [14]. In addition to the distribution, the amount of fat differs between men and women. Japanese women aged 18–22 had greater subcutaneous fat thickness than men [15]. Regarding skin fold thickness, use of a caliper demonstrated that forearm measurements were thinner in women than men starting at age 35 [16], whereas another study found women to have thinner skin fold thickness at the forearm, thigh and calf in a younger age range of 17–24 years [17]. The evolutionary significance and mechanism remain elusive (> Table 93.1).
Biochemical Composition Casual sebum levels determined with a sebumeter in 46 Korean women and 37 Korean men were higher in men at five facial sites sampled [18]. Two studies with 29 and 12 subjects respectively, each with half men and half women, found no difference in casual sebum levels between men and women measured at multiple anatomical locations [19, 20]. Stratum corneum sphingolipid composition had significant age-related differences in women but not men. Ceramides 3 and 6 decreased from pre-puberty to adulthood along with an increase in ceramides 1 and 2. Ceramide 2 decreased and ceramide 3 increased following maturity [21]. The total skin surface lipid content was lower on the forehead, post-auricular area and dorsal forearm in women as compared to men [22]. Sex was demonstrated to be a factor in the quantity and population characteristics of the normal flora of skin. Women carried fewer organisms than men and the prevalence of specific organisms differed between the sexes [23, 24]. Metal content of human hair was found to differ in women but not men in regard to age. Women’s hair had a greater copper concentration than men’s which increased with age in women only [25]. Again, the evolutionary significance of these differences mandates explanation, as they might have functional and mechanistic significance (> Table 93.2).
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_93, # Springer-Verlag Berlin Heidelberg 2010
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Gender Differences in Skin
. Table 93.1 Structure and anatomy (Adapted with permission from Tur E [12]) Findings [Reference]
Obtained by
Subjects
Conclusions
Significant differences Women with thinner skin than men, except for the lower back of young subjects [1].
Echographic evaluation
24 women, 24 men; half 27–31 years, half 60–90 years
Women‘s skin thinner than Ultrasonic men‘s across entire age range of echography 5–90 years [2].
69 women, 54 men; 5–90 years
Cellular epidermis thicker in men Histologic analysis than women [3].
37 women, 34 men; 20–68 years. Skin types II–IV. Smoking status: 32 never, 27 previous, 12 current
Men with thicker epidermis than Histologic analysis women in the age group 50–60 years; men with thicker dermis in the age groups 20–30 and 70–80 years [4].
34 women, 30 men; 20–80 years
Men and women with decreasing epidermal thickness with increasing age between 20–50 years. Men and women with decreasing dermal thickness between 20–40 years [4].
Histologic analysis
34 women, 30 men; 20–80 years
Skin thickness constant in women up to 5th decade, then decreased with advancing age. Skin thickness of men decreased steadily with increasing age [8].
Chemical and histological analysis of skin collagen, skin thickness and collagen density
Collagen: 80 women, 79 men; 15–93 years. Thickness: 107 women, 90 men; 12–93 years. Density: 26 women, 27 men; 15–93 years
Total skin collagen content is greater in men than women at all ages, though the rate of collagen loss is the same in men and women
Dermal thickening after Conjugated estrogen 12 months estrogen therapy [9]. therapy
28 estrogen, 26 placebo; women Skin thickness affected by 51–71 years estrogens
Greater collagen content in hormone treated women vs. non-treated women. Inverse relationship between collagen content and increasing years since menopause in the untreated group. Greater skin thickness in hormone treated group vs. non-treated group [10].
108 postmenopausal women; 52 treated with estradiol and testosterone for 2–10 years, 66 no treatment. Look up age
Histologic analysis X-ray
Increased number of collagen Histological analysis fibers in the hormone treated group 6 months post-treatment, no change in the placebo group. No change in in epidermal thickness, keratin thickness, and elastic fiber content for both groups [11].
41 postmenopausal women: 21 estradiol and cyproterone acetate treatment, 20 placebo
Hormone treatment increases collagen fiber content in postmenopausal women
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. Table 93.1 (Continued) Findings [Reference] Women with greater lipoprotein lipase activity. Women with greater values in the gluteus; men with greater values in the abdomen [13].
Obtained by Hybridization and northern blot of lipoprotein lipase activity and mRNA levels
Subjects 8 women, 11 men; 33–41 years
Up to 12 years of age boys and Caliper girls both with greater than threefold increase in subcutaneous fat and less than doubling of internal fat mass. Girls only with increased relative mass of subcutaneous fat after the age of 12 [14].
1,292 women, 1,008 men; 6–18 years
Women’s subcutaneous fat thicker than men’s [15].
45 women, 41 men; Japanese. 18–22 years
Caliper Ultrasound
Starting at age 35 women have Caliper thinner forearm skin than men. Forearm skinfold thickness decreases starting at age 35 in women and age 45 in men [16].
145 women and men; 8–89 years
Lower skinfold thickness in women at thigh, calf and forearm [17].
42 women, 37 men; 17–24 years
Caliper
No significant differences No difference between men and Histologic analysis women in thickness of stratum corneum [3].
37 women, 34 men; 20–68 years. Skin types II–IV. Smoking status: 32 never, 27 previous, 12 current
No difference in the number of Histologic analysis cell layers in the stratum corneum between men and women. Both men and women with slightly increasing numbers of cell layers in the stratum corneum with increasing age [5].
158 men, 143 women; Japanese. 1–97 years
No difference in the thickness of Ultrasound the epidermis between men and women [6].
29 men, 61 women; Caucasian. 18–94 years, skin type I–III.
No difference in skin thickness Optical coherence between men and women at tomography five anatomic sites, except for women’s skin thinner on the forehead than men in the older group. Thinner epidermal thickness at all six sampled sites in the older group of both men and women compared to the younger group [7].
Young: 13 women, 17 men; 20–40 years; Caucasian. Old: 17 women, 24 men; 60–80 years, Caucasian. "Ethnic group": 6 women, 6 men; 20–40 years; skin types IV–VI
Conclusions Fat distribution and total fat content might be affected by regional sex differences in lipoprotein lipase activity. Variation at mRNA and psottranslational level
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. Table 93.2 Biochemical composition (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Casual sebum levels higher in men than Sebumeter women at five facial sites [18]
46 women, 21–37 years; 37 men, 23–39 years. Korean.
In women but not men stratum corneum sphingolipid composition differs with age [21]
27 women, 26 men; 10–79 years
Ethanolic extracts, biochemical methods of lipid identification
Stratum corneum sphingolipids influenced by female hormones
Women with lower skin surface lipid on Sebumeter forehead, forearm, postauricularly [22].
7 women, 23–26 years; 7 women, 72–77 years. 7 men, 28–29 years; 8 men 71–75 years
Greater metal concentration in women‘s hair. Copper concentrations increase in women with increasing age but not in men [23].
Liquid chromatography; trace metal determination
60 women, 72 men; 6–40 years
Prevalence of specific microorganisms considered to be normal flora different for men and women [24].
Microorganism culture of biologic samples
50 premature infants. 51 healthy babies; Age and sex affect 4–7 days old. 80 children; 3–12 years. composition of skin 166 healthy adults; 18–45 years. normal flora 63 adults; >60 years
Women with fewer microorganisms than men in the groin, axilla and thigh [25].
Microorganism culture of biologic samples
8 men, 8 women.
Sex affects composition of skin flora
No significant differences No difference between men and women Sebumeter in casual sebum level [19]
7 men, 7 women; mean 27 years. 7 men, 8 women; mean 70.5 years
No difference between men and women Sebumeter in sebum rate [20]
6 women, 6 men; 23–25 years. Skin types II–III
Mechanical Properties Results have been diverse from studies seeking to determine if sex is a primary determinant in the barrier function of the skin. Men had greater transepidermal water loss (TEWL) values than women [26–29], or no difference existed between the sexes [19, 20, 30]. Following 5 days of topical caffeine application, 6 of 9 men experienced decreased TEWL values whereas 0 of the 9 women studied did. It was hypothesized that androgens are damaging to barrier function through their ability to increase cAMP levels and because caffeine antagonizes the effects that androgens have on cAMP, its application decreased the negative effects of androgens, demonstrated by decreased TEWL measurements. A difference in skin hydration
and moisture has not been demonstrated between the sexes [20, 31, 32]. In vivo and in vitro data respectively found adhesion of the stratum corneum greater in women than in men [33], or no difference existed [34]. In vivo adhesion properties were assessed by measuring the speed of blister formation induced by controlled suction [33]. Women exhibited longer blistering times than men in the age range of 15–69, after which the difference dissipated. In vitro analysis of skin biopsies from multiple sites did not reveal any difference in stratum corneum adhesion between men and women [34]. Frictional properties as measured by a friction meter [30], skin elasticity as measured by two suction cup methods [35], and torsional extensibility as measured by
Gender Differences in Skin
a twistometer did not produce differing results between men and women [2]. In general accordance with these data, skin elasticity measured by a handheld probe demonstrated that at 9 of 11 sampled anatomical sites, no difference existed between the sexes, except at the volar forearm and forehead. The ratio between viscoelastic properties of the skin and immediate distention was greater in men at the forearm at a load of 500 mbar. The ability of the skin to return to its original position after deformation, as estimated by the ratio between immediate retraction and total distention, was greater in men than in women at the forehead [36]. Measurements of the foot demonstrated that the rate of stretch on traction, estimated from the series elastic element (SEE), was greater in women than in men. Conversely, the SEE on retraction was greater for men than for women on the foot. Skin plasticity of the foot was greater in women than in men [37]. Likewise, following hydration, skin extensibility found to be identical at baseline between the sexes increased in women only. It is inferred that hydration softens the stratum corneum allowing the thickness of the dermis to be the primary determinant in extensibility. In this situation, the thinner dermis of women allowed for a rapid extensibility of female skin (> Table 93.3) [38].
Functional Differences Eccrine Sweating Men have greater sweat rates compared to women [31, 39–41]. This has been found to be true across all age ranges and stages of sexual maturity [40]. Pre-pubertal and pubertal boys as well as adult men were found to have higher mean sweat rates when compared to girls and adult women in like age groups. For males, sweat rate increased with increasing age whereas in females, adult women had lower mean sweat rates than pubertal and pre-pubertal girls. This is challenged by one study which found that when anthropometric variables were accounted for, the greater sweat rate for men no longer existed [42]. This same study concluded that a greater overall sweat production for men was associated with a greater total sweat lactate production, but the lactate concentration did not differ between the sexes as it was proportional in both men and women to total sweat production. Evolutionary mechanisms and significance have not been explored.
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pH The pH of skin has been measured at multiple different sites in men and women with varying results in regards to gender differences. Measurements taken from the volar forearm have revealed no difference between men and women [19], men with lower mean pH values than women [20, 43], and women with lower mean pH values than men [44]. Similarly, measurements from the axilla have produced results of no difference between the sexes [45] or women with lower baseline pH values than men [46]. Men were also found to have lower pH values at five different facial sites sampled as compared to women. These results highlight the significance that sampling different locations can have, and also again emphasize the need for further elucidation regarding the existence and significance of sex differences (> Table 93.4).
Differences in Response to Irritants The reported incidence of irritant dermatitis is greater in women than in men [47], though there has been no consensus from experimental results as to which sex experiences more irritant dermatitis [12]. A review on gender differences in allergic contact dermatitis (ACD) concluded that sex was much less likely an endogenous factor predisposing to ACD but more likely a factor that influences environmental exposure to allergens. It was concluded that exposure history played the central role in determining a predisposition to ACD, with sex being a factor in determining exposure patterns, as opposed to sex determining intrinsic skin characteristics [48]. That sex is not a primary factor in determining response to irritants was highlighted by a study in which neither sex had a tendency toward stronger reactions to 11 different irritants in two groups of men and women, one with and one without hand eczema [49]. What has been shown in experimental results as well as anecdotal reports is that decreased barrier function of the skin and a subjective increase in the severity of symptoms related to skin diseases such as eczema occurs in the time period just prior to the onset of, or during menstruation [50, 51]. This would imply that hormonal factors related to sex do play a role in the reactivity of skin. Experimental results of skin prick testing in over 600 subjects found a small but significant difference with men having an increased response to histamine compared to women, determined by resultant wheel size [52]. This is in contrast to a study of just over 70 subjects in
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. Table 93.3 Mechanical properties (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Men with greater TEWL values [26].
Evaporimeter
8 men, 10 women; Caucasian. 18–28 years
Lower TEWL values in young Evaporimeter women than young men at 10/10 sites sampled. Lower TEWL values in elderly women than elderly men except at chest. Young women with greater TEWL than elderly men at 6/10 sites. Young men with greater TEWL than elderly women at 8/10 sites [27].
34 young men; 23–33 years. 34 young women; 25–35 years. 28 elderly men; 66–74 years. 35 elderly women; 64–76 years
Men with slightly higher basal TEWL than women. Men with decreased TEWL values 7 days post-HEC gel or 0.5% caffeine in HEC but not women [28].
7 women, 26–50 years; 9 men, Androgens are damaging to 28–45 years skin barrier function. Topical caffeine may be reparative to skin barrier dysfunction
Tewameter
Lower baseline TEWL in women Sodium lauryl sulfate than men; similar values in men irritation. Evaporimeter and women after irritation [29].
15 women, 23 men; 18–39 years
Blistering times longer in Time required for blisters women than men ages 15–69. to form by controlled No difference in older ages [33]. suction. Speed of dermalepidermal separation measured
178 women, 15–101 years; 209 men, 16–96 years
The ratio between the Cutometer visocelastic properties of the skin and immediate distention at a load of 500 mbar was greater in men at the volar forearm than for women. The ratio between immediate retraction and total distention greater in men than women at the forehead [36].
8 women, mean age 25; 9 women mean age 75; 8 men, mean age 28; 8 men, mean age 75
Series elastic element (rate of stretch) on traction greater in women than men. Series elastic element greater for retraction in men than women at three locations on the foot. Women with greater foot skin plasticity than men [37].
38 men, 49 women; 35–81 years
Cutometer
Female skin more irritable based on irritation index defined as the difference between irritated and unirritated values over irritated
Gender Differences in Skin
93
. Table 93.3 (Continued) Findings
Obtained by
Women with greater cutaneous Bioengineering methods extensibility only after hydration [38].
Subjects 15 women, 14 men; 23–49 years and 60–93 years
Conclusions The thinner dermis of women can be made more extensible once hydrated
No significant differences No difference in TEWL between Evaporimeter men and women [19].
7 men, 7 women; 23–29 years 7 men, 8 women; 56–84 years
No difference in TEWL or Tewameter stratum corneum hydration between men and women [20].
6 women, 6 men; 23–25 years. Skin types II–III
No difference in TEWL, moisture, or friction between men and women [30].
7 women, 25 years (mean); 7 men, 29 years. 7 women, 75 years; 8 men, 74 years
Bioengineering measurement
No difference in moisture Bioengineering; chronic between men and women [31]. renal insufficiency and healthy subjects
Healthy: 24 women, 21 men Patients: 30 women, 50 men
No difference between men and women in stratum corneum hydration or scaling [32].
Clinical assessment and bioengineering measurement
50 women, 22 men; 21–61 years.
No sex related difference in stratum corneum adhesion [34].
Biopsy; force needed to separate cells measured in vitro
9–34 women and men; number varied based on site sampled. 20–40 years
Capacitance, dynamic skin friction coefficient, or transepidermal water loss did not differ between men and women at 11 different anatomical locations [30].
Capacitance meter; friction meter; evaporimeter; thermistor
7 women, 23–26 years; 7 women, 72–77 years. 7 men, 28–29 years; 8 men 71–75 years
No difference in skin elasticity In vivo suction device between men and women [35].
Young: 8 women, 26 years; 8 men, 28 years. Old: 9 women, 75 years; 8 men, 75 years
No difference in torsional Twistometer extensibility between men and women [2].
69 women, 54 men; 5–90 years
which women produced larger wheels than men following histamine administration by iontophoresis [53]. Sodium lauryl sulfate (SLS) applied daily for 5 days followed by patch testing of the upper back did not demonstrate sex related susceptibility to developing irritant dermatitis [54]. The use of SLS in this experiment produced results that have been interpreted differently by separate groups due to opposing definitions of what constitutes the irritation index. The group who performed the study concluded that women had more irritable skin following SLS application as indicated by having a higher irritation index
determined from TEWL values. At baseline women showed lower TEWL values than men, but following irritation with SLS both sexes had similar TEWL values. The irritation index defined by the authors of the study was the ratio of the difference of the values of irritated and unirritated skin to the value of unirritated skin. Despite the values for irritated skin not differing between men and women, the index was higher in women given the lower baseline unirritated values. The authors concluded this to mean that women had more irritable skin, whereas a review article later challenged that the study showed no sex-related
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. Table 93.4 Functional differences (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Women sweat less than men [32].
Pilocarpine iontophoresis; chronic renal insufficiency and healthy subjects
Healthy: 24 women, 21 men; patients: 30 women, 50 men; 18–75 years
Men with greater sweat rate than women [39].
Scale weight pre and post-test; two separate experiments
Study 1: 40 women, 58 men. Study 2: 56 women, 56 men
Pre-pubertal boys, pubertal boys, Pilocarpine iontophoresis and adult men sweat more than girls and adult women of the same age group. Both sexes with increasing sweat secretion rate with increasing age. Adult men sweat more than pre-pubertal and pubertal boys; adult women sweat less than preprepubertal and pubertal girls [40].
Pre-pubertal: 67 girls, 68 boys; 6–13 years. Pubertal: 80 girls, 39 boys; 9–19 years. Adults: 34 women, 24 men; 20–75 years
Women in both the luteal and Ventilated capsule method; follicular phases sweat less than scale weight pre and men. Local sweat production less in post-test. Laser doppler women than men except at thigh. Cutaneous blood flow similar in men and women except at thigh where it is great in women [41].
10 women, 20–22 years; 6 men, 20–26 years
Women with higher mean pH than pH meter men at forearm [20].
6 women, 6 men; 23–25 years. Skin types II–III
Women with higher pH than men at forearm [43].
Glass electrode and PH meter
12 men, 8 women; 25–49 years
Women with lower mean pH than men at forearm [44].
pH meter
6 men, 31–59 years; 6 women, 26–54 years
Women with lower pH than men in pH meter axilla at baseline [46].
Sweat secretion rate dependent on sex and age. Change with age different for men and women
10 men, 19–29 years; 10 women, 26–55 years No significant differences
No difference between men and Capillary blood sampling; women in mean sweat rate when Capillary tube sampling sweat rate expressed per unit surface are. Blood and sweat lactate concentrations not different between the sexes [42].
6 men, 6 women; college aged
No difference between men and women in pH [19].
pH meter
7 men, 7 women; 23–29 years. 7 men, 8 women; 56–84 years
No difference in skin surface pH in axilla between men and women. No gender difference in sweat pH of axilla [45].
pH meter; pH glass probe; microprocessor pH meter
81 women, 105 men; 18–55 years
Men with greater overall sweat rate than women but difference no longer significant when body surface area accounted for. Greater overall sweat production associated with greater total sweat lactate secretion; no difference in sweat lactate concentration.
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. Table 93.5 Response to irritants (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Women with higher incidence of irritant dermatitis than men [47].
Likely role of occupational factors
Women with more frequent Literature review contact dermatitis to nickel than men, prevalence of piercing greater in women than men [48].
Exposure risk a greater factor than sex in developing contact dermatitis
Transepidermal water loss Evaporimeter higher on the day of minimal estrogen/progesterone secretion compared to the day of maximal secretion. TEWL greater with maximal progesterone secretion than maximal estrogen secretion [50].
9 women, 19–46 years
Eczema worsens in the time Literature review period immediately preceding or during menstruation [51].
Just prior to the onset of menses, skin barrier function is impaired as compared to the days just prior to ovulation
Cyclical patterns of skin disease symptoms related to the menstrual cycle
Larger wheels produced in women secondary to histamine [53].
Histamine administered by iontophoresis
33 women, 38 men; 15–52 years
Men with greater response than women to skin prick test with histamine. Increasing reactivity with increasing age [52].
Skin prick, forearm
307 men, 313 women; mean age 24 years
No significant differences between the sexes regardless of having or not having hand eczema [49].
Irritation tested for 11 irritants 21 women, 21 men, with at several concentrations hand eczema; 21 women, 21 men, without hand eczema. 20–60 years
Men and women with same incidence of cumulative irritant dermatitis [54].
Repeated once daily 7 women, 7 men; 16–65 application of three years concentrations of sodium lauryl sulfate, 5 days, followed by patch testing. Bioengineering measurements
Sex differences in the stratum corneum affect reactivity
No significant differences
differences in SLS irritation if the absolute end value of irritation was used to define the irritation index [12] (> Table 93.5).
Cutaneous Microvasculature The difference in skin blood flow between men and women is hormone-dependent [12]. Differences between men and women depend on sexual maturation and for women, the
Neither sex with stronger tendency to reactions. Occupational exposure may lead to greater irritant exposure
phase of the menstrual cycle [55]. Basal blood flow was lowest in the luteal phase, highest in the preovulatory phase. Compared to the other phases of the menstrual cycle, it was demonstrated that during the luteal phase finger skin perfusion had the greatest cold-induced constriction and the least recovery following [55]. Supporting evidence that female sex hormones influence skin circulation includes an increased occurrence of vasospastic diseases such as Raynaud’s phenomenon in women, an
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Gender Differences in Skin
increased prevalence during the reproductive years, and that these conditions improve during pregnancy [12, 55]. Sex hormones may be directly influencing the blood vessel wall or may be indirectly acting systemically causing a cyclical pattern in women [12]. The sympathetic nervous system is influenced by estrogen. In the presence of estrogen alpha 2-adrenoreceptors are up-regulated [12]. Consequently, laser Doppler flowmetry has shown a decreased basal cutaneous blood flow in women as compared to men provided age under 50 [56–60]. Administration of an alpha 2 antagonist decreased the local response to cooling in women only, supporting the sex difference in alpha 2 adrenoreceptors [61]. Administration of an alpha 1 antagonist decreased the response to cooling in both men and women. Following local heating both men and women experienced an increase in perfusion, though men continued to have a greater tissue blood flow than women [60]. In contrast to the aforementioned difference, laser Doppler has also shown no difference in baseline flux between the sexes measured at the hand [62]. In women, vasodilation in response to local heating occurred at a lower temperature [63]. Women had a greater decrease in laser Doppler flux ipsilaterally and contralaterally to local cooling as compared to men [62]. Additionally, young women when compared to older women and young men had a prolonged response to cooling [59]. However, the maximum cutaneous blood flow subsequent to heating the skin was not different between the sexes, nor was the postocclusive reactivehyperemia response in a study of women aged 20–59 [56]. On the contrary, one study that separated women according to age demonstrated both women over 50 and young men to have greater reactive hyperemia response than young women [59]. Based on the difference demonstrated between sexes in over 300 subjects, one group went so far as to propose that the accepted upper limit of normal capillary refill time be specified based on sex and age. This study was divided into three groups composed of males and females: children, adults 20–49 years, and elderly adults 62–95 years. Pediatric females, pediatric males, and adult males had significantly shorter capillary refill times than adult females, elderly females, and elderly males [64]. Mapping of skin blood perfusion using laser Doppler imagery was done following iontophoresis of acetylcholine, an endothelium-dependent vasodilator, as well as nitroprusside and isoprenaline, two different endothelium-independent vasodilators with different modes of action. Pre-menopausal women had a greater response to nitroprusside, and to a lesser extent acetycholine, than postmenopausal women reflecting a change in skin vasculature with aging [65].
No difference could be demonstrated between men and women in cutaneous blood flow response to topical and intradermal histamine administration the back, volar forearm, and ankle [66]. It was secondarily deduced that no functional difference exists between the sexes in the skin microvascular response to histamine [12]. Changes in oxygen pressure at the skin surface primarily determined by skin blood flow have been measured by assessing alterations in transcutaneous oxygen pressure [12]. Skin surface measurements have demonstrated that women have higher transcutaneous oxygen pressure than men [67, 68]. Supposing women have a thinner epidermis than men, this difference may be accounted for [12]. Transcutaneous oxygen pressure measurements revealead age-related sex differences during postocclusive reactive hyperemia. No difference existed between the younger sexes but adult women had greater values than adult men [69] (> Table 93.6).
Sensory Functions Thermoregulatory Response A difference between the sexes was demonstrated in a study of the physiological response to heat stress. However, when accounting for differences in anthropometric variables such as percent body fat and the ratio of body surface to mass, the effect of gender did not hold [70]. This highlights the impact sex specific variables can have on thermoregulation and the need to account for these when interpreting results [12]. In contrast, despite similar body surface area-to-mass ratios in a cohort of young Japanese subjects, women’s tolerance to cold was superior to men’s in the winter [71]. Differences in the distribution of body fat may have contributed to the difference in cold tolerance despites similar body surface area-to-mass ratios [12]. In opposition to results that women tolerate temperature change better, two other studies found women to be more sensitive to thermal stimuli. One study demonstrated that women reported perceived stimulus to both warm and cold at a lower threshold. This thermosensitivity difference was not affected by the phase of the menstrual cycle women were in [72]. Secondly, women exhibited a greater fast cooling time than men. Fast cooling was taken to represent cooling of the superficial epidermal layers. A thicker male stratum corneum may account for this difference. No difference existed between the sexes for slow cooling, a representation of the deeper epidermis and dermal layers [73].
Gender Differences in Skin
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. Table 93.6 Cutaneous microvasculature (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Skin circulation differed with phases of the menstrual cycle: basal flow lowest in the luteal phase, highest in the pre-ovulatory phase. In the luteal phase, greatest coldinduced constriction and lowest recovery [55].
Bioengineering measurements at four times during the menstrual cycle
31 women, 15–45 years
Basal skin blood flow reduced in women [56].
Bioengineering measurements
56 women, 44 men; 20–59 years
Facial basal skin blood flow reduced in women [57].
Laser Doppler
5 women, 5 men; 25–52 years
Basal skin blood flow reduced in women [58].
Bioengineering measurements, cooling and warming to change sympathetic tone
26 women, 23 men; 22–38 years
Young women with lower reactive hyperemia response compared to women over 50 or young men. Young women with extended response to cooling compared to older women and younger men [59].
Bioengineering measurement. Postocclusive reactive hyperemia and direct and indirect cooling
12 women, 19–39 years; 13 women, 51–67 years; 13 men, 22–47 years
Hormone changes during the menstrual cycle affect skin blood flow and its response to cold
Sympathetic tone is increased
Men with greater mean tissue Laser Doppler blood flow and red cell circulating volume at the face as compared to women at baseline. When heated, men continued to have a greater tissue blood flow than women [60].
5 men, 5 women, 25–52 years
Direct and indirect cooling caused Laser Doppler a greater decrease in cutaneous LD flux in women as compared to men. Injection of an alpha-2 adrenoreceptor antagonist decreased the direct response to cold in women only. Injection of an alpha-1 adrenoreceptor antagonist reduced the indirect response to cooling in men only [61].
6 men, 38–46 years; A sex specific response to cooling 6 women, 32–40 years exists for alpha-2 adrenoreceptors at the level of cutaneous microvasculature
Greater ipsilateral and contralateral Laser Doppler decrease in LD flux in response to local cooling in women [62].
10 men, mean age 33; 10 women, mean age 35
Skin temperature at which local heating produced vasodilation lower in women [63].
Bioengineering measurement
9 women, 6 men; age not specified
Capillary refill times shorter for pediatric females, pediatric males, and adult males as compared to adult women, adult elderly women, and adult elderly men [64].
Time to return of baseline distal phalanx color after 5 s of applied pressure was released
100 children, 2–12 years; 104 adults, 20–49 years; 100 elderly adults 62–95 years
Difference between the sexes in the number of perfused microvessels may account for findings
The currently accepted upper limit of capillary refill time for men and women of all ages should be adjusted according to sex and age
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Gender Differences in Skin
. Table 93.6 (Continued) Findings
Obtained by
Pre-menopausal women with a Laser Doppler perfusion greater response to nitroprusside imager, iontophoresis than post-menopausal women [65].
Subjects 21 women, 13 men; 18–80 years
Conclusions Functional and structural changes in skin vasculature occur in women with aging
No significant differences No difference in baseline flux between men and women [62].
Laser Doppler
10 men, mean age 33 years; 10 women, mean age 35 years
Transcutaneous Oxygen Pressure Significant differences Women with higher values of transcutaneous oxygen pressure [67].
Bioengineering; 23 sites on 7 women, 12 men; face, extremities and trunk 21–36 years
Women with higher values of transcutaneous oxygen pressure [68].
Bioengineering; anterior chest, forearm
18 women, 42 men; 22–88 years
Adult women with greater transcutaneous oxygen pressure during postocclusive reactive hyperemia than adult men; no difference between boys and girls [69].
Bioengineering measurement; postocclusive reactive hyperemia
Adults: 30 women, 37 Indication of hormonal influence. men; 22–60 years. Children before puberty: 34
Histamine caused no change in cutaneous blood flow response [66].
Topical and intradermal administration; bioengineering methods
No significant differences
Thermal Response to Stimulation Women had a greater decrease in finger temperature in response to musical stimulus [74]. It was suggested that a difference in the sensitivity or density of peripheral vascular adrenergic receptors creates a difference between men in women in vascular autonomic sensitivity to music [12]. Women and men may also have a different hemispheric response to auditory stimuli. Electrodermal asymmetry has been likened to an index of hemispheric specialization [12]. Right handed men displayed more asymmetry in the frequency and magnitude of skin conductance between hands, with larger responses on the left hand after hearing tones [75].
Thermal and Pain Sensation, Pressure Sensitivity Mechanical, electrical, chemical and thermal pain stimulus have all been used to study skin sensation in relation to pain. Women had a lower threshold of pricking
10 women, 10 men; 24–34 years
pain sensation at the forearm. The pressure threshold was lower in women than men on the palm and sole, but not at the forearm [76]. No difference existed between men and women in the thermal pain threshold [77]. Possible explanations for the differences in pain sensations between the sexes include anatomical differences in skin thickness, differences in blood flow and the cutaneous vasculature that absorbs heat transmitted to skin, and variations in nervous structure or function [12].
Autonomic Function Neonate girls had greater cutaneous conduction than boys [78]. Skin conductance is one measure of autonomic function. This difference may represent dissimilarity in maturation [12] (> Table 93.7).
Skin Color Skin color differs between the sexes and between different age groups. Studies from Iran [79], India [80], and
Gender Differences in Skin
93
. Table 93.7 Sensory functions (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Women with a greater tolerance to cold in the winter than men [71].
Cold exposure to 12˚C for 1 h at rest in the winter and summer; skin and body temperature
7 women, 8 men; Japanese. 18–26 years
Despite similar body surface area-to-mass ratios, differences in fat distribution between men and women may have contributed to differing tolerance to cold
Women with lower thresholds for warm and cold sensation as compared to men. Results not affected by phase of menstrual cycle [72].
Middlesex Thermal Testing System; reported perceived stimulus produced by thermode
10 men, mean age 31 years; 10 women, mean age 28 years
Women had greater thermosensitivity than men
Fast cooling (representative of superficial epidermal layers) longer in men than women. No difference in cooling time between the sexes for slow cooling (represtative of the deeper epidermal and dermal layers) [73].
Skin temperature; time to cool 7 men, mean age 25 years; 7 women, mean age 24 years
The thicker stratum corneum of men caused a longer fast cooling time as compared to women
Women with a greater decrease in finger temperature in response to musical stimulus [74].
Auditory stimulation: music; skin temperature
60 women, 60 men; young students
Results may be due to a difference between the sexes in autonomic sensitivity to music
Men with more asymmetry Auditory stimulus; magnitude between hands than women. and frequency of skin Women with larger skin conductance responses conductance responses on right hand; men with large responses on left hand [75].
15 women, 15 men; 19–27 years. Right-handed
Auditory stimuli may cause a differing hemispheric response in men and women
Palm and sole more sensitive Pressure threshold to stimulus in women, but measurement not on forearm [76].
68 women, 68 men; 17–30 years
Higher conductance in Skin conductance (autonomic neonate girls as compared to function) boys [78].
20 women, 20 men; neonates: 60–100 h
Difference in maturation may account for differences in conductance
No significant differences Men and women have Heat stress, ergometer; oxygen 12 women, 12 men; 20–28 years differing physiologic uptake, body and skin responses to heat stress, but temperature, sweat rate depend on body surface area and fat content [70]. No difference in thermal pain Thermal sensory analyzer threshold between men and women [77].
19 men, 30 women; 19–59 years. Type III–VI skin types
When percent body fat and ratio of body surface area to mass were accounted for, sex differences no longer existed
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Australia [81] have demonstrated women to have lighter skin. Differences in melanin, hemoglobin, carotene, hormonal influence, and environmental sun exposure may all be involved [12]. The change in skin color that occurs with aging is both similar and different for men and women. In general, both sexes darken with increasing age [81]. Following the onset of puberty both men and women lighten, though females more so [80]. It is hypothesized that hormonal influence alone cannot explain this difference since both estrogen and testosterone cause skin darkening [12]. A difference in environmental exposure to UV light has also been proposed as an explanation, though results of a study of adolescent medial upper arm skin (less sun exposure) challenges this. The aforementioned study found that the forehead (sun exposed) pigmentation of boys was darker than of girls. The medial upper arm (less sun exposed) of girls was darker than boys during early adolescence, similar for the
sexes during adolescence, and significantly lighter for girls than boys in late adolescence [82]. Colorimetric measurements demonstrated elderly men to have redder skin than elderly women, though this did not hold true in a younger population [83]. A separate study of colorimetry also found men to have more red skin than women and found this across all ages 19–73 [84]. Additionally, women had less regional variation in reflectance spectrophotometry and as such were more homogenous in color than men [85] (> Table 93.8).
Hormonal Influence It has already been mentioned that hormones increased the thickness of skin in post-menopausal women [9, 10], are implicated in the change in skin blood flow and transepidermal water loss that occurs during the
. Table 93.8 Skin color (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Lighter skin in women [79].
Spectrophotometry 33 women, 68 men; 8–24 years
Lighter skin in women [80].
Spectrophotometry 566 women, 578 men; 1–50 years Female skin lightens during puberty; male skin darkens. Varying MSH levels differ. Role for environmental and hereditary variables
Women’s skin lighter. Skin color darkens in both sexes with age [81].
Spectrophotometry 461 women, 346 men; 20–69 years
Boys foreheads darker than girls. Skin color Medial upper arms of girls darker reflectance than boys in early adolescence, not different in middle adolescence, and lighter during late adolescence [82].
105 women, 10–16 years; 105 men, 12–18 years
Men’s skin darker and redder than Colorimetric women’s in the elderly; not in the measurements young [83].
8 women, 5 men, 65–88 years; 9 women, 4 men, 18–26 years
Men with redder skin as Colorimetric compared to women at the upper measurements back and forearm. Women 36–73 years with greater luminance of skin on upper back than men of the same age [84].
21 men, 31 women, 19–35 years; 22 men, 23 women, 36–50 years; 22 men, 30 women, 51–65 years; 10 men, 7 women, 66–73 years.
Vascular dissimilarity, differences in tanning
History of sun exposure and MSH levels differ. MSH levels differ Different physiologic changes between the sexes may account for results
Gender Differences in Skin
menstrual cycle [50, 55], and affect the severity of symptoms associated with pathologic skin conditions such as eczema [51]. Hormone replacement also limited the agerelated skin extensibility in menopausal women, whereas women without replacement demonstrated an increase in extensibility [86]. Loss of collagen has been associated with the occurrence of skin thinning post-menopause. Two separate studies demonstrated that collagen content increased 48% [87] and 34% [10] following hormone treatment as compared to non-treated subjects. Additionally, the ratio of type III to type I collagen in the skin decreased with aging [12]. Postmenopausal women receiving hormone replacement therapy had an increased proportion of type III collagen cutaneously [88]. The cutaneous enzyme 17beta-hydroxysteroid dehydrogenase(17B-HSD) was found in vivo to have a different affinity in men and women for interconverting estrone (E1)) and 17 beta-estradiol (E2) in the skin. 17B-HSD catalyzes the reduction of weak steroids to strong ones, e.g., E1 to E2. The skin of women had a tendency to activate estrogen; Vmax for E2 formation 1.7 times larger than for E1 formation. The skin of men tended to
93
deactivate estrogen; Vmax for E1 formation 2.5 times larger than for E2 formation [89] (> Table 93.9).
Pilosebaceous Unit Sebaceous glands are hormone dependent. Androgenic steroids of both adrenal and gonadal origin can directly stimulate sebaceous gland activity. Administration of the appropriate hormone during puberty can cause an increase in their activity [12]. Thyroid stimulating hormone (TSH), corticotrophin (ACTH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act indirectly by stimulating their respective endocrine tissues. Other hormones such as growth hormone (GH) act synergistically with another hormone that the sebaceous gland is sensitive to [12]. For the age range 20–69 years, the average values for sebum secretion were higher in men than women. This was not true for the age range 15–19 years [90]. In the age range 50–70 years secretion in men remained unaltered, whereas women had a significant decrease in sebum secretion likely secondary to decreased ovarian activity [12]. Additionally, the
. Table 93.9 Hormonal influence (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences Decreased age-related Computerized extensibility in skin in hormone suction device replacement treated women [86]. measuring skin deformability and viscoelasticity
43 nonmenopausal women, Skin slackness can be limited by 19–50 years; 25 menopausal not hormone replacement therapy treated, 46–76 years; 46 on hormonal replacement since the onset of menopause, 38–73 years
Hormone replacement treated Hydroxyproline and Postmenopausal women, 35–62 women had 48% increased collagen content; years: 29 untreated, 26 estradiol collagen content as compared to biopsies + testosterone non-treated subjects [87]. Postmenopausal women receiving hormone replacement therapy had an increased proportion of type III collagen [88].
The decreased content of collagen in skin that occurs with aging can be prevented with hormone treatment
Analysis of collagen Postmenopausal women, 41–66 Total collagen increased by types; biopsies years: 14 untreated, 11 estradiol hormone replacement therapy. + testosterone Ratio of type III to type I collagen by hormone treatment
Vmax for 17 beta-estradiol High performance formation 1.7 times larger than liquid for estrone formation in women. chromatography Vmax for estrone formation 2.5 times larger than for 17 betaestradiol formation in men [89].
7 women, 40–72 years; 9 men, 21–75 years; cadavers
Sex differences exist in the affinity of the cutaneous enzyme 17 beta-hydroxysteroid dehydrogenase to interconvert hormones
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. Table 93.10 Pilosebaceous unit (Adapted with permission from Tur E [12]) Findings
Obtained by
Subjects
Conclusions
Significant differences No difference between the sexes in Sebum production sebum secretion ages 15–19. Greater sebum secretion in men than women ages 20–69. Men 50–70 years with no change in secretion; women with significantly decreased sebum output [90]
330 women, 458 men; 15 to Sebum secretion may >69 years change in elderly women due to decreased ovarian activity
Plasma testosterone did not correlate with sebum production [90].
8 women, 28 men
Sebum production and plasma androgen levels
Women’s hair more dense and Phototrichogram, hair count 7 women, 29–49 years; with a lower percentage of telogen after washing 7 men, 25–47 years hair in January as compared to men [91].
composition of sebum is affected by hormonal influence as an age-related decline in the secretion of wax ester starting in young adulthood has been demonstrated [12]. Hair distribution of men and women is one of the more obvious attributes that differs between the sexes. Systemic factors, e.g., hormones, and external factors play an important role in the evolution and phases of follicular growth. This is in addition to mechanisms inherent to the follicles themselves [12]. The evaluation of one study found that in the month of January women’s hair was denser and the percentage of telogen hair was lower as compared to men [12, 91]. This seasonal effect exemplified one difference between men and women in regards to environmental conditions. The hormonally stimulated transformation of vellus to terminal hair, racial, and genetic factors are all components in the diversity of hair patterns of men and women [12]. Body site is also important in the effect of androgens on hair growth. At puberty men’s vellus hair on the face transforms to terminal hair whereas on the scalp the reverse occurs. Despite the same exposure to circulatory hormones, the activity of hair follicles depends on anatomical location. Targets such as the eyelashes have no response to hormonal stimulation whereas the face, scalp, axilla and pubic follicles are main adrogenic targets [12]. Melanocyte-stimulating hormone, prolactin, thyroid hormone, pregnancy, and nutritional state also affect cells targeted by androgens [92] (> Table 93.10).
Conclusion Interpreting the findings in the studies discussed leaves something to be desired in regards to the systematic approach used to study the inherent structure and pathophysiology of skin. The relatively small subject numbers that characterize many of the studies results in a lack of power to support their findings. In this day and age a large number of instruments exist to sample the various functional, mechanical and structural properties of the skin. This is both advantageous and detrimental. Direct comparison between different studies is complicated by the different instruments and methods used to evaluate the same topic. Multiple studies have not only interindividual variability but also intraindividual variability secondary to the different anatomical sites sampled. Additionally, comparison of in vivo and in vitro testing requires extrapolation in reasoning to make a conclusion based on both methods of experimentation. These fundamental techniques of experimentation are further complicated by hormonal factors in many cases, but to what extent continues to need to be elucidated and scientifically, not merely anecdotally, proven. These confounding variables leave much room for further study in larger sample populations and with as stringent experimental protocols as possible. Taken together, the data totality can be interpreted to suggest that the differences are more important than the similarities, suggesting that
Gender Differences in Skin
exploring the mechanisms and significance will provide a better understanding of skin and perhaps gender related skin management.
Cross-references > Determinants
in the Rate of Skin Aging: Ethnicity, Gender, and Lifestyle Influences
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38. Auriol F, Vaillant L, Machet L, et al. Effects of short time hydration on skin extensibility. Acta Derm Venereol (Stockh). 1993;73:344–347. 39. Mehnert P, Brode P, Griefahn B. Gender-related difference in sweat loss and its impact on exposure limits to heat stress. Int J Ind Ergon. 2002;29:343–351. 40. Main K, Nilsson KO, Skakkebaek NE. Influence of sex and growth hormone deficiency on sweating. Scand J Clin Lab Invest. 1991;51:475–480. 41. Inoue Y, Tanaka Y, Omori K, et al. Sex- and menstrual cycle-related differences in sweating and cutaneous blood flow in response to passive heat exposure. Eur J Appl Physiol. 2005;94:323–332. 42. Green JM, Bishop PA, Muir IH, Lomax RG. Gender differences in sweat lactate. Eur J Appl Physiol. 2000;82:230–235. 43. Ohman H, Vahlquist A. In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. Acta Derm Venereol. 1994;74:375–379. 44. Ehlers C, Ivens UI, Moller ML, et al. Females have lower skin surface pH than men. A study on the surface of gender, forearm site variation, right/left difference and time of the day on the skin surface pH. Skin Res Technol. 2001;7:90–94. 45. Burry JS, Coulson HF, Esser I, et al. Erroneous gender differences in axillary skin surface/sweat pH. Int J Cosmet Sci. 2001;23:99–107. 46. Williams S, Davids M, Reuther T, et al. Gender difference of in vivo skin surface pH in the axilla and the effect of a standardized washing procedure with tap water. Skin Pharmacol Physiol. 2005;18:247–252. 47. Wilhelm KP, Maibach HI. Factors predisposing to cutaneous irritation. Dermatol Clin. 1990;8:17–22. 48. Modjtahedi BS, Modjtahedi SP, Maibach HI. The sex of the individual as a factor in allergic contact dermatitis. Contact Dermatitis. 2004;50:53–59. 49. Bjornberg A. Skin reactions to primary irritants. Acta Derm Venereol (Stockh). 1075;55:191–194. 50. Harvell J, Hussona-Safed I, Maibach HI. Changes in transepidermal water loss and cutaneous blood flow during the menstrual cycle. Contact Dermatitis. 1992;27:294–301. 51. Farage MA, Berardesca E, Maibach HI. The effect of sex hormones on irritant and allergic response: possible relevance for skin testing. Br J Dermatol. 2009;160:450–451. 52. Bordignon V, Burastero SE. Age, gender and reactivity to allergens independently influence skin reactivity to histamine. J Investig Allergol Clin Immunol. 2006;16:129–135. 53. Magerl W, Westerman RA, Mohner B, et al. Properties of transepidermal histamine iontophoresis: differential effects of season, gender, and body region. J Invest Dermatol. 1990;94:347–352. 54. Lammintausta K, Maibach HI, Wilson D. Irritant reactivity in males and females. Contact Dermatitis. 1987;17:276–280. 55. Bartelink ML, WOllersheim A, Theeuwes A, et al. Changes in skin blood flow during the menstrual cycle: the influence of the menstrual cycle on the peripheral circulation in healthy female volunteers. Clin Sci. 1990;78:527–532. 56. Maurel A, Hamon P, Macquin-mavier I, et al. Flux microcirculatoire cutane etude par laser-doppler. Presse Med. 1991;20:1205–1209. 57. Mayrovitz HN, Regan MB. Gender differences in facial skin blood perfusion during basal and heated conditions determined by laser Doppler flowmetry. Microvasc Res. 1993;45:211–218. 58. Cooke JP, Creager MA, Osmundson PJ, et al. Sex differences in control of cutaneous blood flow. Circulation. 1990;82:1607–1615. 59. Bollinger A, Schlumpf M. Finger blood flow in healthy subjects of different age and sex in patients with primary Raynaud’s disease. Acta Chir Scand. 1975;465(Suppl):42–47.
60. Mayrovitz HN, Regan MB. Gender differences in facial skin blood perfusion during basal and heated conditions determined by laser Doppler flowmetry. Microvasc Res. 1993;45:211–218. 61. Cankar K, Finderle Z, Strucl M. The role of alpha1- and alpha2adrenoceptors in gender differences in cutaneous LD flux response to local cooling. Microvasc Res. 2004;68:126–131. 62. Cankar K, Finderle Z. Gender differences in cutaneous vascular and autonomic nervous response to local cooling. Clin Auton Res. 2003;13:214–220. 63. Walmsley D, Goodfield MJD. Evidence for an abnormal peripherally mediated vascular response to temperature in Raynaud’s phenomenon. Br J Rheumatol. 1990;29:181–184. 64. Schriger DL, Baraff L. Defining normal capillary refill: variation with age, sex, and temperature. Ann Emerg Med. 1988;17:932–935. 65. Algotsson A, Nordberg A, Winblad B. Influence of age and gender on skin vessel reactivity to endothelium-independent vasodilators tested with iontophoresis and a laser Doppler perfusion imager. J Gerontol A Biol Sci Med Sci. 1995;50:121–127. 66. Tur E, Aviram G, Zeltser D, et al. Histamine effect on human cutaneous blood flow: regional variations. Acta Derm Venereol (Stockh). 1994;74:113–116. 67. Orenstein A, Mazkereth R, Tsur H. Mapping of the human body skin with transcutaneous oxygen pressure method. Ann Plast Surg. 1988; 64:546–550. 68. Glenski JA, Cucchiara RF. Transcutaneous O2 and CO2 monitoring of neurosurgical patients: detection of air embolism. Anesthesiology. 1986;64:546–550. 69. Ewald U. Evaluation of the transcutaneous oxygen method used at 37 C for measurement of reactive hyperaemia in the skin. Clin Physiol. 1984;4:413–423. 70. Havenith G, van Middendorp H. The relative influence of physical fitness, acclimatization state, anthropometric measures and gender on individual reactions to heat stress. Eur J Appl Physiol. 1990;61:419–427. 71. Sato H, Yamasaki K, Yasukouchi A, et al. Sex differences in human thermoregulatory response to cold. J Hum Ergol. 1988;17:57–65. 72. Golja P, Tipton MJ, Mekjavic IB. Cutaneous thermal thresholds - the reproducibility of their measurements and the effect of gender. J Therm Biol. 2003;28:341–346. 73. Jay O, Havenith G. Finger skin cooling on contact with cold materials: an investigation of male and female responses during short-term exposures with a view on hand and finger size. Eur J Appl Physiol. 2004;93:1–8. 74. McFarland RA, Kadish R. Sex differences in finger temperature response to music. Int J Psychophysiol. 1991;11:295–298. 75. Martinez-Selva JM, Roman F, Garcia-Sanchez FA, et al. Sex differences and the asymmetry of specific and non-specific electrodermal responses. Int J Psychophysiol. 1987;5:155–160. 76. Weinstein S, Sersen E. Tactual sensitivity as a function of handedness and laterality. J Comp Physiol Psychol. 1961;54:665–669. 77. Yosipovitch G, Meredith G, Chan YH, et al. Do ethnicity and gender have an impact on pain thresholds in minor dermatologic procedures? A study on thermal pain perception thresholds in Asian ethinic groups. Skin Res Technol. 2004;10:38–42. 78. Weller G, Bell RQ. Basal skin conductance and neonatal state. Child Dev. 1965;36:647–657. 79. Mehrai H, Sunderland E. Skin colour data from Nowshahr City, Northern Iran. Ann Hum Biol. 1990;17:115–120. 80. Banerjee S. Pigmentary fluctuation and hormonal changes. J Genet Hum. 1984;32:345–349. 81. Green A, Martin NG. Measurement and perception of skin colour in a skin cancer survey. Br J Dermatol. 1990;123:77–84.
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88. Savvas M, Bishop J, lauent G, et al. Type II collagen content in the skin of postmenopausal women receiving oestradiol and testosterone implants. Br J Obstet Gynaecol. 1993;100:154–156. 89. Hikima T, Maibach HI. Gender differences of enzymatic activity and distribution of 17beta-hydroxysteroid dehydrogenase in human skin in vitro. Skin Pharmacol Physiol. 2007;20:168–174. 90. Pochi PE, Strauss JS. Endocrinologic control of the development and activity of the human sebaceous gland. J Invest Dermatol. 1974;62: 191–201. 91. Courtois M, Loussouarn G, Hourseau S, et al. Periodicity in the growth and shedding of hair. Br J Dermatol. 1996;134:47–54. 92. Randall VA, Thornton MJ, Messenger AG, et al. Hormones and hair growth: variations in androgen receptor content of dermal papilla cells cultured from human and red deer (Cervus Elaphus) hair follicles. J Invest Dermatol. 1993;101:114S–120S.
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Sensitive Skin and Aging
95 Perceptions of Sensitive Skin with Age Miranda A. Farage
Introduction In the general population, little correlation exists between individuals’ perceptions of the sensitivity of their skin and objective clinical assessments of skin reactivity to irritants [1]. Individuals who exhibit a low threshold of response to a particular irritant may not be susceptible to all other types of irritant stimuli. Nevertheless, a sizeable proportion of people in the general population claim some degree of skin sensitivity. In the UK, for example, a survey of 2,058 men and women found that 38.2% of the men and 51.4% of the women claimed to have sensitive skin [2]. A phone survey conducted in San Francisco among 800 ethnically diverse women aged 18–54 years found that 52% considered themselves to have sensitive facial skin [3]. In two seasonal surveys conducted in France (one in winter among 1,006 individuals and one in summer among 1,001 individuals), 51.7% of the respondents from the winter survey (March) and 58.9% from the summer survey (July) considered their skin to be either sensitive or very sensitive [4, 5]. If respondents who considered their skin to be ‘‘slightly sensitive’’ were included, more than 80% of respondents reported some skin sensitivity in each season. The survey of 1,039 individuals in the state of Ohio, USA found that 68.4% of the respondents overall claimed some degree of skin sensitivity at various anatomical sites: 77% in the facial area, 61% on the body, and 56% in the genital area [6, 7]. Perceptions of skin sensitivity among elderly adults have not previously been examined in detail. This chapter presents an epidemiological survey of perceptions of sensitive skin in adults as they age.
Susceptibility of Aging Skin to Irritants
● ● ● ●
Reduced hydration Increased permeability Flattening of the dermal–epidermal junction Lower elasticity and diminished tensile strength, due changes in the architecture of the collagen and elastin networks ● Reduced dermal thickness (20%) ● Reduced cellularity and vascularity of the dermis ● Slower wound healing Although it is commonly assumed that aging skin is more sensitive to irritation and discomfort, a comprehensive review of clinical assessments of the erythematous response in older people suggests that susceptibility to skin irritation generally decreases with age [10]. For example, a compilation of results of skin patch tests conducted among older people over a period of 4 years demonstrated a trend toward lower reactivity to four common irritants with age [11]. Specifically, older people exhibited significantly lower reactivity to two strong irritants (20% sodium dodecyl sulfate and 100% octanoic acid) and directionally lower reactivity (approaching statistical significance) to two milder irritants (100% decanol and 10% acetic acid); however, the severity of the observed irritant responses was unrelated to people’s perception of the sensitivity of their skin. People aged 65–84 years were less reactive to stinging caused by 5% sodium lauryl sulfate (SLS) than people aged 18–25 years [12]. Pretreatment with 0.25% SLS also had less effect on skin barrier function and susceptibility to irritants in elderly people (mean age, 74.6 years) than in younger adults (mean age, 25.9 years) [13]. Lastly, elderly adults were less reactive to a range of irritants (histamine, DMSO, 48/80 mixture of chloroform–methanol, lactic acid, ethyl nicotinate, and the blistering agent, ammonium hydrazide) [14].
The physiological changes that occur as skin ages might lead one to conclude that older skin is more susceptible to irritant effects. Such changes (reviewed in [8, 9]) include:
Perceptions of Sensitive Skin Among Older adults
● Reduced epidermal thickness and reduced epidermal turnover ● Lower sensory perception
The conclusion that elderly skin is less susceptible to skin irritation is based on objective assessments in patch tests and sting tests. However, because little correlation exists
M. A. Farage, K. W. Miller, H. I. Maibach (eds.), Textbook of Aging Skin, DOI 10.1007/978-3-540-89656-2_95, # Springer-Verlag Berlin Heidelberg 2010
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Perceptions of Sensitive Skin with Age
between objective and subjective assessments of skin sensitivity, objective tests provide little insight on individuals’ perceptions about the sensitivity of their skin. A comparison of the perceptions of skin sensitivity among older and younger adults was carried out through a questionnaire-based survey of 1,039 people in 2006 in Midwestern USA. Results for the entire group have been reported previously [6, 7, 17, 15].
to evaluate the potential differences in perceptions of sensitive skin among individuals with urinary incontinence compared to age- and gender-matched subjects without this condition. No criteria related to skin sensitivity (e.g., hyperreactivity to consumer products, a history of skin or respiratory allergies) were used in recruitment. Ethnic representation within the population reflected that of the location where the study was conducted [16]. The small number of Latinos and Asians did not enable valid conclusions to be drawn for these demographic groups.
Population Demographics The population was recruited from among people participating in consumer product preference tests. Consequently, the surveyed population was predominantly female, and 76% were under the age of 40 (> Table 95.1). The analyses were performed on the following age groups: 30 years; 31–39 years; 40–49 years; 50 years. The two older age groups each comprised over 100 subjects, enabling valid statistical comparisons. One consumer product test was related to urinary incontinence products. This subset of women was used
Perceptions of Skin Sensitivity at Different Anatomical Sites Among Age Groups Sixty-eight percent of the study population described themselves as having sensitive skin to some degree (> Table 95.2): 77% perceived their facial skin to be sensitive, 61% perceived their overall body, and 56%
. Table 95.1 Breakdown of the test population by age, gender, and ethnicity All ages
30
31–39
40–49
50
Number
%
Number
%
Number
%
Number
%
Number
%
1,039a
100%
295
28%
492
48%
128
12%
101
10%
Females
869
84%
261
25%
421
41%
83
8%
84
8%
Males
163
16%
30
3%
70
7%
44
4%
17
2%
Caucasian
684
77%
213
25%
344
40%
58
7%
54
6%
African American
Both genders, all ethnicities
b
Females (n = 869)
108
12%
35
4%
42
5%
10
1%
17
2%
Hispanic
10
1%
2
0%
8
1%
0
0%
0
0%
Asian
13
1%
2
0%
8
1%
1
0%
1
0%
54
6%
9
1%
19
2%
14
2%
12
1%
20
12%
3
2%
8
5%
7
4%
1
1%
Hispanic
8
4%
2
1%
2
1%
3
2%
0
0%
Asian
5
3%
1
1%
3
2%
1
1%
0
0%
12
7%
3
2%
3
2%
5
3%
1
1%
Not given c
Males (n = 163)
African American
Not given
A total of 1,039 individuals filled out sensitive skin questionnaires. Demographic data are summarized for all responders. The percentage of ethnicity subgroups were calculated separately for each gender a Seven subjects did not provide their gender. An additional 22 subjects did not provide their age (20 females and 2 males) b Percentage of females c Percentage of males
95
Perceptions of Sensitive Skin with Age
. Table 95.2 Perceptions of sensitive skin Question: Some people have skin that is more sensitive than others. How would you describe your skin? Overall rating of skin sensitivity Total number of subjects responding
Question: Please rate your skin in each of the following areas Facial area
Genital area
1,033
1,035
1,031
51 (5%)
111 (11%)
19 (2%)
88 (9%)
Moderately sensitive
239 (23%)
245 (24%)
189 (18%)
140 (14%)
Slightly sensitive
421 (41%)
443 (43%)
420 (41%)
352 (34%)
Not sensitive
328 (32%)
234 (23%)
407 (39%)
451 (44%)
Sensitive (any degree)
711 (68%)
799 (77%)
628 (61%)
580 (56%)
Very sensitive
1,039
Body area
Participants were questioned about how they would describe their skin; very sensitive, moderately sensitive, slightly sensitive, not sensitive. On a subsequent page of the questionnaire, participants were asked to rate the skin of three anatomical sites: facial area, body area, and genital area. Responses are shown above for the overall rating (number followed by percentage), and for the ratings at the three anatomical sites
. Table 95.3 Most common reason given for perceiving sensitive skin Question: Select one of the following statements that best describes why you think you have sensitive skin I have sensitive skin because. . .
All
1. Some products cause my skin to break out in a rash (redness and/or swelling)
25% 27% 25%
27%
2. Some products cause burning, stinging, itching, or other unpleasant sensations
25% 23% 27%
23%
29%
3. My skin is sensitive to extreme weather conditions (hot, cold, dry, humid)
36% 34% 37%
35%
33%
4. Items that rub against my skin (such as washcloths and clothing) cause my skin to become sensitive
7%
7%
6%
8%
7%
5. I have sensitive skin due to another reason
7%
9%
6%
7%
10%
Relationship to age group is not significant
30 31–39 40–49 50 21%
p = 0.9015
Participants were asked to choose one reason why they perceived their skin to be sensitive. Analysis for a significant relationship between the response and age were done using Cochran-Mantel-Haenszel statistics
perceived their genital area to be sensitive. The most frequent single cause of skin sensitivity claimed by all age groups was extreme weather conditions (> Table 95.3). About half of each age group also claimed adverse reactions to products. Sensitivity of genital skin was significantly more likely to be reported by those aged 50 or older and the perception of skin sensitivity at this site rose directionally with age (> Fig. 95.1d). A lower percentage of people
aged 40–49 years reported facial skin sensitivity compared to those aged 31–39, respectively (> Fig. 95.1b). Analysis of the results by gender yielded additional insights. In the population as a whole, gender was not associated with overall claims of skin sensitivity (data not shown). However, a significantly higher proportion of women specifically perceived their genital skin to be sensitive (58% of females and 44% of males) (p < 0.03). Moreover, within age groups, the association of age and
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95
Perceptions of Sensitive Skin with Age
. Figure 95.1 (a–d) Age group differences in perceptions of sensitive skin. The percentage of participants who claimed some degree of sensitivity overall (a) or sensitivity of the facial, body, or genital areas (b–d). Correlations between perceptions of sensitive skin and age were assessed by MH chi-square. Paired age-group comparisons were performed by chi-square analysis. aOn the bar of a– 40–49 group significantly lower than 50 group (p = 0.04). bOn the bar of b – 31–39 group significantly higher than 40–49 group (p = 0.03). cOn the bar of b – 40–49 group significantly lower than 50 group (p = 0.02). dOn the bar of d – 30 group significantly lower than 50 group (p = 0.02). eOn the bar of d – 31–39 group significantly lower than 50 group (p = 0.04)
Perceptions of Sensitive Skin with Age
perceived genital skin sensitivity was significant only for women (p ¼ 0.01) (> Fig. 95.2d). Surprisingly, women were not more likely than men in any age group to report facial skin sensitivity.
Ethnic Differences in Perceptions of Sensitivity Skin at Different Anatomical Sites by Age Among Caucasians, people aged 50 and above were more likely to report facial skin sensitivity than those aged 40–49; those aged 30–39 reported more body skin sensitivity than those aged 30 and below; and, as previously noted, those aged 50 and above were more likely than all other age groups to report genital skin sensitivity (> Fig. 95.3). Among African Americans, a slightly higher frequency of skin sensitivity was reported among people aged 50 and above at all anatomical sites, but the differences were not statistically significant, probably because of the smaller sample size. Within age groups, no statistically significant differences in perceived skin sensitivity were found between Caucasians and African Americans at any body site.
Differences in Diagnosed Skin Allergies by Age The proportion of people reporting a medically diagnosed skin allergy increased significantly with age (p ¼ 0.002) (> Fig. 95.4a), but only among those who perceived their skin to be sensitive (p ¼ 0.0006) (> Fig. 95.4b, c). The age-related increase in medically diagnosed skin allergies among those with sensitive skin held true for each specific anatomical site (face, body, or genitalia). Among people who perceived their skin to be sensitive at any site, those aged 50 or older reported a higher frequency of medically diagnosed skin allergies than any other age group (> Fig. 95.4b). Other investigators have reported associations between sensitive skin and dermatologist consultations [4] and between sensitive skin and nickel contact allergy [17]. Possibly, the perception of ‘‘sensitive skin’’ may be part of a syndrome of skin hyperreactivity that includes a higher propensity to developing contact allergy. Alternatively, the need to seek medical treatment for allergic reactions may heighten patients’ awareness of skin sensations and reactions, causing them to perceive their skin to be sensitive. Although the perception of sensitive skin is subjective, these associations suggest that there may be a biological basis for some claims of sensitivity.
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Changes in Perceived Skin Sensitivity Over Time As stated in the beginning of this chapter, of the entire test population, 68% claimed sensitive skin to some degree. Among those who claimed sensitive skin, over 62% of the total population stated that their skin had been sensitive for more than 10 years, and 16% claimed their skin has been sensitive 6–10 years (> Fig. 95.5a). As expected, there was a significant relationship between the duration of perceived sensitive skin and age, with an increased proportion of older subjects responding that their skin has been sensitive longer (p = 0.001). Among the 50 age group, 69% claimed that their skin had been sensitive for more than 10 years, and 20% claimed for 6–10 years. For > Fig. 95.5a, participants were asked: ‘‘How long have you had sensitive skin?’’; for > Fig. 95.5b, responses to the question ‘‘How has your skin sensitivity changed over the years?’’ for the whole population and by age group were collected. Correlations of responses to age were assessed by the Cochran-Mantel-Haenszel statistic (shown above the chart). Forty six percent of people who considered their skin to be sensitive reported that their skin sensitivity increased over time: 37% claimed their skin to be slightly more sensitive than in the past, and 9% claimed their skin to be much more sensitive than in the past (> Fig. 95.5b). People aged 50 and older were more likely to claim that their skin was presently ‘‘much more sensitive’’ (16% frequency) (p ¼ 0.003).
Sensitive Skin and Family History Individuals who have perceived their skin to be sensitive were significantly more likely to report that someone in the family also had sensitive skin (> Fig. 95.6); this was significant for all age groups. A child was the relative most likely identified as also having sensitive skin (reported previously in [18].) The percentage of subjects responding yes and no to the question: ‘‘Does any member of your family have sensitive skin?’’ was calculated. The relationship between the perceived skin sensitivity and a family history of sensitive skin was tested by chi-square analysis.
Irritation Due to Environmental Factors It has been reported that environmental factors such as dry air, cold, and wind, are perceived to contribute to skin
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. Figure 95.2 (a–d) Age group differences in perceptions of sensitive skin among males and females. MH chi-Square was used to test for correlations between perceptions of sensitive skin and age for each gender (shown above the chart). Differences by gender within age groups were tested by chi-Square. aOn the bar of a – women 50 significantly higher than men 50 (p = 0.04). bOn the bar of d – women 30 significantly higher than men 30 (p = 0.05). cOn the bar of d – women 31–39 significantly higher than men 31–39 (p = 0.01)
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95 Perceptions of Sensitive Skin with Age
. Figure 95.3 (a–d) Age group differences in perceptions of sensitive skin among different ethnic groups. Participants who claimed some degree of sensitivity are summarized for each ethnic group. Within each ethnic group, paired comparisons of age groups were conducted by chi-square analysis. aOn the bar of b – among Caucasians, 40–49 group significantly lower than 50 group (p = 0.03). bOn the bar of c – among Caucasians, 30 group significantly lower than 31–39 group (p = 0.03). cOn the bar of d – among Caucasians, 30 group significantly lower than 50 group (p = 0.006). dOn the bar of d – among Caucasians, 31–39 group significantly lower than 50 group (p = 0.02). eOn the bar of d – among Caucasians, 40–49 group significantly lower than 50 group (p = 0.02)
Perceptions of Sensitive Skin with Age
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Perceptions of Sensitive Skin with Age
. Figure 95.4 (a–c) Medically diagnosed skin allergies diagnosed by age group. (a) Percentage of participants who responded affirmatively to the question: ‘‘Do you have any known skin allergies that have been confirmed by a doctor?’’; (b) the percentage of affirmative responses among those who did claim to have sensitive skin; (c) the percentage of affirmative responses among those who did not claim to have sensitive skin. In (b), correlations between confirmed skin allergies and age and differences in confirmed allergies between age groups was assessed by Fisher’s exact test. aOn b – 30 group significantly lower than 50 group (p = 0.005). bOn b – 31–39 group significantly lower than 50 group (p = 0.0006). c On b – 40–49 group significantly lower than 50 group (p = 0.0002)
Perceptions of Sensitive Skin with Age
. Figure 95.5 (a, b) Duration of skin sensitivity and change in skin sensitivity over time
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Perceptions of Sensitive Skin with Age
. Figure 95.6 Family history of sensitive skin
sensitivity [5]. > Table 95.4 summarizes results of the environmental factors for each of the age groups, including the number of subjects who gave a response to the question, and the percentage who claimed that the environmental factor caused skin irritation. In the present investigation, all environmental factors were significantly more likely to be perceived to contribute to skin reactivity by those who claimed to have sensitive skin, regardless of age. Among those who claimed to have sensitive skin, hot weather was more frequently identified as a contributing factor by people aged 50 or older (91% of respondents) than by other age groups (62–67% of respondents). For each environmental factor, paired comparisons were done for each age group (> Table 95.5). Hot weather and rough fabrics were the factors most strongly associated with skin sensitivity among the oldest adults (aged 50 and above) and were specifically associated with genital skin sensitivity in this group; cold weather was most strongly associated with skin sensitivity in midlife (40–49 age group); and stress was the most important factor cited by younger adults. The menstrual cycle was perceived to contribute to skin
sensitivity by women of all age groups except those aged 50 or older.
Perceived Contribution of Household and Personal Products to Skin Sensitivity > Table
95.6 describes perceptions of irritation attributed to household, facial, and personal care products. In all age groups, those who claimed to have sensitive skin were significantly more likely than those who did not claim t
