Proteoglycans in Lung Disease (Lung Biology in Health and Disease)

PROTEOGLYCANS IN LUNG DISEASE Edited by Hari G. Garg Massachusetts General Hospital and Harvard Medical School Boston...

Author: Hari G. Garg

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PROTEOGLYCANS IN LUNG DISEASE

Edited by

Hari G. Garg Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts, U.S.A.

Peter J. Roughley Shriners Hospital for Children Montreal, Quebec, Canada

Charles A. Hales Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts, U.S.A

Marcel Dekker, Inc.

New York • Basel

TM

Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved.

ISBN: 0-8247-0815-6 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http:/ /www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright  2003 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball 20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg 21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva

26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland 27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant'Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders 44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay

56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray 60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J.-P. Martin, and R. Masse 69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer 70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M.-C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O'Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos

86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium–Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology · Pathogenesis · Clinical Manifestations · Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman 92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J.-P. Derenne, W. A. Whitelaw, and T. Similowski 93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey 95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse 118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky

119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck 120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S.-E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind 124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma's Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin 141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield 145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus

148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill 153. Environmental Asthma, edited by R. K. Bush 154. Asthma and Respiratory Infections, edited by D. P. Skoner 155. Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, S. Rak, and R. A. Pauwels 156. Genetic Models in Cardiorespiratory Biology, edited by G. G. Haddad and T. Xu 157. Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder 158. Ventilator Management Strategies for Critical Care, edited by N. S. Hill and M. M. Levy 159. Severe Asthma: Pathogenesis and Clinical Management, Second Edition, Revised and Expanded, edited by S. J. Szefler and D. Y. M. Leung 160. Gravity and the Lung: Lessons from Microgravity, edited by G. K. Prisk, M. Paiva, and J. B. West 161. High Altitude: An Exploration of Human Adaptation, edited by T. F. Hornbein and R. B. Schoene 162. Drug Delivery to the Lung, edited by H. Bisgaard, C. O’Callaghan, and G. C. Smaldone 163. Inhaled Steroids in Asthma: Optimizing Effects in the Airways, edited by R. P. Schleimer, P. M. O’Byrne, S. J. Szefler, and R. Brattsand 164. IgE and Anti-IgE Therapy in Asthma and Allergic Disease, edited by R. B. Fick, Jr., and P. M. Jardieu 165. Clinical Management of Chronic Obstructive Pulmonary Disease, edited by T. Similowski, W. A. Whitelaw, and J.-P. Derenne 166. Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, edited by A. I. Pack 167. Biotherapeutic Approaches to Asthma, edited by J. Agosti and A. L. Sheffer 168. Proteoglycans in Lung Disease, edited by H. G. Garg, P. J. Roughley, and C. A. Hales 169. Gene Therapy in Lung Disease, edited by S. M. Albelda 170. Disease Markers in Exhaled Breath, edited by N. Marczin, S. A. Kharitonov, M. H. Yacoub, and P. J. Barnes 171. Sleep-Related Breathing Disorders: Experimental Models and Therapeutic Potential, edited by D. W. Carley and M. Radulovacki 172. Chemokines in the Lung, edited by R. M. Strieter, S. L. Kunkel, and T. J. Standiford 173. Respiratory Control and Disorders in the Newborn, edited by O. P. Mathew 174. The Immunological Basis of Asthma, edited by B. N. Lambrecht, H. C. Hoogsteden, and Z. Diamant

175. Oxygen Sensing: Responses and Adaptation to Hypoxia, edited by S. Lahiri, G. L. Semenza, and N. R. Prabhakar 176. Non-Neoplastic Advanced Lung Disease, edited by J. Maurer

ADDITIONAL VOLUMES IN PREPARATION

Therapeutic Targets in Airway Inflammation, edited by N. T. Eissa and D. Huston Respiratory Infections in Asthma and Allergy, edited by S. Johnston and N. Papadopoulos Acute Respiratory Distress Syndrome, edited by M. A. Matthay Upper and Lower Respiratory Disease, edited by J. Corren, A. Togias, and J. Bousquet Venous Thromboembolism, edited by J. E. Dalen Acute Exacerbations of Chronic Obstructive Pulmonary Disease, edited by N. Siafakas, N. Anthonisen, and D. Georgopolous Lung Volume Reduction Surgery for Emphysema, edited by H. E. Fessler, J. J. Reilly, Jr., and D. J. Sugarbaker

The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

To my wife, Mithlesh H. G. G. To my wife, Sheila P. J. R. To my wife, Mary Ann C. A. H.

INTRODUCTION

Proteoglycans are critical to the architecture of the lung (and other tissues). All the same, these molecules are, if not ignored, at least not always at the forefront of our thinking! During the last 30 to 40 years, we have come to recognize the complex structure of the lung, as well as the interaction between the unique cells that characterize this organ and the matrix that binds these cells. The proteoglycans are, quite simply, part of the scaffolding that holds together all the pieces of this complex structure. Proteoglycans are not unique to the lungs. We find them in cartilage, kidney, small intestine, and many—indeed, almost all—other tissues. Because of the diversity of their location, proteoglycans quite naturally come in a variety of different types. Proteoglycans contain glycosaminoglycan chains including heparin and hyaluronan. As the editors of this monograph point out in their Preface, the interest in hyaluronan began more than 60 years ago. Likewise, heparin was identified many decades ago. However, the interactions of these molecules, and their role in the lung matrix, were not of intense research interest until recently. This is surprising when one considers that for years heparin was mostly extracted from lung tissue. v

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In 1976, Dr. R. G. Crystal, editor of Biochemical Basis of Pulmonary Function, Volume 2 of the Lung Biology in Health and Disease series, included a chapter titled ‘‘Proteoglycans and Elastic Fibers.’’ Then, in 1989, Dr. D. Massaro, editor of Lung Cell Biology, Volume 41 of the series, presented an update titled ‘‘Proteoglycans of the Lung.’’ This ever-increasing interest in the proteoglycans underscores their importance in the biology of the lung’s extracellular matrix, the scaffolding, and the structural and functional development and integrity of the lung in health and disease. This volume, Proteoglycans in Lung Disease, edited by Drs. H. G. Garg, P. J. Roughley, and C. A. Hales, represents the interest in this field of proteoglycans as it relates to the lung. It is presented by a cadre of scientists whose expertise ranges from fundamental biology to the clinical relevance of these molecules. In addition, and just as important, the authors provide the readers with a detailed panoramic review of the role the proteoglycans play in biology and in lung pathology. As stated by the editors, the content of this volume is a step toward the ultimate goal to provide new therapeutic targets. As the Executive Editor of the Lung Biology In Health and Disease series, I am as proud to introduce this volume as I am grateful to its contributors for the opportunity to do so. Claude Lenfant, M.D. Bethesda, Maryland

PREFACE

During breathing, the lung undergoes a constant change in volume. This function places special demands on the pulmonary connective tissue skeleton, particularly lung alveoli, where the gas exchange process takes place. For efficient functioning of the lung, the walls of the alveoli should be: (a) thin, to obtain proper gas exchange, (b) firm, to prevent the collapse of the alveolus, and (c) flexible, to cope with expansion during breathing. The extracellular matrix maintains the functions of the lung by supporting its architecture that contains proteoglycans, collagens, and noncollagenous proteins. In normal tissue a balance is maintained between synthesis and degradation of proteoglycans, and this balance is disturbed by either injury or disease. Many lung diseases are amenable to correction or cure whereas others, such as adult respiratory distress syndrome, are killers. Proteoglycans are complex macromolecules having polysaccharide chains that are called glycosaminoglycans (GAGs). These are unbranched polymers of repeating disaccharide units that are highly negatively charged due to the presence of carboxylate and sulfate groups on their sugar residue. Most GAGs are covalently linked via a tetrasaccharide to a protein backbone, but hyaluronan is an exception, being neither sulfated nor linked to protein as part of a proteoglycan. All types of glycosaminoglycans—hyaluronan, chondroitin 4–sulfate, chonvii

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droitin 6–sulfate, dermatan sulfate, heparin, heparan sulfate, and keratan sulfate—are found in the lung. Hyaluronan was first implicated in a lung disorder about 60 years ago. This finding stimulated interest in the biological role of hyaluronan and other proteoglycans in lung diseases. Intense research efforts in the past 20 years have clearly shown that hyaluronan and proteoglycan macromolecules become altered in diseased states. The exact role of proteoglycan in lung diseases is not yet known. This book presents 18 chapters describing lung proteoglycans and the alteration of their metabolism in different diseases. Chapter 1 focuses on the chemistry and functions of these macromolecules. Chapter 2 provides an overview of hyaluronan in lung function. Proteoglycans in an organ can be studied using biochemical and/or morphological techniques. Chapter 3 discusses these methods and their sensitivity. The side chains of proteoglycans are highly hydrophilic, and therefore have the ability to attract ions and fluid into the matrix. This property affects tissue viscoelasticity. Lung tissue viscoelasticity has also been attributed to the movement of fibers within the connective tissue matrix. Chapter 4 discusses the effect of mechanical strain on the distribution and properties of lung proteoglycans. Maintenance of a normal extracellular matrix is essential for the function of a normal lung. Changes in hyaluronan occur during development. Chapter 5 discusses hyaluronan and how its binding proteins occur in relation to lung biology during development and in response to a variety of insults. Chapter 6 reviews the currently known intracellular signaling pathways activated after hyaluronan interacts with its receptors. Pleural mesothelioma is an uncommon complication of asbestos exposure, originating from mesothelial cells of pleura, peritoneum, or tunica vaginalis testis. Chapter 7 discusses the importance of hyaluronan alterations in malignant mesothelioma. Within a relatively short period of time after birth, the lung changes from a liquid-filled to an air-filled dry organ. This is almost opposite to what is experienced during edema development. Chapter 8 describes the role of matrix proteoglycans in development of pulmonary edema. Small proteoglycans are important during remodeling of the lung in physiological as well as pathophysiological conditions due to their effects on matrix maintenance and on cell and cytokine activities. Therefore, they are deeply involved in disease processes such as inflammation associated with fibrosis. Chapter 9 focuses on the role of small proteoglycans in the formation of fibrosis. Chapter 10 discusses the role of versican in the cell biology of pulmonary fibrosis, and Chapter 11 reviews the role of decorin in the structural remodeling of chronic asthma. Emphysema is a condition of the lung characterized by abnormal permanent enlargement of the air spaces distal to the terminal bronchioles, accompanied by

Preface

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destruction of their walls and without obvious fibrosis. Chapter 12 summarizes the role of proteoglycans in the development and pathogenesis of emphysema. One of the main functions of bronchial mucus is to protect the lung from airborne particles by trapping them and facilitating their clearance by ciliary movement. Chapter 13 describes proteoglycans in normal and pathological bronchial mucus. In blood vessels, proteoglycans constitute only a minor component but are critically involved in a variety of physiological events that occur within the vascular wall. Chapter 14 focuses on the importance of vascular proteoglycans. Bronchiectasis refers to the pathological lung condition in which the walls of medium-sized bronchi are damaged and then dilated. Chapter 15 discusses the degradation of lung matrix proteoglycans in bronchiectasis. Hypoxia is associated with interstitial lung diseases, including pulmonary hypertension, aggressive tumor progression, and fibrosis. The relative proportions of collagen and proteoglycan reflect the type of lung disease. Chapter 16 reviews the effect of hypoxia on glycosaminoglycan synthesis by lung cells. Understanding of proteoglycan interaction in the lung will undoubtedly lead to a better comprehension of disease pathogenesis and may provide new therapeutic targets. Chapter 17 addresses the relationship among integrins, proteoglycans, and lung diseases. Because vascular remodeling with smooth muscle cell hypertrophy and hyperplasia contributes to the high pulmonary vascular resistance seen in primary and secondary pulmonary hypertension, interest continues in possible therapeutic agents to reverse vascular remodeling. Chapter 18 describes in detail the chemical structural modification of the glycosaminoglycan heparin in order to develop heparin derivatives as a potential therapeutic agent to reverse vascular remodeling. In summary, this book presents significant information concerning the participation of proteoglycans in different lung diseases, with the ultimate goal of providing therapeutic targets. This book offers an overview for pulmonologists of the specific roles of proteoglycans in lung pathology. It also gives medical students and nonspecialist researchers in the pulmonary field up-to-date information on the structure and metabolism of proteoglycans and their role in the normal and diseased lung. Hari G. Garg Peter J. Roughley Charles A. Hales

CONTRIBUTORS

Vera Demarchi Aiello, M.D., Ph.D. Laboratory of Pathology, Heart Institute (InCor), University of Saõ Paulo Medical School, Saõ Paulo, Brazil K. Ramakrishnan Bhaskar, Ph.D. Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A. Horace M. Delisser, M.D. Associate Professor, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Giancarlo De Luca, M.D. Full Professor of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy Erik Eklund, Ph.D. Department of Cell and Molecular Biology, Biomedical Center, Lund University, Lund, Sweden Hari G. Garg, D.Sc., Ph.D.

Associate Biochemist, Pulmonary Research Laboxi

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ratories, Department of Medicine, Massachusetts General Hospital, and Principal Associate, Harvard Medical School, Boston, Massachusetts, U.S.A. Paulo Sampaio Gutierrez, M.D., Ph.D. Laboratory of Pathology, Heart Institute (InCor), University of Saõ Paulo Medical School, Saõ Paulo, Brazil Charles A. Hales, M.D. Chief, Pulmonary and Critical Care Unit, Massachusetts General Hospital, and Professor, Harvard Medical School, Boston, Massachusetts, U.S.A. Sara R. Hamilton Department of Biochemistry, London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada Mary Sau-Man Ip, M.D., F.R.C.P. (Edin.), F.R.C.P. (Lond.), F.R.C.P. (Glas.) Professor, University Department of Medicine, The University of Hong Kong, Hong Kong S.A.R., China Hannu Ja¨rvelaäinen, M.D., Ph.D. Associate Professor, Department of Medical Biochemistry, University of Turku, Turku, Finland George Karakiulakis, Ph.D. Department of Pharmacology, School of Medicine, Aristotle University, Thessaloniki, Greece Robert J. Linhardt, Ph.D. Professor, Departments of Medicinal Chemistry, Chemistry, and Chemical Engineering, University of Iowa, Iowa City, Iowa, U.S.A. Mara S. Ludwig, M.D. Professor, Department of Medicine, Meakins–Christie Laboratories, McGill University, Montreal, Quebec, Canada Anders Malmstrom, Ph.D. Professor, Department of Cell and Molecular Biology, Biomedical Center, Lund University, Lund, Sweden Gunnar Martensson, M.D., Ph.D. Senior Consultant, Department of Pulmonary Medicine, Go¨teborg University, Go¨teborg, Sweden Giuseppe Miserocchi, M.D. Professor of Physiology, Department of Experimental and Environmental Medicine and Biotechnology, University of MilanBicocca, Monza, Italy Daniela Negrini, Ph.D. Professor of Physiology, Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy

Contributors

xiii

Paul W. Noble, M.D. Associate Professor, Section of Pulmonary and Critical Medicine, Department of Medicine, Yale University School of Medicine, New Haven, Connecticut, U.S.A. Eleni Papakonstantinou, Ph.D. Department of Pharmacology, School of Medicine, Aristotle University, Thessaloniki, Greece Alberto Passi, M.D. Associate Professor, Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy Dirkje S. Postma, M.D., Ph.D. Professor, Department of Pulmonology, University Hospital Groningen, Groningen, The Netherlands Anthony E. Redington, M.D. Senior Lecturer in Respiratory Medicine, Academic Department of Medicine, Castle Hill Hospital, University of Hull, Cottingham, East Yorkshire, England Clive R. Roberts, Ph.D. Associate Professor, Faculties of Dentistry and Medicine, University of British Columbia, Vancouver, British Columbia, Canada Michael Roth, Ph.D. Associate Professor, University Hospital Basel, Basel Switzerland, and Institute of Respiratory Medicine, Royal Prince Alfred Hospital, University of Sydney, Camperdown, New South Wales, Australia Peter J. Roughley, Ph.D. Scientist, Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada Rashmin C. Savani, M.B.ChB., F.A.A.P., F.R.C.P.(C). Associate Professor, Division of Neonatology, Department of Pediatrics, Children’s Hospital of Philadelphia, and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Daisy Kwok-Yan Shum, Ph.D. Associate Professor, Department of Biochemistry, The University of Hong Kong, Hong Kong S.A.R., China Anders L. Thylen, M.D., Ph.D. Senior Consultant, Department of Pulmonary Medicine, Sahlgrenska University Hospital, Go¨teborg, Sweden Wim Timens, M.D., Ph.D. Professor of Pathology, Department of Pathology and Laboratory Medicine, University Hospital Groningen, Groningen, The Netherlands

xiv

Contributors

Ellen Tufvesson, M.Sc. Department of Cell and Molecular Biology, Biomedical Center, Lund University, Lund, Sweden Eva A. Turley, Ph.D. Senior Scientist, Departments of Oncology and Biochemistry, London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada Ymke M. van der Geld, Ph.D. Department of Pathology and Laboratory Medicine, University Hospital Groningen, Groningen, The Netherlands Jeanette F. M. van Straaten, Ph.D. Department of Pathology and Laboratory Medicine, University Hospital Groningen, Groningen, The Netherlands Fu-Sheng Wang, Ph.D. Cancer Research Labs, London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada Gunilla Westergren-Thorsson, Ph.D. Associate Professor, Department of Cell and Molecular Biology, Biomedical Center, Lund University, Lund, Sweden Thomas N. Wight, Ph.D. Professor and Chair, Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington, U.S.A. Jiahua Xu, Ph.D. Assistant Professor, Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, U.S.A.

CONTENTS

Series Introduction Preface Contributors 1. Proteoglycans of the Lung Peter J. Roughley I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Proteoglycans Glycosaminoglycans Proteoglycan Metabolism Extracellular Matrix Proteoglycans Basement Membrane Proteoglycans Cell Surface Proteoglycans Intracellular Proteoglycans Conclusions References

v vii xi 1 1 1 2 4 5 11 12 15 15 16

xv

xvi 2.

3.

4.

5.

Contents Hyaluronan in Lung Function: An Overview Paul W. Noble

23

I. II. III. IV. V. VI. VII. VIII.

23 24 24 25 26 28 30 31 31

Introduction Hyaluronan in Normal Lung Development Hyaluronan in Diseased Lung Hyaluronan in Models of Lung Injury and Repair Hyaluronan Depolymerization in Lung Injury CD44 Is the Major Hyaluronan Receptor in the Lung Functions of Hyaluronan in Lung Injury and Repair Conclusions References

Morphological Tools for Studying Lung Proteoglycans Paulo Sampaio Guitierrez and Vera Demarchi Aiello

37

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

37 39 39 40 41 42 48 49 49 50 50 51 51

Introduction Histochemistry for Proteoglycans Enzymatic Treatment Immunohistochemistry Probe for Hyaluronan Pulmonary Vascular Hypertension ‘‘In Situ’’ Hybridization Electron Microscopy Proteoglycan Morphometry Other Types of Microscopy Magnetic Resonance Imaging Summary References

Proteoglycans, Lung Physiology, and Mechanical Strain Mara S. Ludwig

55

I. Introduction II. Proteoglycans in the Lung III. Role of Proteoglycans in Determining Lung Tissue Mechanical Behavior IV. Effects of Mechanical Strain on Proteoglycans V. Implications for Lung Disease VI. Conclusion References

55 56

Hyaluronan and Its Receptors in Lung Health and Disease Rashmin C. Savani and Horace M. Delisser

56 63 66 68 68 73

Contents

xvii

I. II. III. IV. V. VI.

Introduction Hyaluronan Hyaladherins HA and Its Receptors in Wound Healing HA and Its Receptors in Inflammation HA and Its Receptors in Vasculogenesis and Angiogenesis VII. HA and Its Receptors in Lung Health and Disease VIII. Summary and Conclusions References 6. Hyaluronan and Hyaladherin Signaling in the Lung Sara R. Hamilton, Fu-Sheng Wang, and Eva A. Turley I. II. III. IV. V. VI.

Introduction Hyaluronan Hyaladherins Hyaluronan and Lung Diseases A Role for HA in Regulating Leukocyte Trafficking and Macrophage Activation Summary References

7. Hyaluronan in Malignant Mesothelioma Anders L. Thyleń and Gunnar Martensson I. II. III. IV. V. VI.

Introduction Hyaluronan HA in Malignant Mesothelioma HA Analysis Alterations of HA in Serum Immunohistochemical Characterization of HA-Producing Malignant Pleural Mesothelioma VII. HA as a Prognostic Factor in MPM VIII. Serum HA Versus Tumor Volume IX. Conclusions References 8. Matrix Proteoglycans in Development of Pulmonary Edema Daniela Negrini, Alberto Passi, Giancarlo de Luca, and Giuseppe Miserocchi I. The Pulmonary Interstitial Space

73 74 76 80 81 83 84 92 92 107 107 108 113 117 119 121 123 135 135 136 136 136 137 138 139 139 139 140 143

143

xviii

Contents II. III. IV. V.

9.

10.

Lung Proteoglycans and Hyaluronan Pulmonary Interstitial Pressure Microvascular Fluid Exchanges Pip Pulmonary in the Transition Toward Hydraulic and Lesional Edema VI. Lung Proteoglycans During Developing of Interstitial Edema VII. Proteoglycans in Interstitial Lung Edema Induced by Hypoxia Exposure VIII. An Example of ‘‘Spontaneous Recovery’’ from Lung Edema: The Newborn Lung IX. Conclusions References

144 147 149

The Role of Small Proteoglycans in the Formation of Fibrosis Gunilla Westergren-Thorsson, Ellen Tufvesson, Erik Eklund, and Anders Malmstro¨m

169

I. Introduction II. Small Proteoglycans: A Family of Structurally Different Macromolecules III. Impact of Small Proteoglycans on Matrix Assembly IV. Core Proteins of Small Proteoglycans: Interactions with Cytokines and Their Receptors V. Polysaccharide Substitution of Small Proteoglycans VI. Metabolism of the Small Proteoglycans VII. Different Pathological Disorders Involving the Small Proteoglycans VIII. Normal Physiology of the Lung IX. Small Proteoglycans and Lung Pathology X. Conclusions and Future Perspectives References

169

Versican in the Cell Biology of Pulmonary Fibrosis Clive R. Roberts I. II. III. IV.

Introduction Proteoglycans Functions of Versican in Provisional Matrix Platelet-Derived Growth Factor and Transforming Growth Factor Beta V. Parallels Between Pulmonary Fibrosis and Wound Healing in Other Systems

153 155 159

164 164

170 172 173 174 174 176 176 177 181 183 191 191 194 199 202 203

Contents VI.

xix Conclusions References

11. Decorin in Asthma Anthony E. Redington I. Introduction II. Molecular Biology of Decorin III. Interactions Between Decorin and the Extracellular Matrix (ECM) IV. Interactions Between Decorin and TGFβ V. Airway Fibrosis in Asthma VI. Expression of Decorin and TGFβ in Normal and Asthmatic Airways VII. Therapeutic Implications References 12. Role of Proteoglycans in the Development and Pathogenesis of Emphysema Ymke M. Van Der Geld, Jeanette F. M. Van Straaten, Dirkje S. Postma, and Wim Timens I. II. III. IV. V.

Introduction Pulmonary Interstitium in Emphysema Proteoglycans in Emphysema Pathogenesis of Pulmonary Emphysema Conclusions References

13. Proteoglycans in Normal and Pathological Bronchial Mucus K. Ramakrishnan Bhaskar I. II. III. IV. V. VI. VII.

Introduction Cellular Sources of Bronchial Mucus Proteoglycans in Normal Bronchial Mucus Bronchoalveolar Lavage (BAL) from Healthy Volunteers In Vitro Culture of Airway Mucosal Explants Glycoconjugates Secreted by Airway Epithelial Cells in Culture Proteoglycans in Pathological Bronchial Mucus References

205 207 213 213 214 215 217 220 221 228 228

241

241 243 247 254 259 260 269 269 270 271 271 273 275 279 285

xx 14.

Contents Vascular Proteoglycans Hannu Ja¨rvelaïnen and Thomas N. Wight I. II. III. IV. V. VI.

15.

Degradation of Lung Matrix Proteoglycans in Bronchiectasis Daisy Kwok-Yan Shum and Mary Sau-Man Ip I. II. III. IV. V.

16.

291 292 295 300 307 308 308

323

Introduction Central Role of the Neutrophil in Bronchiectasis Neutrophil Elastase and Antielastases Degradation of Lung Matrix Proteoglycans Summary References

323 323 324 325 329 329

Effect of Hypoxia on Glycosaminoglycan Synthesis by Lung Cells Eleni Papakonstantinou, George Karakiulakis, and Michael Roth

335

I. II. III. IV.

17.

Introduction Proteoglycans: Structure and Classification Proteoglycans Identified in Blood Vessels Functions of Proteoglycans Found in the Vascular Wall Proteoglycans in Vascular Diseases Conclusion References

291

Introduction Role of Glycosaminoglycans in Lung Cell Biology Effect of Hypoxia on Glycosaminoglycans Glycosaminoglycans as New Therapeutic Agents or Targets References

335 339 340 344 344

Integrins, Proteoglycans, and Lung Disease Jiahua Xu

351

I. Introduction II. Cellular Function of Integrins III. Integrins in Common Lung Diseases References

351 352 357 365

Contents 18. Heparin as a Potential Therapeutic Agent to Reverse Vascular Remodeling Hari G. Garg, Charles A. Hales, and Robert J. Linhardt I. II. III. IV.

Introduction Heparin Structure Low-Molecular-Weight Heparins Differences in the Structure of Heparin and Heparan Sulfate V. Heparin and Pulmonary Hypertension VI. Antiproliferative Activity of Heparin and Its Derivatives VII. Summary and Conclusion References Author Index Subject Index

xxi

377 377 379 380 381 382 384 391 392 399 455

1 Proteoglycans of the Lung

PETER J. ROUGHLEY Shriners Hospital for Children Montreal, Quebec, Canada

I.

Introduction

The lung is a complex organ consisting of multiple cell and tissue types, and one therefore expects many proteoglycans to be present. Unfortunately, the complement of lung proteoglycans has not been fully characterized and it is not possible to produce a definitive list of which proteoglycans are present. This review presents a description of those proteoglycans known to be present in the lung and those expected to be present, and provides information on their structure, heterogeneity, and probable function. II. Proteoglycans Proteoglycans belong to the family of glycoproteins and are characterized by the presence of one or more sulfated glycosaminoglycan chains on a protein core. Like all glycoproteins, they may also possess N-linked or O-linked oligosaccharides. Because of the presence of glycosaminoglycan chains, proteoglycans are sometimes referred to as being glycanated rather than glycosylated. 1

2

Roughley

Proteoglycans have been classified by many means, with original classifications being based on the type of glycosaminoglycan substitution (e.g., chondroitin sulfate proteoglycan) or the tissue of origin (e.g., cartilage proteoglycan). Both systems are inappropriate, as the type of glycosaminoglycan present on a given proteoglycan may vary among different cells, and a given proteoglycan may be present in multiple tissues. Current classifications are based on the tissue location of the proteoglycan (extracellular, cell membrane-associated, or intracellular) and the gene family to which it belongs. Proteoglycans are extremely variable in structure, particularly with respect to their glycosaminoglycan chains. As there is no template for glycosaminoglycan synthesis, their structure can vary with parameters such as cell type, age, and health status. In addition, not all potential glycosaminoglycan attachment sites need be glycanated by all cells, and different cells may initiate the synthesis of different glycosaminoglycans at the same attachment site. For some proteoglycans, variability may also exist in the core protein structure, owing to either alternative splicing of exons in the gene or proteolytic processing following synthesis. It is also possible to have nonglycanated proteoglycans. This can arise by a number of mechanisms: incomplete glycosaminoglycan substitution of the core protein during synthesis (e.g., perlecan); alternative splicing of the exon(s) encoding the glycosaminoglycan attachment region(s) (e.g., versican); and proteolytic cleavage of a terminal glycosaminoglycan attachment region (e.g., biglycan). Proteoglycans in which glycosaminoglycan synthesis may be absent or present are commonly termed “part-time” proteoglycans. In some cases proteoglycan function may be independent of glycosaminoglycan substitution, whereas in others the absence of the glycosaminoglycan chain(s) may modify function.

III. Glycosaminoglycans Glycosaminoglycans (GAGs), formerly known as mucopolysaccharides, belong to one of four families (1): hyaluronan, chondroitin sulfate/dermatan sulfate, heparan sulfate/heparin, and keratan sulfate (Table 1). Hyaluronan (hyaluronic acid, HA) is by far the longest glycosaminoglycan and is not sulfated (2). It is composed of repeating disaccharide units of glucuronic acid and N-acetyl glucosamine, and is synthesized at the plasma membrane of most cells by a hyaluronan synthase (HAS). Mammals possess three HAS genes (3), whose products may vary in their rate of HA synthesis and the size of the HA they produce (4). In all cases HA synthesis takes place at the inner surface of the plasma membrane, and the HA is extruded directly through the membrane to the extracellular space (5). In contrast, synthesis of the other glycosaminoglycans takes place within the Golgi apparatus of the cell on a proteoglycan core protein and requires a variety of glycosyl transferases and sulfotransferases (6). Although HA is never synthe-

Proteoglycans of the Lung

3

Table 1 Glycosaminoglycans Glycosaminoglycan

Hexose

HA CS DS

GlcA GlcA GlcA IdoA GlcA IdoA Gal

HS/Heparin KS

Sulfation a — — — 2 — 2 6

Hexosamine GlcNAc GalNAc GalNAc GalNAc GlcNAc GlcNAc GlcNAc

Sulfation a — 4,6 4,6 4,6 3,6,N 3,6,N 6

a

Sulfation is indicated by the position of the substituted hydroxyl group (2, 3, 4, or 6) or by attachment to the hexosamine amino group (N). Abbreviations: HA, hyaluronan; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; GlcA, glucuronic acid; IdoA, iduronic acid; Gal, galactose; GlcNAc, N-acetyl glucosamine; GalNAc, N-acetyl galactosamine.

sized as a proteoglycan, some of it may ultimately exist as a proteoglycan via the covalent interaction of members of the inter-α-trypsin inhibitor family (7). HA may also interact noncovalently with members of the aggregating proteoglycan family (e.g., aggrecan) within the extracellular matrix. Chondroitin sulfate (CS) is composed of a repeating disaccharide backbone of glucuronic acid and N-acetyl galactosamine, with the latter residue being sulfated at either the 4- or 6-position hydroxyl groups, or occasionally at both positions. CS with 4-sulfation was originally termed CS-A whereas that with 6-sulfation was termed CS-C, though it is now appreciated that the majority of CS chains possess sulfation at both sites, but to varying degrees depending on the cell of origin. Dermatan sulfate (DS) is a modified form of CS in which glucuronic acid is converted to iduronic acid by an epimerase; hence its original name of CS-B. The iduronic acid residues of DS may be sulfated at their 2-position hydroxyl groups, and the degree of epimerization of DS may vary considerably with cell type. Heparan sulfate (HS) is the most structurally heterogeneous of the glycosaminoglycans (8). It is initially synthesized with repeating disaccharides of glucuronic acid and N-acetyl glucosamine, but undergoes extensive modification, including epimerization of glucuronic acid to iduronic acid and subsequent 2sulfation, deacetylation, and subsequent N-sulfation of the glucosamine residues, and sulfation of the 6-position (and occasionally the 3-position) hydroxyl groups of the glucosamine residues. As with DS, the extent of polymer modification of HS varies considerably with cell type. Heparin is the most highly modified form of HS. It is produced by the mast cell and occurs exclusively on the serglycin proteoglycan core. In the early literature heparan sulfate was known as heparitin sulfate or heparin monosulfate.

4

Roughley

Keratan sulfate (KS) is the only glycosaminoglycan not to possess a uronic acid residue. Instead, it is composed of repeating disaccharides of galactose and N-acetyl glucosamine. The N-acetyl glucosamine is commonly sulfated at its 6position hydroxyl group, and the galactose may be sulfated at the same position. KS and DS are most commonly associated with proteoglycans of the extracellular matrix, whereas HS is most commonly associated with proteoglycans of basement membranes or at the cell surface. Heparin, in its proteoglycan form, is stored intracellularly. CS may be associated with proteoglycans at all locations.

IV. Proteoglycan Metabolism All proteoglycans described to date are encoded by a single gene, though in some cases multiple messages may arise from this gene owing to alternative splicing of the initial transcript (e.g., aggrecan, versican, and CD44). Core protein synthesis then takes place in the endoplasmic reticulum of the cell, followed by GAG synthesis in the Golgi apparatus (6). Synthesis of the CS and HS families of GAGs commence with a common linkage tetrasaccharide of xylose-galactosegalactose-glucuronic acid, with the xylose being attached to a serine residue in the core protein. The serine residue is commonly followed by an adjacent glycine residue and preceded by an acidic residue for recognition by the xylosyl transferase. Subsequently, the linkage tetrasaccharide is extended by either the enzymes involved in CS/DS synthesis or those involved in HS/heparin synthesis, which are located in different regions of the Golgi apparatus (6). It is thought that sequences within the core protein are responsible for direction to the appropriate site (9–11). Synthesis of KS is different from the other GAGs. It commences with a linkage that is common to many N-linked or O-linked oligosaccharides, with substitution to asparagine or serine/threonine residues, respectively (12). The linkage region is then extended by polylactosamine synthesis and concomitant sulfation to yield KS. The fine structure of the GAG chains depends on the level of expression of the different enzymes involved in their synthesis by a given cell. For many steps several isoforms of the glycosyl transferases or sulfotransferases exist, which give rise to different ultimate structures for the GAG chains. Following their synthesis, the proteoglycans are packaged into Golgiderived vesicles (6). Usually these vesicles translocate to the cell surface, where they fuse with the plasma membrane, causing release of those molecules destined for the extracellular matrix and retention at the outer surface of the plasma membrane of those that had been integrated with the vesicle membrane. The exception to this process is with serglycin, which remains within the cell in storage granules. Once the proteoglycan has reached its final destination it may be turned over by several mechanisms involving proteolysis and/or endocytosis. Cell-asso-


5

ciated proteoglycans may be endocytosed directly, with the endosomes subsequently fusing with lysosomes containing a variety of proteinases, glycosidases, and sulfatases that can degrade both the core protein and GAG chains of the proteoglycan (13). Some extracellular proteoglycans may also be catabolized via endocytosis following their release from extracellular matrix or basement membrane interactions and subsequent interaction with a cell surface receptor that mediates endocytosis (e.g., decorin) (14). However, many extracellular proteoglycans are catabolized via their loss from the matrix following proteolysis and subsequent removal from the tissue via the lymphatic and/or circulatory systems. The degradation products are ultimately endocytosed by liver endothelial cells for degradation via lysosomal enzymes. Gene defects in the enzymes responsible for lysosomal glycosaminoglycan degradation can result in incomplete catabolism and storage of the partially degraded material within the cells. This group of disorders is known as the mucopolysaccharidoses (15). Some cell surface proteoglycans may follow the same route of degradation following proteolytic cleavage of their ectodomain close to the plasma membrane. Some regions of the extracellular matrix proteoglycans that interact with other matrix components are relatively stable toward proteolysis and may survive in the matrix for many years (e.g., aggrecan), particularly in avascular connective tissues such as cartilage (16). This limited proteolysis contributes to the molecular heterogeneity of such proteoglycans. HA turnover may take place via either endocytosis or loss from the tissue, with transport via the lymphatic system and circulation for ultimate catabolism in the liver in the latter pathway (2,17).

V.

Extracellular Matrix Proteoglycans

There are two major families of proteoglycans present in the extracellular matrix of all tissues—the large aggregating proteoglycans, and the small, leucine-rich repeat proteoglycans (Table 2)—though different tissues vary in the family members that they contain. A. Aggregating Proteoglycans

The aggregating proteoglycans are characterized via their ability to interact noncovalently with HA to form proteoglycan aggregates. This interaction takes place via an amino terminal globular (G1) region of the core protein and has led to the suggestion that the family be termed hyalectans (18). There are currently four members of the family—aggrecan, versican, neurocan, and brevican (Table 2)— though only aggrecan and versican are likely to be present in the lung, as neurocan and brevican are restricted to the brain. All family members also possess a carboxy terminal globular (G3) region of their core proteins which has lectinlike

6

Roughley

Table 2

Extracellular Proteoglycans

Proteoglycan Aggrecan Versican Neurocan Brevican Decorin Biglycan Fibromodulin Lumican Keratocan Epiphycan Mimecan Perlecan Agrin Bamacan

Chromosome 15q26 5q12–14 19p12–13.1 (1q25–31) a 12q23 Xq28 1q32 12q22 12q22 12q21 9q22 1q36 1p32-ter (10q25) a

Exons 19 15 15 (14) a 8 8 3 3 3 7 7 94

Alternative names CSPG2, PG-M CSPG3 DSPG2 DSPG1

DSPG3, PGLb Osteoglycin HSPG2

(31) a

a

Brackets indicate data inferred for the human gene from analysis of the mouse gene.

homology, and this has led to the suggestion that the family be termed lecticans (19). 1.

Aggrecan

Aggrecan is characteristic of hyaline cartilage and connective tissues that are subject to compression, e.g., intervertebral disk, and is present in the airway cartilages of the lung. Its core protein contains ⬎2000 amino acids and can be divided into seven functional regions (20). In addition to the terminal G1 and G3 regions, the aggrecan core protein contains a third globular (G2) region adjacent to the G1 region, and is separated from it by a short interglobular domain (IGD). The function of the G2 region is unclear, for although it shares sequence similarity to the HA-binding domain of the G1 region, it does not interact with HA (21,22). The G2 and G3 regions are separated by a long extended domain possessing most of the GAG chains in the proteoglycan (Fig. 1), which may number ⬎100. This extended domain can be separated into three regions—a KS-rich region adjacent to the G2 region; a central CS-rich (CS-1) region; and a second CS-rich (CS-2) region adjacent to the G3 region. Part of the KS-rich region and all of both the CS-1 and CS-2 regions are encoded by one large exon in the aggrecan gene (exon 12) (23). The CS-1 and CS-2 regions are characterized by multiple repeat sequences that differ in both amino acid sequence and CS attachment site spacing between the two regions. The CS-1 region is of interest as it exhibits polymorphic


7

Figure 1 Extracellular matrix proteoglycans. The figure depicts the proteoglycans with fully extended core proteins whose length is proportional to the number of amino acid residues that they possess. The position of glycosaminoglycan chain attachment is indicated by vertical lines (which are not proportional to chain length). The amino terminus (N) of each core protein is on the left of the figure. Abbreviations: KS, keratan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate.

variation in humans, with the number of repeats so far identified ranging from 13 to 33 (24). The G3 region of aggrecan also exhibits structural variation, owing to alternative splicing of some of its subdomains. It is unclear whether these structural differences in the G3 region are of any functional consequence. The function of aggrecan relates to its ability to generate high osmostic pressures within the tissue and so resist compressive deformation (25). This property in part relates to the ability of aggrecan to form large aggregates and in part to its high content of glycosaminoglycan chains. This latter property is reduced in individuals with the shortest CS-1 region variants, and some evidence suggests that such individuals may be susceptible to intervertebral disk degeneration (26). The importance of glycosaminoglycan sulfation on the properties of aggrecan is perhaps better illustrated in individuals with diastrophic dysplasia (27), where a congenital dwarfism develops as a result of a mutation in an intracellular sulfate

8

Roughley

transporter required for glycosaminoglycan sulfation during synthesis. Other chondrodysplasias have been described in mice and chickens owing to mutations in the aggrecan gene that interfere with glycosaminoglycan substitution by truncating core protein synthesis (28). Inadvertent proteolysis can also impair aggrecan function, as degradation products that do not possess a G1 region are rapidly lost from the tissue. Such proteolysis can occur during inflammation, as cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) are known to stimulate aggrecan degradation by chondrocytes. Such degradation has been associated with aggrecanases (members of the ADAMTS family of proteinases) and matrix metalloproteinases (MMPs) (29,30). 2.

Versican

Versican was named for its supposed versatile function owing to the presence of multiple structural domains in its core protein (31). Unlike aggrecan, it is present in most connective tissues, including those of the lung. In contrast to aggrecan, versican does not possess a G2 region, does not exhibit alternative splicing of its G3 region, has a much lower degree of substitution by CS in the central glycosaminoglycan attachment region, and possesses no KS-rich domain (Fig. 1). The CS attachment region of the versican gene is encoded by two large exons (exons 7 and 8) that exhibit alternative splicing (32). Four possible isoforms can result in which both exons are present (V0 ), only exon 8 is present (V1 ), only exon 7 is present (V2 ), or neither exon is present (V3 ). The V1 isoform is ubiquitously expressed and has ⬃2400 amino acids and 16 CS chains. The V0 isoform possesses nearly 3400 amino acids and may have about eight additional CS chains. It is more commonly associated with embryonic development of connective tissues and was originally termed PG-M (33). The other versican isoforms are most commonly associated with the brain. The domains encoded by exons 7 and 8 are referred to as the GAGα and GAGβ regions, respectively, and hence the common V1 isoform of versican possesses only the GAGβ region. The precise function of versican is unclear though it is thought to be involved in tissue hydration in mature tissues. In the embryo it is thought to play a role in influencing cell migration and interaction during organogenesis. It might play a similar role during wound healing and in fibrosis. 3.

Link Protein

The interaction between the aggregating proteoglycans and HA is noncovalent and potentially reversible, which could have dire consequences for the tissue. In vivo, undesired dissociation is prevented by a link protein that interacts with both the G1 region of the hyalectan and HA (34). In some respects the link protein may be considered the smallest member of the hyalectan family, as it is structur-


9

ally similar at both the protein and gene organization levels to the G1 region (35). It is not, however, a proteoglycan. The best-studied link protein is that which interacts with aggrecan in cartilage. The link protein in an aggrecan-derived proteoglycan aggregate is relatively resistant to proteolysis, with the exception of its amino terminus. Proteolytically modified link protein accumulates in the cartilage matrix with age, attesting to its relatively slow turnover. Analysis of the site of link protein cleavage provides evidence for the action of several proteinases, including MMPs (36). The importance of the link protein in cartilage function is evident from mice which bear a null mutation in the link protein gene (37). Such mice show severe abnormalities in cartilage development and endochondral ossification. A second link protein gene has recently been described (38), and this raises the question of whether different tissues or different hyalectans use different link proteins to stabilize their proteoglycan aggregates. B. Leucine-Rich Repeat Proteoglycans

The leucine-rich repeat proteoglycans are characterized by a central region of adjacent leucine-rich repeats flanked by cystine-bonded domains and an amino terminal domain. To date seven members of this family have been characterized (Table 2), together with several related leucine-rich repeat proteins that are not glycanated (39,40). The family members may be divided into two groups depending on whether they are substituted with CS/DS or KS chains. Each subfamily may be further subdivided according to the number of leucine-rich repeats (10 or 6) within their central region or their gene organization. The most widespread leucine-rich repeat proteoglycans are decorin, biglycan, fibromodulin, and lumican; keratocan, epiphycan, and mimecan show tissue-selective expression and have yet to be identified in the lung. This family of proteoglycans have relatively short core proteins with 300–400 amino acids (Fig. 1). 1. Decorin and Biglycan

Decorin and biglycan are substituted with CS or DS chains in their amino terminal region. In the case of decorin one chain is present, whereas biglycan has two chains (hence its name). Both proteoglycans possess 10 leucine-rich repeats in their central region, and also possess two or three N-linked oligosaccharides in this region (41). Both proteoglycans are encoded by genes possessing eight exons, which show conservation in their splice junctions (42,43). The two proteoglycans are unique among proteoglycans in possessing a short propeptide between their signal peptide and the mature core protein (44). The propeptide is removed following secretion of the proteoglycans from the cells by the same proteinase that cleaves the carboxy propeptide from the fibrillar collagens (45). It is not clear whether this process is fortuitous or by design, or whether or not it influences proteoglycan function. Biglycan can also undergo proteolytic processing within

10

Roughley

its amino terminal region within the extracellular matrix, the consequence of which is to separate the CS/DS chains from the remainder of the core protein (46). The resulting “nonglycanated” biglycan then accumulates in the tissue. The functional consequence of this event is also unclear. Decorin is commonly thought of as a collagen-binding proteoglycan. Indeed, its name was derived from its surface decoration of collagen fibrils when viewed in the electron microscope. The interaction of decorin with collagen is mediated by the core protein and is independent of the presence of the CS/DS chain (47). The presence of decorin alters the kinetics of fibril formation and the diameter of the resulting fibril. The importance of decorin in collagen fibrillogenesis is best illustrated by the decorin-null mouse, which has a phenotype with abnormal skin fragility and collagen fibril organization (48). Decorin may also function in the extracellular matrix by modulation of growth factor action (49). Of particular note is its ability to interact with transforming growth factor-β (TGFβ) and limit its function. This property is of therapeutic interest in the treatment of fibrosis (50). Decorin has also been shown to interact with the epidermal growth factor (EGF) receptor and initiate signaling (51). The function of biglycan is less clear. In vitro it is able to interact with collagen fibrils under selected conditions (52), but it is not clear that it fulfills this role in vivo. It does, however, share with decorin the ability to interact with TGFβ via its core protein (49). Biglycan-null mice develop an osteoporotic phenotype that is quite distinct from the decorin-null mice (53). Thus, it is clear that decorin and biglycan fulfill different functional roles. 2.

Fibromodulin and Lumican

Fibromodulin and lumican are KS proteoglycans, with the sites of KS substitution being in the central leucine-rich repeat region. In some cell types KS synthesis does not occur, and both fibromodulin and lumican are then produced in a nonglycanated form possessing only N-linked oligosaccharides (54,55). When the proteoglycan forms of the molecules are produced, they may possess one to four KS chains (56,57). Depending on the cell type involved, the length of the KS chains and their degree of sulfation may vary considerably. Both proteoglycans are encoded by genes possessing three exons (54,58). Fibromodulin and lumican are collagen fibril-binding proteoglycans, though they interact at sites that are distinct from decorin (59–61). Fibromodulin’s name relates to its ability to influence collagen fibrillogenesis, and its deficiency in the fibromodulin-null mouse results in collagen fibrils that are more heterogeneous in size, though the resulting phenotype is relatively mild (62). Lumican was named for its original discovery in the cornea and its presumed role in maintaining corneal transparency. Lumican is, however, widespread in its tissue distribution and is prevalent in the lung (63).


11

A functional role for lumican in several connective tissues is supported by the phenotype of the lumican-null mouse (64,65).

VI. Basement Membrane Proteoglycans Basement membranes have been associated with both HS proteoglycans and CS proteoglycans (18), and to date three proteoglycans that are structurally unrelated have been identified—perlecan, agrin, and bamacan (Table 2). Of these, perlecan is by far the best studied and is thought to be ubiquitous in its basement membrane distribution. Perlecan may exist as either an HS proteoglycan or a CS proteoglycan, whereas agrin is described as an HS proteoglycan and bamacan as a CS proteoglycan (66,67). Perlecan is the largest of the well-characterized proteoglycans, with its core protein possessing ⬃4400 amino acids in the human and multiple structural domains (68). This complexity in protein structure is reflected in the gene which possesses 94 exons (69). Upon examination by rotary shadowing electron microscopy, these multiple domains have the appearance of pearls on a string, and hence the name perlecan. The major sites for glycosaminoglycan attachment reside in the extreme amino terminal domain of the perlecan core protein (Fig. 1) (70). These three sites are usually occupied by HS, though CS substitution has also been described. Sites for GAG substitution have also been described near the carboxy terminus of the core protein (71). Perlecan has also been identified within the extracellular matrix of some tissue, remote from basement membranes. Of particular note is its presence in cartilage (72), where no basement membranes exist. At present it is not clear whether perlecan remains in the tissue in its intact form, or whether it undergoes proteolytic processing with the accumulation of selected fragments. It is not clear whether perlecan has a specific role to play in tissues or multiple roles (73). Certainly its complex core protein structure provides the potential to interact with numerous proteins. In basement membranes it has often been described as a filtration barrier, limiting the flow of macromolecules or cells between two tissue compartments. HS degradation on perlecan by the action of heparanases has been associated with the ability of some metastatic cancer cells to penetrate basement membranes (74). Perlecan is also able to interact with basic fibroblast growth factor (bFGF) via its HS chains, a property shared with other HS proteoglycans. In this manner it can regulate interaction of this growth factor with its receptor and so modulate tissue metabolism. This may be a principal role of perlecan in the extracellular matrix. The multiple functions of perlecan are evident from the tissue disruption that occurs in both perlecan-null mice (75,76) and humans with mutations in the perlecan gene, where problems occur in

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organ development owing to basement membrane perturbation and in the skeleton owing to abnormal chondrocyte differentiation. In humans, gene mutations have been associated with chondrodystrophic myotonia (77) and dyssegmental dysplasia (78). VII. Cell Surface Proteoglycans There are two major families of cell surface proteoglycans (79)—the syndecans and the glypicans (Table 3). There are currently four members of the syndecan family which are integral membrane proteins, and six members of the glypican family which are attached via a GPI anchor. It is not clear how many of these are present in the lung. There are also other unrelated cell surface proteoglycans, such as betaglycan and CD44, that may be present in the lung. Both are integral membrane proteins. A. Syndecans

All the syndecans are type I membrane-spanning glycoproteins (80), with a single transmembrane domain near the carboxy terminus of their core protein (Fig. 2). They all possess relatively small cytoplasmic domains that have terminal conserved regions and a central variable region, which allows both common and unique intracellular interactions to occur (81). The cytoplasmic region has been associated with interaction with both the cytoskeleton and protein kinases. The

Table 3 Cell-Associated Proteoglycans Proteoglycan

Chromosome

Syndecan 1 Syndecan 2 Syndecan 3 Syndecan 4 Glypican 1 Glypican 2 Glypican 3 Glypican 4 Glypican 5 Glypican 6 Betaglycan CD44 Serglycin

2p23–24 8q23 (1p32–36) a 20q12 2q35–37

a

Exons 5

Xq26 Xq26 13q32 13q32

(5) a 5 9 9 8 9 8 9

11p13 10q22

21 3

Alternative names Syndecan Fibroglycan N-syndecan Amphiglycan, ryudocan Glypican Cerebroglycan OCI-5 K-glypican

TGFβRIII Epican, Hermes, H-CAM Heparin

Brackets indicate data inferred for the human gene from analysis of the rat gene.


13

Figure 2 Cell-associated proteoglycans. The figure depicts the proteoglycans with fully extended core proteins whose length is proportional to the number of amino acid residues that they possess. The position of glycosaminoglycan chain attachment is indicated by vertical lines (which are not proportional to chain length). The amino terminus (N) of each core protein is on the left of the figure. Abbreviations: CS, chondroitin sulfate; HS, heparan sulfate; Hep, heparin. The location of the transmembrane domains in syndecan, ⴝ), the location of the GPI plasma membrane anchor in glypican betaglycan and CD44 (ⴝ (䉬), and the site of insertion for protein encoded by alternatively spliced exons in CD44 (䉱) are also indicated.

extracellular domain varies in size and amino acid sequence among all family members. All these ectodomains are characterized by the presence of attachment sites for HS near the amino terminus of the core protein, which is remote from the plasma membrane. Some family members (syndecans 1 and 3) also possess additional GAG substitution sites close to the plasma membrane that may be involved in CS attachment. The function of syndecans is most commonly associated with its HS chains and their interaction with heparin-binding growth factors (e.g., bFGF) or extracellular proteins (e.g., fibronectin and laminin) (82,83). Such interactions may influence cell metabolism directly by inducing changes in the interactions of the cytoplasmic tail. In the case of bFGF, interaction with HS facilitates binding to its natural receptor and subsequent intracellular signaling. Interaction of the HS chains with extracellular matrix or basement membrane components may also

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be important for cell adhesion. The possibility of unique functions for the syndecans has begun to be studied in knockout mice. Perhaps surprisingly, the syndecan-1-null mouse shows little phenotype (79), even though this is the predominant family member on epithelial cells. Presumably, there is some redundancy in function with other cell surface HS proteoglycans. However, the knockout mice did exhibit abnormal wound healing in both skin and cornea owing to an inability of keratinocytes and keratocytes to migrate, indicating that unique functions do exist. B. Glypicans

All members of the glypican family are bound to the cell surface via a GPI link between the carboxy terminus of their core proteins and the plasma membrane (Fig. 2). In contrast to the syndecans, the attachment sites for HS are toward the carboxy terminal end of the core proteins, and the HS chains are presumably in close proximity to the plasma membrane. It is likely that glypicans and syndecans can have some distinct roles at the cell surface, even though both can interact with HS-binding proteins. First, glypicans contain no cytoplasmic domain that could be involved in direct signaling; secondly, the extracellular domain of the glypicans can be shed from the cell by lipase action, whereas shedding of the syndecan extracellular domain requires proteolysis. The unique role of glypicans is best illustrated in the Simpson-Golabi-Behmel syndrome, where mutations in the glypican-3 gene result in a wide spectrum of tissue abnormalities (84). C. Betaglycan

Betaglycan is a type I membrane-spanning glycoprotein with a short carboxy terminal cytoplasmic tail, an adjacent transmembrane domain, and a large extracellular domain (85). It has been shown to be substituted with either HS or CS chains near the center of its extracellular domain (Fig. 2). It is commonly known as the type III TGFβ receptor and plays a crucial role in TGFβ signaling (86), though it is not itself a signaling receptor. Instead, it acts as a TGFβ-binding protein that presents TGFβ to the signaling type II receptor. Betaglycan interacts with all forms of TGFβ toward the amino terminal end of the core protein, with the interaction being independent of the glycosaminoglycan chains. As with other membrane-bound proteoglycans, the extracellular domain of betaglycan can be shed by proteolysis. This soluble domain still retains TGFβ-binding properties, but may now impede TGFβ signaling rather than promote it, by competing with the membrane-bound betaglycan. As with other cell membrane HS proteoglycans, betaglycan may also be involved in the interaction of heparin-binding proteins via its HS chains. Hence, it may also participate in bFGF signaling in a manner similar to the syndecans and glypicans.


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D. CD44

CD44 is a type I membrane-spanning glycoprotein that acts as a receptor for HA (87,88). Its extracellular domain is composed of three regions—a membrane proximal portion that is extremely variable in length owing to alternative splicing of the gene; a CS attachment domain; and an amino terminal domain that is involved in the interaction with HA (Fig. 2). This latter domain shares structural features in common with the HA-binding domains of link protein and the G1 region of the aggregating proteoglycans. The shortest form of CD44, possessing none of the 10 adjacent alternatively spliced exons, is very common, and has been termed CD44H (for its identification on hematopoietic cells). Larger forms of CD44 vary with tissue and cell type (89), and some may possess HS chains if exon 8 of the gene is expressed (90). In addition to its function as an adhesion molecule for extracellular matrix components, CD44 may also participate in intracellular signaling via its cytoplasmic tail. It has been reported that the interaction with large polymeric HA promotes adhesion, whereas interaction with small HA fragments may promote signaling (87). VIII. Intracellular Proteoglycans Serglycin is the only proteoglycan destined for specific storage within cells. It is present within the storage granules of many hematopoietic cells and exists as either a heparin proteoglycan or a CS proteoglycan (91). The presence of heparin is unique to the mast cell. Serglycin possesses a short core protein with about eight GAG chains attached to its central region (Fig. 2), which is composed of adjacent serine-glycine repeats and is encoded by a three-exon gene (92,93). When CS is present it is often oversulfated, being rich in N-acetyl galactosamine residues that are both 4- and 6-sulfated. Within the storage granules, the highly anionic serglycin may form complexes with cationic proteins that are also present and limit their function. The serglycins can also serve extracellular roles following their release from the storage granules. The best-characterized of these is the role of heparin as an anticoagulant via its interaction with antithrombin III (94). However, it is now appreciated that some HS proteoglycans may also fulfill this role via specifically modified regions of their HS chains. IX. Conclusions The lung undoubtedly possesses multiple proteoglycans including aggrecan, versican, decorin, biglycan, fibromodulin, lumican, perlecan, agrin, syndecans, glypicans, betaglycan, CD44, and serglycin. The presence of these proteoglycans will vary among the different tissues of the lung and at different ages, and, when

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present, the structure of the proteoglycans may vary by both core protein and glycosaminoglycan modifications. In addition, proteoglycan structure and abundance may vary as a consequence of lung disorders. We are only just beginning to understand the consequence of such change on both proteoglycan function and the lung, but there is no doubt that this family of macromolecules is of considerable importance in maintaining normal lung function. Acknowledgment The author is funded by the Shriners of North America. References 1. Jackson RL, Busch SJ, Cardin AD. Glycosaminoglycans: Molecular properties, protein interactions, and role in physiological processes. Physiol Rev 1991; 71:481– 539. 2. Fraser JRE, Laurent TC, Laurent UBG. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997; 242:27–33. 3. Spicer AP, McDonald JA. Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family. J Biol Chem 1998; 273:1923–1932. 4. Itano N, Sawai T, Yoshida M, Lenas P, Yamada Y, Imagawa M, Shinomura T, Hamaguchi M, Yoshida Y, Ohnuki Y, Miyauchi S, Spicer AP, McDonald JA, Kimata K. Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J Biol Chem 1999; 274:25085–25092. 5. Weigel PH, Hascall VC, Tammi M. Hyaluronan synthases. J Biol Chem 1997; 272: 13997–14000. 6. Prydz K, Dalen KT. Synthesis and sorting of proteoglycans: commentary. J Cell Sci 2000; 113:193–205. 7. Bost F, Diarra-Mehrpour M, Martin JP. Inter-α-trypsin inhibitor proteoglycan family: a group of proteins binding and stabilizing the extracellular matrix. Eur J Biochem 1998; 252:339–346. 8. Stringer SE, Gallagher JT. Heparan sulphate. Int J Biochem Cell Biol 1997; 29: 709–714. 9. Esko JD, Zhang LJ. Influence of core protein sequence on glycosaminoglycan assembly. Curr Opin Struct Biol 1996; 6:663–670. 10. Dolan M, Horchar T, Rigatti B, Hassell JR. Identification of sites in domain I of perlecan that regulate heparan sulfate synthesis. J Biol Chem 1997; 272:4316–4322. 11. Doege K, Chen XC, Cornuet PK, Hassell J. Non-glycosaminoglycan bearing domains of perlecan and aggrecan influence the utilization of sites for heparan and chondroitin sulfate synthesis. Matrix Biol 1997; 16:211–221. 12. Funderburgh JL. Keratan sulfate: structure, biosynthesis, and function. Glycobiology 2000; 10:951–958. 13. Yanagishita M, Hascall VC. Cell surface heparan sulfate proteoglycans. J Biol Chem 1992; 267:9451–9454.


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Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999; 68:729–777. Mali M, Jaakkola P, Arvilommi A-M, Jalkanen M. Sequence of human syndecan indicates a novel gene family of integral membrane proteoglycans. J Biol Chem 1990; 265:6884–6889. Woods A, Oh ES, Couchman JR. Syndecan proteoglycans and cell adhesion. Matrix Biol 1998; 17:477–483. Lyon M, Gallagher JT. Bio-specific sequences and domains in heparan sulphate and the regulation of cell growth and adhesion. Matrix Biol 1998; 17:485–493. Tumova S, Woods A, Couchman JR. Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. Int J Biochem Cell Biol 2000; 32: 269–288. Pilia G, Hughes-Benzie RM, MacKenzie A, Baybayan P, Chen EY, Huber R, Neri G, Cao A, Forabosco A, Schlessinger D. Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome. Nat Genet 1996; 12:241–247. Ponce-Castan˜eda MV, Esparza-Lo´pez J, Vilchis-Landeros MM, Mendoza V, Lo´pezCasillas F. Murine betaglycan primary structure, expression and glycosaminoglycan attachment sites. Biochim Biophys Acta Protein Struct Mol Enzymol 1998; 1384: 189–196. Attisano L, Wrana JL, Lo´pez-Casillas F, Massague´ J. TGF-β receptors and actions. Biochim Biophys Acta Mol Cell Res 1994; 1222:71–80. Bajorath J. Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions. Proteins 2000; 39:103–111. Kremmidiotis G. CD44. J Biol Regul Homeost Agents 1999; 13:234–239. Kugelman LC, Ganguly S, Haggerty JG, Weissman SM, Milstone LM. The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44. J Invest Dermatol 1992; 99:381–385. Cooper DL, Dougherty GJ. To metastasize or not? Selection of CD44 splice sites. Nat Med 1995; 1:635–637. Kresse H, Hausser H, Scho¨nherr E. Small proteoglycans. Experientia 1993; 49:403– 416. Avraham S, Stevens RL, Nicodemus CF, Gartner MC, Austen KF, Weis JH. Molecular cloning of a cDNA that encodes the peptide core of a mouse mast cell secretory granule proteoglycan and comparison with the analogous rat and human cDNA. Proc Natl Acad Sci USA 1989; 86:3763–3767. Humphries DE, Nicodemus CF, Schiller V, Stevens RL. The human serglycin gene. Nucleotide sequence and methylation pattern in human promyelocytic leukemia HL60 cells and T-lymphoblast Molt-4 cells. J Biol Chem 1992; 267:13558–13563. Lindahl U. “Heparin”—from anticoagulant drug into the new biology. Glycoconjugate J 2000; 17:597–605.

2 Hyaluronan in Lung Function An Overview

PAUL W. NOBLE Yale University School of Medicine New Haven, Connecticut, U.S.A.

I.

Introduction

Hyaluronan (HA) is a ubiquitous glycosaminoglycan found in almost all tissues. HA was first characterized from the vitreus of the eye (1) and shown to contain a hexuronic acid, an amino sugar, and acetyl groups with no sulfoester content. HA was subsequently isolated from umbilical cord, and glucuronic acid and glucosamine sugar constituents were identified (2). The actual linkages of the repeating disaccharide motif (-β-1,4-glucuronic acid-β1,3-N-acetylglucosamine-) n was described in 1954 (3). The number of repeat disaccharides can approach 30,000 (a molecular mass of 10 ⫻ 10 6 daltons) in tissues such as synovial fluid. In the lung, HA can exist as a soluble polymer or complexed with proteoglycans. In the lung, the major proteoglycan with HA side chains is versican. The polyionic nature of HA polymers results in an avidity for water, and HA has been suggested to have a role in regulating edema formation and solute transport in the lung interstitium (4). In addition to regulating physiological processes in the normal lung, HA undergoes dynamic regulation under conditions of tissue injury and inflammation. The purpose of this review is to discuss the emerging roles of HA in lung injury, inflammation, and repair. 23

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Noble II. Hyaluronan in Normal Lung Development

HA is an important constituent of developmental interstitium in fetal lungs and is thought to mediate cell migration. The polyionic nature of HA allows the formation of open, hydrated matrices that provide channels for migrating cells. Histochemical staining of HA using a biotinylated binding probe consisting of a mixture of proteoglycan and link protein from cartilage that is specific for HA (5,6), has shown that large amounts of HA are present in the interstitium during early embryonic development. However, as development progresses, this interalveolar HA disappears, and in the adult, HA is restricted to the regions surrounding the major blood vessels, bronchi, and bronchioles. HA is localized in the subepithelial regions of the bronchioles. HA turnover has been examined in the perinatal period (4). HA concentration is highest in the youngest fetuses. HA concentration decreases just before birth and then increases to adult levels by 4 days after birth. These data suggest that HA facilitates morphogenesis before term and the major role in the neonatal lung is most likely in regulation of fluid balance in the interstitium. The mechanisms that regulate HA homeostasis in the developing lung are unknown. The molecular cloning of a family of hyaluronan synthase (HAS) genes has initiated investigations into the role of hyaluronan in developmental processes (7). Three HAS cDNAs have been isolated encoding vertebrate HAS proteins 1, 2, and 3 (7–10). HAS2 is expressed constitutively throughout embryonic development, whereas HAS1 and -3 are expressed early and late in gestation, respectively (9). Targeted deletion of HAS2 leads to an embryonic lethal phenotype characterized by a severe cardiac abnormality (11). The characteristic transformation of cardiac endothelial cells into mesenchyme is impaired. These data demonstrate the critical importance of HA in development and confirm earlier suggestions that HA has a fundamental role in regulating cell migration.

III. Hyaluronan in Diseased Lung HA content has been quantitated in bronchoalveolar lavage fluid (BALF) under a variety of pathologic lung conditions. In normal BALF, HA content is low, suggesting that potent endogenous mechanisms exist for regulating HA turnover. Tissue HA enters the bloodstream in significant amounts through the lymph and is rapidly absorbed into liver endothelial cells and degraded (12). HA metabolism is deranged in a variety of pathologic conditions involving the lungs. HA content in BALF has been shown to be elevated in both inflammatory and fibrotic lung conditions as well as in acute and chronic lung diseases. HA content has been measured in patients with acute lung injury in the adult respiratory distress syndrome (ARDS). ARDS is defined pathologically as diffuse alveolar damage. The

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alveolar space is filled with protein-rich fluid with fibrin a significant component. HA content as well as deposition is increased in lung tissue and BALF from patients with ARDS. The increase is not due to extravasation from the bloodstream. HA content in BALF has been shown to correlate with the extent of lung injury and is inversely related to survival. HA has been proposed to contribute to the formation of interstitial edema in ARDS owing to the hydrophilic properties of the polyanion core structure. In addition to having a role in the pathophysiology of acute lung injury, HA has also been shown to accumulate in BALF in interstitial lung diseases. Interstitial lung diseases are a group of disorders characterized by the accumulation of inflammatory cells and extracellular matrix in the lung interstitium. HA accumulation has been evaluated in sarcoidosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis. HA content has been shown to correlate with abnormal physiology. In addition, increased HA content has been shown to distinguish active, symptomatic hypersensitivity pneumonitis from inactive disease. HA accumulation has been suggested to be a marker of fibrosis. HA production is most abundant in mesenchymal cells such as fibroblasts and smooth muscle cells. The concept that has been put forth is that increased production of HA correlates with fibroblast stimulation of extracellular matrix production. HA content has also been studied in patients with asthma. HA content in BALF was shown to be elevated in patients with persistent symptomatic asthma when compared to patients with inactive disease. The authors suggested that HA production may be a marker of airway remodeling associated with chronic persistent asthma. In summary, HA content is increased in a variety of lung diseases that have in common increased production and deposition of extracellular matrix. What is unclear is whether HA content is an epiphenomenon of the inflammatory response or a cause of abnormal lung repair.

IV. Hyaluronan in Models of Lung Injury and Repair As described above, a number of lung diseases are characterized by increased HA content in the lung. However, the physiological and biological significance of HA accumulation in lung inflammation and fibrosis is uncertain. Intratracheal instillation of bleomycin sulfate has been used as an animal model of lung injury, inflammation, and repair. Bleomycin sulfate has two main structural components: a bithiazole component, which partially intercalates into the DNA helix, parting the strands; and pyrimidine and imidazole structures, which bind iron and oxygen, forming an activated complex capable of releasing damaging oxidants in close proximity to the polynucleotide chains of DNA. In addition, bleomycin is capable of causing cell damage independent from its effects on DNA breakage by induction of lipid peroxidation (13). Depending on the dosage and route of administra-

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Figure 1 Schematic of the relationship among inflammatory cell influx, hyaluronan content, and collagen content after intratracheal bleomycin treatment.

tion, bleomycin has been used as a model of acute or chronic lung injury and repair culminating in patchy interstitial fibrosis (13). Over the past 25 years, extensive investigations from a number of laboratories into the role of ECM turnover in bleomycin-induced lung injury and fibrosis have been undertaken (14– 18). An interesting paradigm has emerged for ECM production following lung injury due to bleomycin. As shown in Figure 1, following lung injury, there is an influx of inflammatory cells and peak lung injury is between days 5 and 9 in rats and mice (19,20). Interestingly, maximal HA content in the lung occurs at the time of this peak injury (20). The accumulation of HA has been shown to occur in both the alveolar and interstitial spaces, and has been proposed as an important mechanism for edema formation because of the hydrophilic properties of HA (19). HA content increases between 10- and 30-fold following bleomycininduced injury when water content is accounted for (20). The lung has a tremendous capacity to clear HA that appears to commence at the time of peak injury such that over the ensuing 7–10 days, HA levels return to near baseline levels. The biological significance of the rise and fall in HA content is unknown. Collagen deposition then begins once the HA content is restored to near baseline levels. It has been suggested, but remains speculative, that HA content must fall before collagen accumulation is significant. V.

Hyaluronan Depolymerization in Lung Injury

HA is a normal constituent of basement membrane and makes up ⬃10% of the proteoglycan content of the lung (21). In its native form, HA exists as a highmolecular-weight polymer typically ⬎10 6 daltons. HA plays a role in maintaining


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the structural integrity of tissues such as the joint, where it is responsible for maintaining the viscosity of joint fluid. In addition to existing as a soluble polymer, HA is bound to large proteoglycans such as aggrecan and versican to form proteoglycan aggregates. The HA in these proteoglycan aggregates is important in cartilage organization, and recent data suggest that HA is essential for maintaining cartilage integrity (22,23). In the lung, versican is the major proteoglycan that binds HA (24). At sites of inflammation, such as during inflammatory arthritis or in wound healing, HA becomes depolymerized into lower-molecular-weight forms. There appear to be two mechanisms for depolymerizing HA: enzymatic and nonenzymatic (21). The enzymes that degrade HA are hyaluronidases, chondroitinases, and hexosaminidases (21). Most hyaluronidases are lysosomal enzymes and require an acid pH for maximal activity (25). However, the hyaluronidase PH-20 found in sperm is active at pH 7. Recently, a soluble form of PH20 has been identified (26–29). In addition, a novel hyaluronidase (HYAL2) that generates HA fragments of 10,000–20,000 daltons has been described and shown to be expressed in fibrotic lung injury (30,31). HA can also be degraded into smaller fragments by exposure to reactive oxygen intermediates (ROI) (32). This is believed to be an important mechanism for generating HA fragments at sites of inflammation (33). Interestingly, hyaluronidases are endoglucosaminidases, whereas ROIs fragment HA randomly at internal glycoside linkages. HA degradation products appear to have biological functions distinct from the native high molecular weight polymer. Oligosaccharides of ⬍20 disaccharides have been shown to be angiogenic (34). Low- and intermediate-molecular-weight HA (20,000–450,000 daltons) have been shown to stimulate gene expression in macrophages, endothelial cells, eosinophils, and certain epithelial cells (35–38). HA degradation is purported to contribute to scar formation (39). Fetal wounds heal without scar formation, and wound fluid HA is high molecular weight (40,41). When hyaluronidase is added to generate HA fragments there is increased scar formation (39). Collectively, these data support the concept that high-molecular-weight HA promotes cell quiescence and supports tissue integrity, whereas generation of HA breakdown products is a signal that injury has occurred and initiates an inflammatory response. Interestingly, whether it be wound healing, liver injury, or lung injury, there is a potent mechanism for clearing HA following tissue injury. This suggests that while the generation of HA breakdown products may be important in initiating the inflammatory response, removal of these fragments may be critical for the resolution of the repair process. The mechanisms by which HA accumulates in the lung have been studied. Elegant work from Swedish investigators has demonstrated that growth factors such as PDGF and TGF-β that accumulate following bleomycin-induced lung injury, stimulate lung fibroblasts to produce HA. However, whether increases in

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HA are due to increased production or decreased degradation has been difficult to sort out because the genes encoding HA synthases had been unknown. However, as stated above, three isoforms of HA synthase have recently been cloned (42). The three isoforms exhibit different patterns of expression in developing embryos (7–9). HAS1 is expressed early from day 1 to day 5, HAS2 is expressed throughout development, and HAS3 is expressed in late development. The three isoforms have been mapped to three distinct chromosomes, suggesting that they arose from gene duplication (43). Targeted deletion of HAS2 has recently been reported (11). HAS2 deletion results in an embryonic lethal condition, whereas HAS1 and HAS3 mice develop normally. The HAS2 deletion appears to present a similar phenotype to that described for a naturally occurring versican knockout (44). There are major abnormalities in heart and blood vessel development. Interestingly, in vitro data suggest that HAS1 and HAS2 produce high-molecularweight HA, whereas HAS3 produces lower-molecular-weight HA (10).

VI. CD44 Is the Major Hyaluronan Receptor in the Lung CD44 is a polymorphic type I transmembrane glycoprotein whose diversity is determined by differential splicing of at least 10 variable exons encoding a segment of the extracellular domain, termed exons v1–10, and cell type–specific glycosylation (45). Most cells express the standard isoform which is an 85-kDa protein that undergoes posttranslational modification (45). CD44 was identified as an HA-binding protein, and HA-CD44 interactions have been suggested to play an important role in development, inflammation, T cell recruitment and activation, and tumor growth and metastasis (45). Although glycosaminoglycan side chains associated with some CD44 isoforms can also bind a subset of heparinbinding growth factors, cytokines, and ECM proteins such as fibronectin, most of the functions ascribed to CD44 thus far can be attributed to its ability to bind and internalize HA (46). Most cells including stromal cells such as fibroblasts and smooth muscle cells, epithelial cells, and immune cells such as neutrophils, macrophages, and lymphocytes all express CD44 (46). In an animal model of tumor metastasis, CD44 was shown to have a critical role in the ability of tumor cells to form lung metastases (47). Using a novel approach, Stamenkovic and colleagues transfected murine mammary tumor cells with a recently described soluble form of CD44 (47). Soluble CD44 released into cell supernatants blocked the interaction of endogenous CD44 with HA substratum. Soluble CD44 also prevented uptake of HA. In effect, CD44 released by the infiltrating tumor cell competes with tumor cell CD44 for binding to HA in the basement membrane. Remarkably, this resulted in the ablation of lung metastases. In addition, the tumor cells that were transfected with soluble CD44, and failed to infiltrate the matrix, underwent apoptosis. These data suggest that HA-


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CD44 interactions may be critical for tumor metastasis. More recently, CD44 has been shown to be associated with MMP-9 on the surface of tumor cells (48). It has been suggested that this interaction may be important in allowing tumor cells to migrate across the basement membrane (48). In addition, the interaction of CD44 with MMP-9 has been suggested as a novel mechanism for the activation of TGFβ in vitro (49). Two studies using antisense oligonucleotides have suggested a direct role for CD44 in HA catabolism in wound healing (50) and in cartilage formation (23). Recent studies have suggested an important role for CD44 in inflammatory states such as rheumatoid arthritis (51) and the extravasation of T-cells to sites of tissue inflammation (52–54). Mice with a targeted deletion of standard CD44 and all isoforms have been reported (55). Suprisingly, the mice were viable and developed normally. When challenged with the infectious organism C. parvum, an organism that forms granulomas, there was excessive granuloma formation in the CD44 knockout mice (55). To investigate the role of CD44 in regulating HA turnover in lung injury and repair, we first examined HA deposition by immunohistochemistry and measured HA content in whole-lung extracts and BALF by ELISA in wild-type and CD44-deficient mice following treatment with intratracheal bleomycin. As shown in Figure 2, HA content measured by ELISA rises fourfold in the wild-type mice and peaks at day 7. HA is then cleared by mechanisms that are not established such that levels approach baseline by day 14. The lung thus has an endogenous

Figure 2 Hyaluronan content in bronchoalveolar lavage fluid from wild-type and CD44deficient mice treated with intratracheal bleomycin. (From Ref. 71.)

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Figure 3 Immunohistochemical staining of hyaluronan from lung sections of wild-type and CD44-deficient mice at day 10 following intratracheal bleomycin treatment. Staining was performed with a biotinylated hyaluronan binding protein. (From Ref. 71.)

capacity for clearing HA following noninfectious lung injury. CD44-deficient mice demonstrate a marked difference in HA turnover. HA content rises to a peak similar to that observed in the wild-type mice; however, HA levels continue to rise through day 14 until death. Examination of the lung tissue in the CD44deficient animals reveals that they succumb to unrelenting inflammation. These data suggest that CD44 is required for clearance of HA and that failure to clear HA is associated with persistent inflammation and death. We examined the pattern of HA deposition by immunohistochemistry using a biotinylated hyaluronan binding protein (HABP) (56). We stained lung tissues from wild-type and CD44-deficient mice at day 14 following a single treatment with intratracheal bleomycin. As shown in Figure 3, there is increased HA staining in the interstitium compared to the wild-type mice at day 14. Intermixed within the HA staining are the large contingent of inflammatory cells. These data confirm the HA content data and suggest that failure to clear HA is associated with persistent inflammation. These data support a critical role for CD44 in HA clearance in lung injury. VII. Functions of Hyaluronan in Lung Injury and Repair Evidence is accumulating that HA functions as more than a structural scaffold and is a dynamic molecule that influences cell behavior (57–59). West and colleagues have shown that oligosaccharides derived from high-molecular-weight HA stimulate angiogenesis in vivo and endothelial proliferation in vitro (34). More recently, a number of laboratories have shown that HA can induce the expression of genes in fibroblasts, kidney epithelial cells, eosinophils, and cells derived from amniotic membranes (60–63). In general, HA fragments are more


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effective than the larger precursor molecules. Several studies have suggested that CD44 mediates this signaling (37). Work from our own laboratory has suggested that HA fragments in the 200,000-dalton range induce the expression of a number of inflammatory mediators including chemokines, cytokines, growth factors, proteases, and nitric oxide in macrophages (35,64–69). Similar results have been demonstrated by other laboratories (60–63). We have provided data that CD44 is important in mediating HA induction of chemokine expression in macrophages by partially inhibiting HA fragment signaling in the presence of anti-CD44 antibodies. However, HA signaling still occurs in the absence of the standard form of CD44, suggesting that CD44 is not essential and that other receptors are involved or can be compensated for in the CD44-deficient animal (70). Collectively, these data suggest that HA breakdown products can function as signaling molecules in a variety of cell types and CD44-dependent and -independent signaling pathways exist. VIII. Conclusions HA is an extracellular matrix molecule that has not received much attention because of its simple structure and perceived role as merely providing the glue to maintain tissue structural integrity. Recent studies and the cloning of a family of HA synthases have led to an explosion of new investigations into the exciting roles of this simple sugar in regulating the biology of lung repair following injury. References 1. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem 1934; 107:629–634. 2. Meyer K, Palmer JW. On glycoproteins II. The polysaccharides of vitreous humor and of unbilical cord. J Biol Chem 1936; 114:689–703. 3. Weissman B, Meyer K. J Am Chem Soc 1954; 76:1753–1757. 4. Allen SJ, Gunnar Sedin E, Jonzon A, Wells AF, Laurent TC. Lung hyaluronan during development: a quantitative and morphological study. Am J Physiol 1991; 260: H1449–1454. 5. Underhill CB, Nguyen HA, Shizari M, Culty M. CD44 positive macrophages take up hyaluronan during lung development. Dev Biol 1993; 155:324–336. 6. Green SJ, Tarone G, Underhill CB. Distribution of hyaluronate and hyaluronate receptors in the adult lung. J Cell Sci 1988; 90:145–156. 7. Spicer AP, Olson JS, McDonald JA. Molecular cloning and characterization of a cDNA encoding the third putative mammalian hyaluronan synthase. J Biol Chem 1997; 272:8957–8961. 8. Spicer AP, Augustine ML, McDonald JA. Molecular cloning and characterization of a putative mouse hyaluronan synthase. J Biol Chem 1996; 271:23400–23406.

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42. Weigel PH, Hascall VC, Tammi M. Hyaluronan synthases. J Biol Chem 1997; 272: 13997–14000. 43. Spicer AP, Seldin MF, Olsen AS, Brown N, Wells DE, Doggett NA, Itano N, Kimata K, Inazawa J, McDonald JA. Chromosomal localization of the human and mouse hyaluronan synthase genes. Genomics 1997; 41:493–497. 44. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol 1998; 202:56–66. 45. Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol 1993; 54:271–335. 46. Sherman L, Sleeman J, Herrlich P, Ponta H. Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. Curr Opin Cell Biol 1994; 6:726–733. 47. Yu Q, Toole BP, Stamenkovic I. Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function. J Exp Med 1997; 186:1985–1996. 48. Yu Q, Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion. Genes Dev 1999; 13: 35–48. 49. Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 2000; 14:163–176. 50. Kaya G, Rodriguez I, Jorcano JL, Vassalli P, Stamenkovic I. Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev 1997; 11:996–1007. 51. Mikecz K, Brennan FR, Kim JH, Glant TT. Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat Med 1995; 1:558–663. 52. Camp RL, Scheynius A, Johansson C, Pure E. CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation. J Exp Med 1993; 178:497–507. 53. DeGrendele HC, Estess P, Siegelman MH. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 1997; 278:672–675. 54. Siegelman MH, DeGrendele HC, Estess P. Activation and interaction of CD44 and hyaluronan in immunological systems. J Leukoc Biol 1999; 66:315–321. 55. Schmits R, Filmus J, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A, Simard JJ, Ohashi PS, Paige CJ, Gutierrez-Ramos JC, Mak TW. CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity. Blood 1997; 90:2217–2233. 56. Underhill CB, Zhang L. Analysis of hyaluronan using biotinylated hyaluronan-binding proteins. Meth Mol Biol 2000; 137:441–447. 57. Toole BP. Hyaluronan is not just a goo. J Clin Invest 2000; 106:335–336. 58. Lee JY, Spicer AP. Hyaluronan: a multifunctional, megadalton, stealth molecule. Curr Opin Cell Biol 2000; 12:581–586. 59. Camenisch TD, McDonald JA. Hyaluronan: is bigger better? Am J Respir Cell Mol Biol 2000; 23:431–433.


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60. Termeer CC, Hennies J, Voith U, Ahrens T, Weiss JM, Prehm P, Simon JC. Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 2000; 165:1863–1870. 61. Ohkawara Y, Tamura G, Iwasaki T, Tanaka A, Kikuchi T, Shirato K. Activation and transforming growth factor-beta production in eosinophils by hyaluronan. Am J Respir Cell Mol Biol 2000; 23:444–451. 62. Oliferenko S, Kaverina I, Small JV, Huber LA. Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth. J Cell Biol 2000; 148: 1159–1164. 63. Fitzgerald KA, Bowie AG, Skeffington BS, O’Neill LA. Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells. J Immunol 2000; 164:2053–2063. 64. Horton MR, Burdick MD, Strieter RM, Bao C, Noble PW. Regulation of hyaluronaninduced chemokine gene expression by IL-10 and IFN-gamma in mouse macrophages. J Immunol 1998; 160:3023–3030. 65. Noble PW, McKee CM, Cowman M, Shin HS. Hyaluronan fragments activate an NF-kappa B/I-kappa B alpha autoregulatory loop in murine macrophages. J Exp Med 1996; 183:2373–2378. 66. Horton MR, McKee CM, Bao C, Liao F, Farber JM, Hodge-Dufour J, Pure E, Oliver BL, Wright TM, Noble PW. Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages. J Biol Chem 1998; 273:35088–35094. 67. Horton MR, Shapiro S, Bao C, Lowenstein CJ, Noble PW. Induction and regulation of macrophage metalloelastase by hyaluronan fragments in mouse macrophages. J Immunol 1999; 162:4171–4176. 68. Horton MR, Olman MA, Noble PW. Hyaluronan fragments induce plasminogen activator inhibitor-1 and inhibit urokinase activity in mouse alveolar macrophages: a potential mechanism for impaired fibrinolytic activity in acute lung injury. Chest 1999; 116:17S. 69. Noble PW, Lake FR, Henson PM, Riches DW. Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alphadependent mechanism in murine macrophages. J Clin Invest 1993; 91:2368–2377. 70. Khaldoyanidi S, Moll J, Karakhanova S, Herrlich P, Ponta H. Hyaluronate-enhanced hematopoiesis: two different receptors trigger the release of interleukin-1 beta and interleukin-6 from bone marrow macrophages. Blood 1999; 94:940–949. 71. Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure´ E, Henson PM, Noble PW. Resolution of lung inflammation by CD44. Science 2002; 296:155–158.

3 Morphological Tools for Studying Lung Proteoglycans

PAULO SAMPAIO GUTIERREZ and VERA DEMARCHI AIELLO Heart Institute (InCor) University of Saõ Paulo Medical School Saõ Paulo, Brazil

I.

Introduction

Proteoglycans (PGs) in a tissue or an organ can be studied using either biochemical or morphological tools, both of which have molecular biology counterparts. The general scheme employed to isolate PGs biochemically from tissues is given in Table 1 (1). The morphological tools commonly used in the study of lung PGs are reviewed in this chapter and summarized in Table 2. The tissue macerate samples analyzed by biochemical techniques are relatively large; the histological sections are usually 3–5 µm thick or less, as in electron microscopy. The biochemical methods are usually more sensitive and specific and allow reliable quantification. In addition, they provide information about the size of protein or PG’s glycosaminoglycan chains, the degree of sulfonation, and other characteristics of the macromolecules. In contrast, there are many limitations in evaluating the content of PGs using morphologic instruments, as discussed below. The major advantage of microscopic analyses is that they reveal the precise location of the PGs and thus provide information about their biology. In regard to the lungs, it is not only important to know the amount and characteristics of a PG, but also to recognize the organ compartment in which it is located; for 37

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Table 1 1. 2. 3. 4. 5.

Sequence of Biochemical Techniques Used to Isolate Proteoglycans

Tissue sample chopped/ground in Wiley Mill under liquid nitrogen Extraction with 4 M guanidium chloride, 0.05 M sodium acetate, pH 5.8, with protease inhibitors Centrifugation DEAE ion exchange chromatography Proteoglycans fractionation

Source: Ref. 1.

example, whether they are deposited in the alveolar septa or in the pulmonary vasculature. Therefore, it is easy to understand that these approaches complement each other, and whenever possible, should be used together. By examination with the naked eye, it is usually possible to recognize types of tissues, such as muscle or skin, or processes, such as infection or neoplasia, but not specific macromolecules, except if a significant accumulation is present. For example, scars formed almost exclusively by collagen are well defined macroscopically. Tissues with large amounts of proteoglycans, like cartilage, have a bright white-bluish color. The brightness is also a feature of mucous and normal mesothelial membranes, including pleura, probably due to PGs or hyaluronan. In the lungs, the bronchial cartilages are the sole areas in which the presence of these macromolecules can be identified macroscopically. In standard histological preparations for light microscopy, certain components of the tissue react with dyes. The specificity of the reactions varies from

Table 2 Summary of the Main Morphological Techniques to Study Proteoglycans in Lung Tissue 1. 2.

3. 4. 5. 6.

Tissue sample cut in 3- to 5-µm-thick sections (light microscopy) or ⬃70 nm (electron microscopy) Staining: hematoxylin and eosin/cationic dyes (sugar moiety) Controlled pH and electrolyte concentration—may allow identification of different types of GAGs Pretreatment with specific enzymes (hyaluronidase, chondroitinase, etc.) and comparison with untreated sections may help in the identification of GAGs Identification of the protein core: immunohistochemistry (see Table 3) Detection of hyaluronan: specific probe Identification of cells producing PGs: “in situ” hybridization Electron microscopy: cationic products and enzymic treatment may be used

Studying Lung Proteoglycans

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generic stainings, such as the widely disseminated, extremely useful hematoxylin and eosin method, to those that allow the identification of a bacterial genus (e.g., Ziehl-Neelsen for Mycobacterium), or a molecule or molecule family, like Masson’s trichrome or Sirius red for collagen. There are also methods in which one of the participants in a highly specific chemical interaction is labeled and applied to a tissue section; if the other ligand is present in the sample, it will be detected, since the label will be fixed and seen at the microscope. Examples of such methods include immunohistochemistry, in situ hybridization, and probes for hyaluronan. II. Histochemistry for Proteoglycans The sugar moiety permits the histochemical detection of PGs. Since glycosaminoglycans (GAGs) are highly charged polyanions, several staining methods that involve cationic dyes (e.g., toluidine blue, ruthenium red, Alcian blue) (2,3) allow the detection of these substances. Nevertheless, these dyes react not only with GAGs but with other polyanions (e.g., carboxylated groups) as well. To increase the specificity of the reaction, it is necessary to control pH and the electrolyte concentration (2) whenever it is important to detect and differentiate PGs from other tissue components. The polyanions that usually aggregate in bulky, light microscope–visible amounts are mostly hyaluronan and large PGs (such as aggrecan, versican, etc.), thus the presence of these sugar complexes can be fairly well evaluated via routine staining. As a matter of fact, our personal experience suggests that hematoxylin and eosin staining is acceptable for demonstrating the presence of GAGs in most instances; these molecules have affinity for hematoxylin, and stain in a light bluish color. The histochemical method developed by Movat (4) mixes previously described reagents in order to stain, in a single slide, PGs (Alcian blue), collagen, and elastin (ferric hematoxylin, as in Verhoeff ’s method). Very beautiful, elucidative histological preparations can be obtained, particularly in vascular tissues. However, this method also has some pitfalls. First, it is skill dependent—for example, colors vary with the histotechnologist’s expertise; even with a single professional, shades may vary considerably from one slide to another. Finally, interactions among the many reagents may interfere with the findings. Thus, this method is useful for illustrations but should be avoided when evaluating the nature and extent of tissue components. III. Enzymatic Treatment A method that is commonly used to determine and quantify the types of glycosaminoglycans present in a tissue macerate, after purification, involves incubation

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with hyaluronidase, chondroitinase AC and/or ABC, or heparitinase. Each of these enzymes digests one type of GAG. One can then run gel electrophoresis and compare treated and untreated samples. The difference between samples corresponds to the amount of compound that was removed by the action of the enzymes. This technique can be adapted to microscopy. Two adjacent histological sections are almost identical—the thickness of each section is 3–5 µm (much less if it is an electron microscopy preparation), and significant differences do not exist within a tissue at such narrow widths. If enzyme-treated and untreated adjacent sections are stained according to any of the above-mentioned methods, it is possible to compare them to determine not only the existence of glycosaminoglycans in the tissue, but also the types of GAGs involved (5,6).

IV. Immunohistochemistry One way to know whether or not a substance is present in a histological section is to consider it as an antigen and add a specific antibody, which has somehow been labeled. After washing the slide, a positive reaction will indicate that the antibody has attached to the tissue and that the substance under investigation is present. The signal strength can be amplified by using an unlabeled primary antibody and a labeled secondary antibody. Instead of labeling each antibody, it is easier to label only antibodies against antibodies. Again, this secondary antibody will attach itself to the tissue only if the primary antibody has already done so (indicating that the target substance is present). A few types of tagged secondary antibodies are sufficient for identifying many primary antibodies, and thus many substances. The primary antibodies are produced in a chosen animal species, generally in mice or rabbits, and may be polyclonal (sera of animals treated with the antigen) or monoclonal (secreted in hybridomas). The secondary antibodies are made in another species, such as in sheep. The most common methods used to label the secondary antibodies are to link them to a fluorescent reagent (immunofluorescence) or to enzymes that catalyze reactions with an uncolored substrate to yield a colored final product. Immunoperoxidase is an example of the latter. In this case, the secondary antibody is biotinylated; biotin has an affinity for avidin or streptavidin, which has been linked to peroxidase. The reaction finishes with the addition of diaminobenzidine that is acted upon by the peroxidase to render a colored product. Another enzyme utilized in immunohistochemistry is phosphatase. Immunohistochemistry may be adapted to electron microscopy: in the most common method, electron-dense gold is attached to the secondary antibody, either directly or with protein A. The primary antibodies may be produced against the whole molecule or, more usually, against one epitope of it. There are particular situations in which this fact must be borne in mind when interpreting the results.

Studying Lung Proteoglycans Table 3 1. 2. 3.

4.

5.

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Main Steps in the Immunoperoxidase Reaction for Proteoglycans

The tissue section is digested with enzymes that digest the glycosaminoglycan chains, thus exposing protein core. Antibody against a PG a is added. It will attach to the section only if the PG is present in the tissue. Otherwise, the antibody will be washed away. Biotinylated antibody against the primary antibody (produced in a different animal species) is added. It will attach only if the primary antibody (thus the PG) is present in the tissue. Otherwise, the secondary antibody will be washed away. Incubation with avidin or streptoavidin linked to peroxidase. If the biotinylated secondary antibody is present, biotin will react with avidin, and peroxidase will be fixed in the tissue sample. Diaminobenzidine, a substrate for peroxidase, is added. If peroxidation occurs, a colored product will be deposited in the place where the PG is situated. Endogenous tissue peroxidase must have been previously blocked by treating the section with H 2 O 2 , usually before the incubation with the primary antibody.

a

The antibody may be produced against the protein core, against an epitope of it (more used), or against the whole PG. In the last case, incubation with enzymes that degrade GAGs should not be applied.

Concerning proteoglycans, the more commonly used antibodies are raised against epitopes of the protein core. The GAG chains hinder their attachment. Prior treatment with enzymes that degrade GAGs, such as heparinase (7) or chondroitin ABC lyase (8–10), is therefore necessary. Another possibility is to target hyaluronan receptors, including CD44 (11). There is a correspondence between immunohistochemistry and the Western blot; the same primary and secondary antibodies are utilized. The advantage of Western blots over immunohistochemistry is sensitivity. The main disadvantage is that it does not localize the target substance. For example, with immunoperoxidase, versican was the main PG deposit identified in the lesions associated with bronchiolitis obliterans organizing pneumonia (BOOP), adult respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) (12), and granulomatous diseases (10). In addition, decorin was demonstrated within matrix-producing cells (11), a finding that would be difficult to obtain with another method. The main steps in the identification of proteoglycans by immunoperoxidase staining are summarized in Table 3. V.

Probe for Hyaluronan

It is difficult to obtain an antibody against hyaluronan owing to its low antigenicity. Thus, a probe for its detection has been introduced (13,14). Instead of employing an antigen-antibody linkage, it takes advantage of another specific reac-

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tion: that between link protein and hyaluronan. This protein is required for the stability of attachment of the large PG core to hyaluronan. The link protein is obtained from bovine nasal cartilage. To prepare it, the cartilage is solubilized, lyophilized, and submitted to the action of trypsin, which yields small fragments. These are biotinylated and passed through a column of hyaluronan previously attached to a Sepharose gel. The molecules with affinity for hyaluronan are retained; the remnants are washed away. Hyaluronan is then separated from the retained molecules—which will function as the probe—by a chemical treatment. Once the probe is prepared, the detection of hyaluronan is quite simple. The tissue section is incubated with the probe, and after washing, the revelation phase, which is similar to an immunoperoxidase reaction, ensues. Among other findings, this probe has been employed to show the presence of hyaluronan in bronchial and bronchiolar epithelium, as well as in the adventitia of large pulmonary blood vessels (14).

VI. Pulmonary Vascular Hypertension: An Example of the Utilization of Immunohistochemistry for Proteoglycans and Probe for Hyaluronan To illustrate the use of immunohistochemistry for proteoglycans and the probe for hyaluronan, a few cases of human vascular pulmonary hypertension are shown in Figures 1 and 2. Pulmonary hypertension is a clinical condition distinguished by elevated pressure in the pulmonary artery, which may or may not be due to a recognizable cause. Increased pulmonary blood flow (as in patients with congenital cardiovascular shunts), destruction of lung parenchyma (as in patients with chronic lung emphysema), and chronic pulmonary venous congestion are among the most common circumstances associated with secondary pulmonary hypertension. In the primary form of the disease, on the other hand, the pathogenesis is not well defined, and the morphological presentation may vary considerably (15). Most forms of pulmonary hypertension are associated with occlusive arterial lesions, characterized by changes in the three layers of the vessel wall: the intima, media, and adventitia. The most striking and prevalent feature is hypertrophy of the media, often accompanied by variable degrees of intimal proliferation and adventitial thickening. The neointima observed in peripheral pulmonary arteries may have a myxoid appearance, being highly cellular and showing cells immersed in a loose, proteoglycan-rich basophilic matrix or, alternatively, it may present few cells in a fibrotic medium. Other types of lesions are the plexiform and angiomatoid (also called the “dilatation lesions”) (16), and fibrinoid necrosis of muscular branches. Among these types of lesions, plexiform lesions are the most frequent. They appear at the branching points of muscular arteries and repre-


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Figure 1 Adjacent histological sections of a medium-sized pulmonary artery with medial hypertrophy and intimal thickening. (A) Miller’s elastic stain: the limits of the medial (M) and Intimal (I) layers are well delineated by the elastic laminae and the vessel lumen (L) is partially filled with red blood cells. (B, C, and D). Immunostainings: the intimal lesion shows strong labeling for versican (B) and weak, focal positivity for biglycan (C) and decorin (D). (E) The probe for hyaluronan also demonstrated strong positivity in the intimal lesion. Objective magnification: 6.3⫻.

44

Figure 1 Continued



45

Figure 2 Adjacent histological sections of an intraacinar pulmonary branch showing a plexiform lesion (PL), characterized by a tuft of small vessels originating from an occluded parent vessel (arrow in A—Miller’s elastic stain). (B, C, and D) Immunostainings. The plexiform lesion shows strong labeling for versican (B) and decorin (D) but is negative for biglycan (C). (E) The probe for hyaluronan demonstrated focal positivity inside the same lesion. Objective magnification: 16⫻.

sent dilated arterial branches filled by a plexus of small vessels or capillaries. The common finding of remnants of necrotic medial layer at the site of dilatation led to the hypothesis that plexiform lesions are a consequence of the repair of necrotizing arteritis (fibrinoid necrosis of muscular arteries). Angiomatoid lesions are clusters of thin-walled, dilated vessels, which are usually distal to a parent vessel with occlusive intimal proliferation.

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Figure 2 Continued



47

As with systemic arteries, the peripheral pulmonary arteries show a common response to injury: signaling from the endothelial cells to the smooth muscle cells (the “effector units” of the arterial walls) promotes hypertrophy of the medial layer by increasing the cell volume and deposition of matrix proteins (e.g., collagen, proteoglycans, and elastin). Later, modified medial cells migrate to the intima, proliferate, and synthesize great amounts of matrix proteins, which occlude the vascular lumen (17). However, the role that matrix constituents play in vascular remodeling is more than simply filling spaces. Triggered by stimuli from the endothelial cells, matrix proteases, their inhibitors, and matrix components themselves all modulate cell function. The breakdown of elastin initiates this process, allowing the phenotypically modified smooth muscle cells to migrate through holes in the internal elastic lamina. The byproducts of elastin degradation maintain the course of remodeling by promoting cell proliferation and migration (18,19). Increased deposition of the glycoprotein fibronectin, an adhesion molecule, has been detected in highly cellular proliferative lesions of the intimal layer and also in plexiform lesions from peripheral pulmonary arteries (20). Fibronectin is probably associated with the migration of the dedifferentiated smooth muscle cells and acts as a scaffold for the future deposition of other matrix proteins, such as collagen. GAGs and PGs may modulate fibrosis and cell growth, either directly, like heparan sulfate (17) and heparin (21), or by binding growth factors, such as decorin (18,22). The histological presentation of the pulmonary vascular occlusive lesions may be quite similar, regardless of the nature of the underlying injury; however, in some circumstances, lesions may evolve differently and show some of the following distinct features. Congenital Cardiac Shunts. The noxious stimulus is the increased pulmonary arterial flow operating on an immature vascular bed. In parallel with the reactive hypertrophy of the medial layer and intimal proliferative lesions, a disturbance occurs in the process of postnatal development and remodeling of vessels. This disturbance in the remodeling leads to a decrease in the number of arteries relative to the number of alveoli and to early muscularization of the most peripheral arterial branches (23). Dilatation lesions appear eventually (24), in some infants even in the first year of life, if the cardiac defect is not surgically corrected. Chronic Obstructive and Interstitial Pulmonary Disease. Chronic lung emphysema involves the destruction of great amounts of alveolar walls. Pulmonary hypertension in such cases is the consequence of the reduction in number of lung vessels and of the chronic vasoconstriction due to hypoxia. The preacinar arteries usually show variable degrees of medial hypertrophy and intimal fibrosis (25), but the presence of plexiform lesions is not a feature in such cases. Chronic Pulmonary Venous Obstruction. In this condition, increased

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Gutierrez and Aiello pressure in the venous compartment of the pulmonary vasculature is transmitted to the arterial level, resulting in morphologic changes in both territories. States that impede the egress of blood from the lung include the condition known as “pulmonary veno-occlusive disease,” rheumatic and congenital stenosis of left-sided heart valves, and chronic left ventricular diastolic dysfunction. The morphological presentation of pulmonary hypertension in such cases is characterized by medial hypertrophy of both pre- and intra-acinar arteries, coexisting with thickening of the venous walls and lymphatic dilatation (15). Intimal lesions usually occur in arteries as well as in veins, showing variable degrees of fibrosis; however, plexiform lesions are exceedingly rare. Chronic Pulmonary Thromboembolism. Although frequently regarded as a type of primary pulmonary hypertension, the morphologic lesions associated with this particular form of pulmonary vascular disease are secondary to thromboembolic occlusion of arteries. Typically, this condition features organizing thrombosis (15) and colanderlike lesions, besides medial hypertrophy and intimal proliferation in small peripheral arterial branches. Plexiform lesions may be absent. Parasitic Diseases. Several parasites, in the form of eggs or adult worms, may embolize to pulmonary vessels, causing occlusion and lung infarction. Among these, the Schistosoma spp. may be associated with plexogenic pulmonary arteriopathy when there is concomitant portal hypertension (26). The arteries that are occluded by eggs may show granulomatous reaction but are too small in number to explain the frequent morphological alterations in other arteries, such as intimal proliferation and plexiform lesions.

In the cases we analyzed, we found that in the intimal thickening of medium-size branches PG deposition was represented mostly by versican and hyaluronan, with focal accumulation of biglycan and decorin (Fig. 1). Versican and biglycan have also been detected in the intima of both atherosclerotic and nonatherosclerotic coronary arteries (8). In plexiform lesions, decorin and versican were present; hyaluronan stained focally, and biglycan was almost negative (Fig. 2). The presence of versican and hyaluronan could reinforce the idea that the plexiform lesion is part of a repairing process, since these molecules were also reported in chronic aortic dissection (9). On the other hand, decorin is considered to be a modulator of TGF-β (22), and could influence the action of this key mediator of fibrosis and cell growth in plexiform lesions. VII. “In Situ” Hybridization If one wants to know about the presence of a nucleic acid sequence in a tissue section, a labeled probe consisting of its complementary sequence can be used,


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in a method similar to immunohistochemistry. Since there is hybridization between the nucleic acid sequence and the probe, this method is called “in situ” hybridization (ISH). For technical reasons, fluorescence is less often utilized, and radioisotopic probes, besides those with an attached enzyme, are common. Unless one is looking for any kind of infection or gene mutation, the main advantage of ISH is that this method allows one to determine, in a set of cells, which of them are producing (i.e., have mRNA for) a specific protein. In the case of PGs, this could be the core protein. Northern blots, for RNA, or Southern blots, for DNA, are methods that use probes like those of the ISH technique on material prepared from tissue macerates. Halfter and coworkers showed that collagen XVIII is a heparan sulfate proteoglycan, and demonstrated with ISH its production in chick embryo (27). So far, ISH has not been used to study lung PGs. VIII. Electron Microscopy The demonstration of PGs in standard preparations for transmission electron microscopy is irregular. The PGs look somewhat like a “dotted background” in the pictures, or, in greater magnifications, as the classical “brushlike” structures. This appearance is due mainly to large PG chains, as small PGs are not evident. As in light microscopy, cationic dyes enhance the detection of PGs, so they are used in most studies. Ruthenium red, cuprolinic blue, and cuprumeronic blue are the most commonly applied stains, preferentially by addition to the fixative solution—usually, glutaraldehyde. Again, pH and electrolyte concentration may influence the staining, and enzymatic treatment may be applied. Anyway, a concern remains because the findings depend on the dye used, and there is always some degree of interpretation involved. The appearance of proteoglycans in transmission electron microscopy has been variously described as fernlike, scroll-like, crystalloid fibrils or granules (stellate particles). This variability is probably due not only to actual differences in PG types and sizes but also to the limitations of the method. The majority of the published articles about proteoglycan ultrastructure deal with tissues in which they are abundant (e.g., cartilage, cornea, aorta), but there are some studies about PGs in the lungs (6,28). In spite of the problems, electron microscopic detection of hyaluronan has been utilized, in addition to the specific probes (13), for the sometimes very difficult differential diagnosis between pleural mesothelioma (in which this GAG is prominent) and adenocarcinoma (29). IX. Proteoglycan Morphometry To avoid subjective descriptions, morphometrical techniques can be used to obtain quantitative data. The number of cells of a certain type, or the amount of a

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material in a tissue, i.e., any structure, can be evaluated. For example, Dingemans et al. quantified PG chains in electron microscopy specimens (30). The number of cells positive for a specific substance may be counted. Morphometric analysis of PGs is not always easy. Since they are components of the extracellular matrix, “positive cells” cannot be quantified, except in instances such as when dealing with CD44 (11), transmembrane PG, or with basement membrane compounds (7). There are morphometric studies that quantitate interstitial PGs as the extension of area occupied by them (31); nevertheless, considering that these molecules interact with the other matrix components, including collagen and hyaluronan, the interpretation can be tricky. The amount or percentage of positive areas can be measured, but the intensity of staining may vary from case to case and from field to field. If the aim is detection of large, aggregating PGs, e.g., aggrecan or versican, which are attached to hyaluronan, it is sometimes worthwhile repeating the reaction after treatment with hyaluronidase. In our personal opinion, the troubles with morphometry are particularly important in regard to immunohistochemical preparations; although less precise in a sense, histochemical results may be more reliable.

X.

Other Types of Microscopy

There are two other types of microscopy that have been applied to the study of proteoglycans: confocal laser microscopy and atomic force microscopy. The first provides three-dimensional images of tissues. A substance may be labeled with fluorescent dyes, and its position detected. Tissue sections or live cells in culture can be visualized. In atomic force microscopy, a tip held by a cantilever tracks a surface. The cantilever movements, reflected to a position-sensitive photodiode with a resolution level that can reach 1 nm, cause the formation of an image (32). Atomic force microscopes also work as force sensors (32). Interactions between sponge PGs macromolecules (33) and between PGs and collagen in rat cornea and tendon (5) have been analyzed with this instrument.

XI. Magnetic Resonance Imaging Imaging methods have been developed with the goal of providing information similar to structural findings in living beings. Preferably these methods are noninvasive and avoid the need for biopsies. Magnetic resonance spectroscopy can indicate not only pathological processes, but also, in some cases, substances or groups of substances present in an organ. It has been used for the detection of PGs in human Graves’ ophthalmopathy (34).


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XII. Summary Four general methods that can be used to detect proteoglycans in tissue sections are as follows: 1. Histochemical staining—as highly anionic molecules, PGs have a strong chemical affinity for cationic dyes, such as Alcian blue, toluidine blue, ruthenium red, and others; PGs are also slightly stained in blue with hematoxylin and eosin, which is often sufficient for an adequate detection of these compounds. 2. Enzymatic treatment—the degradation of certain GAGs followed by subsequent comparison with similar untreated sections allows the verification of their prior presence and the types involved. 3. Immunohistochemistry—the specificity of the antigen-antibody linkage permits the detection of PGs in tissue sections by a labeling method that uses antibodies specific for each proteoglycan type (specific for core protein, or core protein epitopes). 4. “In situ” hybridization—probes that contain antisense sequences of the nucleic acids encoding the core protein allow one to identify cells or cell types that are producing these proteins in a tissue. Aside from these methods, hyaluronan may also be studied using a probe made of the link protein extracted from cartilage. Collectively, these methods may be applied in light, electron, and atomic force microscopy. Magnetic resonance spectroscopy may be used to localize and quantify proteoglycans in living beings. Acknowledgments The antibodies for biglycan and versican were kindly provided by Dr. Larry Fisher, from the National Institute for Dental Research, Bethesda, MD, and Dr. Erkki Ruoslahti, from the Cancer Research Center, Burnham Institute, La Jolla, CA, respectively. The probe for hyaluronan was kindly provided by Dr. Lucia O. Sampaio, from the Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de Saõ Paulo, Saõ Paulo, Brazil. References 1. Garg HG, Lyon NB. Structure of collagen fibril-associated, small proteoglycans of mammalian origin. Adv Carbohydr Chem Biochem 1991; 49:239–261. 2. Scott JE. Proteoglycan histochemistry: a valuable tool for connective tissue biochemists. Collagen Res Rel 1985; 5:541–575.

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3. Scott JE. Alcian blue: now you see it, now you don’t. Eur J Oral Sci 1996; 104: 2–9. 4. Movat H. Demonstration of all connective tissue elements in a single section. Arch Pathol 1955; 60:289–295. 5. Raspanti M, Congiu T, Alessandrini A, Gobbi P, Ruggieri A. Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study. Eur J Histochem 2000; 44:335–343. 6. Ferrara TB, Fox RB. Effects of oxygen toxicity on cuprolinic blue-stained proteoglycans in alveolar basement membranes. Am J Respir Cell Mol Biol 1992; 6:219– 224. 7. Nackaerts K, Verbeken E, Deneffe G, Vanderschueren B, Demedts M, David G. Heparan sulfate proteoglycan expression in human lung-cancer cells. Int J Cancer 1997; 74:335–345. 8. Gutierrez PS, Ferguson M, O’Brien K, Nikkari ST, Alpers CE, Wight TN. Immunolocalization of versican, decorin and biglycan in atherosclerotic human coronary arteries. Cardiovasc Pathol 1997; 6:271–279. 9. Gutierrez PS, Reis MM, Aiello VD, Higuchi ML, Stolf NAG, Lopes EA. Distribution of hyaluronan and dermatan/chondroitin sulphate proteoglycans in human aortic dissection. Connect Tiss Res 1998; 37(3–4):151–161. 10. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycans in granulomatous lung diseases. Eur Respir J 1997; 10:2731–2737. 11. Penno MB, August JT, Baylin SB, Mabry M, Linnoila RI, Lee VS, Croteau D, Yang XL, Rosada C. Expression of CD44 in human lung tumors. Cancer Res 1994; 54: 1381–1387. 12. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycan deposition in pulmonary fibrosis. Am J Resp Crit Care Med 1996; 154:1819–1828. 13. Azumi N, Underhill CB, Kagan E, Sheibani K. A novel biotylated probe specific for hyaluronate: its diagnostic value in diffuse malignant mesothelioma. Am J Surg Pathol 1992; 16:16–21. 14. Green SJ, Tarone G, Underhill CB. Distribution of hyaluronate and hyaluronate receptors in the adult lung. J Cell Sci 1988; 89:145–156. 15. Pietra GG. The pathology of primary pulmonary hypertension. In: Rubin LJ, Rich S, eds. Primary pulmonary hypertension. New York: Marcel Dekker, 1997:19–61. 16. Wagenvoort CA. Plexogenic arteriopathy. Thorax 1994; 49(Suppl):S39–S45. 17. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med 1994; 330:1431–1438. 18. Rabinovitch M. Pulmonary hypertension: updating a mysterious disease. Cardiovasc Res 1997; 34:268–272. 19. Zaidi SHE, You XM, Ciura S, O’Blenes S, Husain M, Rabinovitch M. Suppressed smooth muscle proliferation and inflammatory cell invasion after arterial injury in elafin-overexpressing mice. J Clin Invest 2000; 105:1687–1695. 20. Aiello VD, Higuchi ML, Lopes EA, Lopes AAB, Barbero-Marcial M, Ebaid M. An immunohistochemical study of arterial lesions due to pulmonary hypertension in patients with congenital heart defects. Cardiol Young 1994; 4:37–43. 21. Snow AD, Bolender RP, Wight TN, Clowes AW. Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am J Pathol 1990; 137:313–330.


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22. Kolb M, Margetts PJ, Sime PJ, Gauldie J. Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung. Am J Physiol Lung Cell Mol Physiol 2001; 280:L1327–L1334. 23. Rabinovitch M. Problems of pulmonary hypertension in children with congenital cardiac defects. Chest 1988; 93(Suppl 3):119S–126S. 24. Heath D, Edwards JE. The pathology of hypertensive pulmonary vascular disease. Circulation 1958; 18:533–544. 25. Harley Jr RA. Tobacco. In: Dail DH, Hammar SP, eds. Pulmonary pathology. 2nd ed. New York: Springer-Verlag, 1993:831–845. 26. Guimara˜es A, Guimara˜es I. Pulmonary Schistosomiasis. In: Sharma OP, ed. Lung disease in the tropics. New York: Marcel Dekker, 1991:319–339. 27. Halfter W, Dong S, Scurer B, Cole GJ. Collagen XVIII is a basement membrane heparan sulfate proteoglycan. J Biol Chem 1998; 273:25404–25412. 28. Erlinger R. Glycosaminoglycans in porcine lung: an ultrastructural study using cupromeronic blue. Cell Tissue Res 1995; 281:473–483. 29. Hammar SP, Bockus DE, Remington FL, Rohrbach KA. Mucin-positive epithelial mesotheliomas: a histochemical, immunohistochemical, and ultrastructural comparison with mucin-producing pulmonary adenocarcinomas. Ultrastruct Pathol 1996; 20: 293–325. 30. Dingemans KP, Teeling P, Lagendijk, Becker AE. Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. Anat Rec 2000; 258:1–14. 31. Huang J, Olivenstein R, Taha R, Hamid Q, Ludwig M. Enhanced proteoglycan deposition in the airway wall of atopic asthmatics. Am J Respir Crit Care Med 1999; 160:725–729. 32. Bowen WR, Doneva T, Hilal N, Wright CJ. Atomic force microscopy: images and interactions. Microsc Anal 2001; 46:13–18. 33. Dammaer U, Popescu O, Wagner P, Anselmetti D, Guntherodt HJ, Misevic GN. Binding strength between cell adhesion proteoglycans measured by atomic force microscopy. Science 1995; 267:1173–1175. 34. Ohtsuka K, Hashimoto M. H-magnetic resonance spectroscopy of retrobulbar tissue in Graves ophthalmopathy. Am J Ophthalmol 1999; 128:715–719.

4 Proteoglycans, Lung Physiology, and Mechanical Strain

MARA S. LUDWIG McGill University Montreal, Quebec, Canada

I.

Introduction

Proteoglycans (PGs) and glycosaminoglycans (GAGs) are an integral part of the lung extracellular matrix (1). They subserve a number of different biological functions (2). Recently, data have accumulated demonstrating that these molecules play an important role in determining the viscoelastic properties of the lung parenchyma (3), much as has been shown for other tissues, such as cartilage (4,5). In addition, proteoglycan metabolism is affected by mechanical strain, and the lung is an organ that is constantly subject to varying mechanical stresses (6,7). The purpose of this chapter is to describe recent data that document the importance of proteoglycans in determining lung physiology, and to present evidence that production of proteoglycans is affected by mechanical strain. These observations may have important implications for various lung diseases in which alterations in PGs contribute to abnormal physiology and in which altered mechanical strain is a potential pathologic mechanism.

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Proteoglycans are now well described in the lung extracellular matrix (1). Versican is the hyalectin, or large aggregating proteoglycan, that has been demonstrated in the lung (8). Hyaluronic acid has been shown to be present in the lung in a number of studies (9,10). In the peripheral lung, lumican has been shown to be the predominant small PG (11) (Fig. 1), although biglycan and decorin have also been described in airway and blood vessel wall and in alveolar tissue (12). Finally, the basement membrane heparan sulfate proteoglycan (HSPG), perlecan, has been identified in lung samples (13,14). These molecules participate in a number of key biological functions. Versican, along with hyaluronic acid, forms large supramolecular aggregates that are important in the formation of the open matrix required for cell migration and proliferation (2). Small, leucine-rich PGs such as decorin interact with fibrillar collagen and influence collagen fiber assembly (15). Heparan sulfate PGs are major components of the basement membrane and play a role in mediating cellmatrix and cell-cell interactions (16). Finally, proteoglycans affect tissue hydration because of the hydrophilic nature of their GAG side chains. III. Role of Proteoglycans in Determining Lung Tissue Mechanical Behavior A. Theoretical Basis

Lung parenchymal tissues display prominent viscoelastic behavior. Viscoelastic materials demonstrate hysteresis and stress relaxation (17). Hysteresis refers to

Figure 1 Western blot analysis of lumican in extracts of human lung. In all samples (lanes 1–6), a broad component within the molecular range of 65–90 kDa was detected. Blots were incubated with antilumican antiserum, and subsequent color development was for 5 min. (From Ref. 11.)

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the phenomenon wherein a material subjected to cyclic loading demonstrates a stress-strain relationship during the loading process that is different from that observed during the unloading process. Stress relaxation is the decrease in tension that occurs when a material is stretched and maintained at its new constant length. When lung tissues are oscillated, as occurs during normal tidal breathing, energy is dissipated to overcome the viscoelastic properties of the parenchymal tissues. These characteristics of the lung tissues were described many years ago by Bayliss and Robertson (18), among others (19–22), and were more recently defined in animal studies in which alveolar capsules were used to directly measure alveolar pressure (23,24) and in human studies in which multiple frequency volume oscillations were used to measure complex lung impedance (25,26). The anatomic elements potentially responsible for this viscoelastic behavior are several, and include the collagen-elastin-proteoglycan matrix, the surface film and the contractile elements in the lung periphery (27). The tissue matrix represents a composite of collagen fibers, elastic fibers, and PGs and GAGs. Collagen and elastin fibers are essentially elastic in nature (17). However, when fibers are arranged in a network, the network may display hysteretic properties (28). There are several reasons to postulate, however, that the viscoelastic characteristics of the parenchymal tissues may be attributed, at least in part, to PGs and GAGs. The glycosaminoglycan side chains of these molecules are highly hydrophilic, and therefore have the ability to attract ions and fluid into the matrix and affect tissue viscoelasticity (2). Lung tissue viscoelasticity has also been attributed to the movement of fibers within the connective tissue matrix. Mijailovich et al. (29,30) have proposed that energy dissipation occurs not at the molecular level within collagen or elastin, but rather at the level of fiber-fiber contact and by the shearing of GAGs which provide the lubricating film between adjacent fibers. Suki et al. (31) have developed the idea that the mechanical properties of the tissue matrix can be understood if one models matrix behavior as a polymer. At higher frequencies, viscoelastic behavior occurs because of conformational changes in the matrix molecules. External stresses cause a velocity gradient in the solution that continuously alters the configuration of the various polymer molecules. At lower frequencies the process of “reptation” becomes more important, wherein step changes in molecular conformation create distortions which, as the molecules reassume their equilibrium shape over time, dissipate energy. One possible anatomic correlate for these polymerlike molecules would be the PG and GAG molecules that comprise the “ground substance” of the matrix. B. Studies in Other Organ Systems

Some information is available regarding the role of GAGs in determining viscoelastic behavior in other systems that may be pertinent to the lung. A number of investigators have used specific degradative enzymes to digest glycosaminoglycan side chains to see whether mechanical behavior of the tissues is altered under

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these conditions. Schmidt and coworkers (4) have shown in articular cartilage preparations exposed to chondroitinase, a degradative enzyme that digests chondroitin sulfate and dermatan sulfate, that the kinetics of the creep response (creep is also a characteristic of viscoelastic materials) are influenced by the GAG content of the tissue. They hypothesized that GAGs may act to “brake” the load applied when a sudden tensile stress is applied to the cartilage. In further studies published by this group, changes in shear modulus and energy dissipation were noted when articular cartilage was exposed to either chondroitinase ABC or hyaluronidase (5). More recently, Gandley et al. (32) have examined the stiffness of rat mesenteric arteries and determined that exposure to chondroitinase ABC resulted in increased arterial wall stiffness. C. Studies in the Lung

We have recently published data showing that exposure of excised lung parenchymal strips to specific degradative enzymes results in alterations in tissue mechanical behavior (3). Mechanical behavior was sampled during sinusoidal oscillations of the strip in a Krebs-filled organ bath before and after enzyme exposure. Tissue strips exposed to chondroitinase ABC for 16 h showed a relative increase in resistance and hysteresivity (a measure of the energy dissipated to that stored per cycle, and an index of the mechanical friction in the tissues) as compared to control strips exposed to Krebs solution alone (Fig. 2). Measurements of static elastance were also affected. Strips exposed to heparitinase I, which degrades heparan sulfate, were similarly affected (Fig. 3). Conversely, exposure to hyaluronidase had no effect on the mechanical behavior of the parenchymal tissues. These data support the hypothesis that chondroitin sulfate and heparan sulfate are important modulators of tissue mechanical behavior. To address the hypothesis as to whether this effect was simply due to alterations in tissue hydration with enzyme degradation, we measured wet-to-dry weight ratios in these tissues with and without enzyme exposure. Whereas chondroitinase affected wet-to-dry ratios, heparitinase did not; however, the mechanical effects of these two enzymes were similar. This argues that the effect on tissue mechanical behavior was not simply an issue of the amount of fluid in the tissue. Rather, the ability of GAGs to affect biomechanical behavior may lie in the interactions between these molecules and other matrix molecules and/or the cell membrane. Chondroitin sulfate (CS)/dermatin sulfate (DS) is covalently attached to decorin, which is present at the d band of the collagen fibril (15). CS/DS attached to decorin interacts electrostatically across the fibril, thereby aiding in collagen organisation (33). Removal of CS/DS may have resulted in the disruption of the collagen fibril, which could lead to increased friction and energy dissipation during fiber-fiber interactions. A further possibility is that disruption of CS attached to the large aggregat-


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Figure 2 Changes in resistance (R), dynamic elastance (Edyn), and hysteresivity (η) in control and chondroitinase-treated tissues. The decrement in R and the increase in η were significantly different between the control and treated groups (*p ⬍ .001). (From Ref. 3.)

ing PG, versican, may have affected viscoelastic behavior. Versican, together with hyaluronic acid, forms large pericellular aggregates. Disruption of these structures may have affected mechanical behavior. The change in mechanics with heparan sulfate degradation may relate to the role of HS in mediating cell-matrix interaction and in maintaining the integrity of the basement membrane (2,16). The lack of hyaluronidase effect is perhaps more difficult to reconcile. HA is a large GAG that has no covalent interaction with a proteoglycan core protein. It

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Figure 3 Changes in resistance (R), dynamic elastance (Edyn) and hysteresivity (η) in control and heparitinase-treated tissues. The change in η was significantly greater in the strips exposed to the enzyme (p ⬍.001). The change in R after heparitinase I treatment did not achieve statistical significance (p ⫽ .06). (From Ref. 3.)

forms aggregates with versican and plays an important role in tissue water balance (10). The failure of HA degradation to alter tissue mechanics reinforces the idea that viscoelastic behavior is not simply a function of tissue hydration, but rather lies in the interaction of these molecules with other matrix components. There are two reports by other investigators examining the effect of hyaluronidase on lung parenchymal properties, which similarly showed minimal effect of this enzyme. Sata and colleagues (34) measured the effects of hyaluroni-


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dase treatment on stress-strain curves in parenchymal strips previously exposed to collagenase or elastase and found no change in several measures of tissue mechanics. Martin and Sugihara (35) found no effect of hyaluronidase on peak force, slope of the length-tension curve, or hysteresis ratio in alveolar wall preparations. Gleisner and Martin (36) and Shimura et al. (37) published data on lung parenchymal strips in which GAGs and/or matrix proteins were nonspecifically leached out of the tissues by prolonged immersion in phosphate-buffered saline. These authors demonstrated that the peak tissue tension decay (TTD), after extension of the strip, was affected by removal of GAGs from the tissue. Moreover, the rate of TTD was correlated with the rate of GAG loss. Furthermore, they observed that agents that specifically enhanced HS loss resulted in the greatest change in TTD, suggesting a relative importance of this particular GAG in determining mechanical behavior. We have also conducted studies in models in which the PG matrix is altered, to determine whether changes in physiology occur coincident with changes in the structural makeup of the extracellular matrix. The first of these two models is that of bleomycin-induced fibrosis. This model has been used extensively in an attempt to characterize the alterations in lung structure and function that occur in lung fibrosis (38,39). Bensadoun and colleagues (8,40) have recently shown that proteoglycans, in particular, versican, are increased in the lung interstitium in both granulomatous and nongranulomatous forms of human pulmonary fibrosis. In our study (41) we administered bleomycin intratracheally and then studied the mechanical behavior of isolated parenchymal strips from animals sacrificed at various time points postexposure. In addition, we characterized changes in the matrix using histochemistry to identify collagen and elastic fibers, and immunohistochemistry to identify small leucine-rich PGs, in order to correlate changes in structure with changes in lung function. Changes in lung mechanics were maximal at 2 weeks postbleomycin exposure, a time point at which collagen fibers had not yet increased in number. Moreover, alterations in the various physiologic parameters measured, i.e., elastance, resistance, and hysteresivity, were significantly correlated with changes in the small PG biglycan (Fig. 4). These data argue that it is changes in PGs that drive the abnormal mechanics typical of this disease process, at least in the early stages. A further model we have utilized is that of the maturing lung. The mechanical properties of the lung parenchyma change markedly in the first few weeks to months postpartum (42,43). Dramatic morphologic and biochemical changes also occur in the alveolar wall. In addition to changes in collagen and elastic fiber content, hyaluronic acid, chondroitin sulfate, and heparan sulfate PGs vary (44,45). We used this naturally evolving system to try to address the question of whether mechanical changes might be related to specific alterations in the extracellular matrix. In one study (46) we measured viscoelastic properties of excised parenchymal strips from baby, young, and adult rats in the organ bath.

Figure 4 Correlations between the mechanical parameters resistance (R), elastance (E), and hysteresivity (η), and the volume fraction of biglycan in alveolar wall in control (C) and bleomycin (BM)-exposed parenchymal strips at 7, 14, and 28 days postexposure. Linear regression for all datapoints is shown. (From Ref. 41.)


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Collagen, elastic fibers, and alveolar wall thickness were quantitated morphologically; hyaluronic acid was measured using radioimmunoassay. Resistance (R) and elastance (E) were greatest in strips from baby rats and least in strips from adult rats. Changes in strip mechanics were positively correlated with the thickness of the alveolar wall and negatively correlated with the measures of collagen and elastic fiber content. Hyaluronic acid (HA) content fell coincidentally with maturational decreases in R and E. We interpreted these data as showing that components of the alveolar wall other than collagen and elastin, e.g., HA and/or other PGs, account for maturational changes in lung tissue mechanical behavior. IV. Effects of Mechanical Strain on Proteoglycans A. Background

Attention has been directed recently to the effects of mechanical strain on cellular systems. “Mechanotransduction” describes the biological phenomenon wherein cells can alter protein metabolism in response to mechanical stimuli. The intracellular cytoskeleton connects with the extracellular matrix via cell surface receptors known as focal adhesion complexes (Fig. 5). Because of this “molecular continuum” (47) cells can respond to mechanical stimuli applied to the surrounding matrix. A cell, with its intracellular cytoskeletal components, therefore represents a dynamic structure that can respond to compression or tension (47). Further, the cell has an internal prestress generated by the cytoskeleton. Hence, external loads are applied to a system that already has a basal tone. A number of studies have examined the effects of mechanical forces on ECM remodeling. Studies in cartilage have documented an increase in message for collagen and the aggregating PG, aggrecan, in response to cyclic compression (48,49). The aggrecan response can be detected as early as 1 h (50). In vivo

Figure 5 The intracellular cytoskeleton connects with the underlying extracellular matrix. (From Ref. 47.)

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studies in hamsters have shown that animals subjected to a sedentary life style have decreased cartilage PG and GAG content (51). Robbins et al. (52) have also demonstrated that in tendon, cyclic mechanical stimulation resulted in an increase in aggrecan, biglycan, and versican mRNA. Other studies in rabbit ligament have shown that compression as well as tension significantly increased aggrecan mRNA levels, but not those of versican, decorin, biglycan, or fibromodulin (53). B. Studies in the Lung In Vitro

The effects of excessive mechanical strain on extracellular matrix production have been studied using experimental systems which either stretch cell monolayers or strain three-dimensional gels of mixed cell cultures. Xu et al. (54,55) have documented that cylic mechanical strain of mixed fetal lung cell cultures resulted in a reduction of message for both collagen type I and biglycan, and an increase in collagen type IV. There was a modest qualitative increase in versican mRNA. Biglycan protein was increased, as was the expression of a large chondroitin/dermatan sulfate PG thought to be versican. We examined the effects of excessive mechanical strain on monolayers of adult human lung fibroblasts cultured on collagen I–coated plates (7,56). After 24 h of stimulation, cell layer associated versican protein was increased in response to 30% strain of the membrane on which cells were cultured (Fig. 6). The mRNA levels for versican were also increased after 12 and 24 h of mechanical strain. No change in either protein or message was observed for the small PGs, biglycan, decorin, or lumican. The change in versican was not due to an increase in cell number as there was no change in total cell count in response to mechanical strain. C. Studies in the Lung In Vivo

Ventilation-induced lung injury (VILI) is now a well-recognized complication of mechanical ventilation (57). The mechanism of VILI is not completely understood, but can be attributed in part to the effects of excessive airway pressure and alveolar overdistension (58–60). Atelectrauma, i.e., injury related to the shear forces generated by repetitive opening and closing of lung units, may also contribute (57). In effect, VILI may be considered an in vivo version of excessive mechanical strain. Excessive mechanical ventilation causes lung injury that is characterized by alterations in lung tissue mechanical behavior. Webb and Tierney (58) originally showed that dynamic compliance decreased substantially in rats ventilated with high inflation pressures and zero positive end expiratory pressure (PEEP). Tremblay and coworkers (59) documented changes in static lung compliance in isolated rat lungs ventilated for 2 h with high tidal volumes and zero PEEP. Less is known about changes in the dynamic mechanical properties of the lung tissues. One potential mechanism to explain the alteration in tissue properties may include


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Figure 6 Increase in versican expression in cultured human fibroblasts exposed to 30% mechanical strain. The top panel is a representative immunoblot of cell layer extracts from strained cells. The bottom panel represents the mean densitometric values from all the experiments at each time point. (*p ⬍ .05.)

mechanical stimulation–induced alterations in ECM proteins. Changes in lung tissue mechanics with excessive ventilation might, in part, be explained by the effects of altered ventilation on ECM components. Recent studies have described alterations in matrix components in response to abnormal ventilation regimes. Berg et al. (61) described increased procollagen and fibronectin mRNA after ventilation with high levels of PEEP. Parker and colleagues (62) found that ventilation with high peak airway pressures led to

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increased mRNA expression of these factors as well as laminin B, another extracellular matrix glycoprotein. We have recently completed studies in which the effect of different ventilation regimens on lung tissue mechanics and tissue proteoglycans were examined in an in vivo rat model (6). Animals were ventilated with tidal volumes of varying amplitudes at different PEEPs. Dynamic lung tissue mechanics were measured using a small-animal ventilator that has the capacity to deliver a multifrequency signal to the lungs and thereby allow measurement of complex impedance (63). After physiologic measurements were completed, the lung tissue was examined by immunohistochemistry, and protein extraction and Western blotting were done to determine whether changes occurred in extracellular matrix proteoglycans coincident with the changes in mechanical behavior. After 2 h of mechanical ventilation, elastance and tissue damping, a measure equivalent to tissue resistance, were significantly increased in rats ventilated with large amplitude tidal volumes and zero PEEP as compared to controls. Versican, heparan sulfate PG (HSPG), and biglycan were all increased in rat lungs ventilated with this regimen. We used immunohistochemistry to anatomically localize changes in these molecules. HSPG and versican staining became prominent in the alveolar wall and air space; biglycan was identified in the airway wall. Hence, in this study, alterations in lung tissue mechanics with excessive mechanical ventilation were accompanied by changes in all major subclasses of proteoglycans.

V.

Implications for Lung Disease

Proteoglycans are altered in a number of different lung diseases. Bensadoun and coworkers (8,40) have recently published data on human fibrotic disease, examining the role of versican in both granulomatous and nongranulomatous fibrotic processes. In specimens from patients with diseases such as idiopathic pulmonary fibrosis, bronchiolitis obliterans with organizing pneumonia, and sarcoidosis, immunohistochemical analysis showed prominent deposition of versican. Staining for biglycan was also observed, especially in samples from patients with sarcoidosis. Westergren-Thorssen and coworkers (12) studied the bleomycin-induced lung fibrosis model in the rat. They showed that biglycan mRNA was increased as was biglycan protein. Both decorin message and protein were decreased. Our data, cited above, showed increases in both biglycan and fibromodulin protein in this model (41). In addition to these immunohistochemical studies, we also extracted PG from the tissues and, using Western blotting, demonstrated that versican and HSPG were also increased in lung tissue samples from these bleomycin-exposed animals (64) (Fig. 7). Changes in the mechanical behavior of the lung tissues are well documented in this disease process (65,66). Alterations in PG molecules may be responsible. Our data in bleomycin-induced lung fibrosis in rats (41) showing a positive correlation between increases in tissue


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Figure 7 Increases in versican in respose to bleomycin exposure in rat lung extracts. The top panel shows representative immunoblots, the bottom panel densitometric quantitation of data from six rats 7, 14, or 28 days postbleomycin (BM) or saline control (CON). (**P ⬍ .001 vs. control; #P ⬍ .001 vs. 7 and 28 days postexposure). (From Ref. 64.)

resistance and elastance, and volume fraction of proteoglycan support this hypothesis. Some data on alterations in proteoglycans in lung tissue are available in patients with emphysema. Van Straaten and colleagues (67) examined lung tissue from emphysema patients undergoing therapeutic resections. In patients with severe emphysema they showed diminished staining for decorin, biglycan, and HSPG. Emphysema is a disease characterized by marked alterations in lung mechanics. Again, alterations in PG may contribute. A further consideration is the contribution of PG to the abnormal physiology seen in response to excessive mechanical strain, such as during ventilationinduced lung injury. As described above, increased mechanical strain can result in altered PG metabolism (7,56). Insofar as PG alters tissue viscoelasticity, this finding has further potential implications. Versican binds to hyaluronic acid (HA) and forms large aggregates (68). Versican participates in the formation of pericellular matrices through its interaction with cell surface–bound HA (69).

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Pericellular matrices may also be formed through versican chondroitin sulfate side chain interactions with CD44 (70). These pericellular matrices could be important in protecting the cell from the traumatic effects of excessive mechanical strain. When the plasma membrane is subjected to mechanical strain it undergoes unfolding, elastic deformation, or stress failure (71). The pericellular matrix may act to prevent such deformation, by dispersing the effect of the physical force applied at the membrane level before it can be transmitted via the cytoskeleton to the nucleus (72). Hence, the increase in versican in response to mechanical strain may serve as an “adaptive” response. The altered pericellular layer with its altered viscoelastic properties may function as a type of “shock absorber” to protect the cell from excessive mechanical strain and subsequent plasma membrane disruption. Another example of this potential adaptive response could be the airway wall remodeling seen in asthma. Asthmatic airways are likely subject to excessive mechanical strain because of increases in baseline tone and/or because of the excessive transmural pressures generated during normal ventilation. We have published data showing that versican, lumican, and biglycan were all increased in the subepithelial layer of the airway wall of mild atopic asthmatics sampled using endobronchial biopsies (73). Roberts (74) has reported that versican, decorin, biglycan, and HA were localized in airways in postmortem tissue from patients with severe asthma for whom asthma was considered to be the cause of, or a significant contributor to, death. It is possible that these changes in the airway wall could represent an adaptive response by the cell in order to try to minimize the trauma caused by the excessive variations in mechanical stress to which the asthmatic airway wall is subject. Studies to try to directly address this hypothesis are warranted. VI. Conclusion Proteoglycans are important in contributing to both normal and abnormal lung physiology. They influence lung tissue viscoelastic behavior and are altered in lung diseases characterized by abnormal physiology. They can also be affected by excessive mechanical strain and may contribute to the pathophysiology of ventilation-induced lung injury. However, they may also serve a protective role, by limiting the cellular injury that occurs as a consequence of excessive mechanical strain. References 1. Roberts CR, Wight TN, Hascall VC. Proteoglycans In: Barnes PJ, Grunstein MM, Leff AR, Woolcock AJ, eds. The Lung: Scientific Foundations, 2nd ed. Philadelphia: Lippincott-Raven, 1997:757–767.


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2. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998; 67:609–652. 3. Al Jamal R, Roughley PJ, Ludwig MS. Effect of glycosaminoglycan degradation on lung tissue viscoelasticity. AJP: Lung Cell Mol Physiol 2001; 280:306–315. 4. Schmidt MB, Mow VC, Chun LE, Eyre DR. Effects of proteoglycan extraction on the tensile behavior of articular cartilage. J Orthop Res 1990; 8:353–363. 5. Zhu W, Mow VC, Koob TJ, Eyre DR. Viscoelastic shear properties of articular cartilage and the effects of glycosidase treatments. J Orthop Res 1993; 11:771–781. 6. Al-Jamal R, Ludwig MS. Changes in proteoglycans and lung tissue mechanics during excessive mechanical ventilation in rats. AJP: Lung Cell Mol Physiol 2001; 28: L1078–L1087. 7. Al-Jamal R, Roughley PJ, Ludwig MS. The effect of mechanical strain on extracellular matrix composition in cultured human lung fibroblasts (abstr). Am J Respir Crit Care Med 2000; 161(suppl 3):A478. 8. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycan deposition in pulmonary fibrosis. Am J Respir Crit Care Med 1996; 154:1819–1828. 9. Bray BA, Hsu W, Turino GM. Lung hyaluronan as assayed with a biotinylated hyaluronan-binding protein. Exp Lung Res 1994; 20:317–330. 10. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997; 242:27–33. 11. Dolhnikoff M, Morin J, Roughley PJ, Ludwig MS. Expression of lumican in human lungs. Am J Respir Crit Care Med 1998; 19:582–587. 12. Westergren-Thorsson G, Hernnas J, Sarnstrand B, Oldberg A, Heinegard D, Malmstrom A. Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats. J Clin Invest 1993; 92:632–637. 13. Murdoch AD, Liu B, Schwarting R, Tuan RS, Iozzo RV. 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. 14. Yurchenco PD, JC Schittny. Molecular architecture of basement membranes. FASEB J 1990; 4:1577–1590. 15. Scott JE. Proteoglycan-fibrillar collagen interaction. Biochem J 1998; 252:313–323. 16. Liu W, Litwack ED, Stanley MJ, Langford JK, Lander AD, Sanderson RD. Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions. J Biol Chem 1998; 273:22825–22832. 17. Fung YC. Biomechanics. New York: Springer Verlag, 1993. 18. Bayliss LE, GW Robertson. The viscoelastic properties of the lungs. Q J Exp Physiol 1939; 29:27–47. 19. Mount LE. The ventilation flow-resistance and compliance of rat lungs. J Physiol 1955; 127:157–167. 20. Hughes R, May AJ, Widdicombe JG. Stress relaxation in rabbits’ lungs. J Physiol 1959; 146:85–97. 21. Hildebrandt J. Pressure-volume data of cat lung interpreted by a plastoelastic, linear viscoelastic model. J Appl Physiol 1970; 28:365–372. 22. Bachofen H, Hildebrandt J. Area analysis of pressure-volume hysteresis in mammalian lung J Appl Physiol 1971; 30:493–497.

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23. Fredberg JJ, Keefe DH, Glass GM, Castile RG, Frantz ID III. Alveolar pressure nonhomogeneity during small-amplitude high-frequency oscillation. J Appl Physiol 1984; 57:788–800. 24. Ludwig MS, Dreshaj I, Solway J, Munoz A, Ingram RH, Jr. Partitioning of pulmonary resistance during constriction in the dog: effects of volume history. J Appl Physiol 1987; 62:807–815. 25. Kaczka DW, Ingenito EP, Israel E, Lutchen KR. Airway and lung tissue mechanics in asthma. Effects of Albuterol. Am J Respir Crit Care Med 1999; 159:169–178. 26. Kaczka DW, Ingenito EP, Suki B, Lutchen KR. Partitioning airway and lung tissue resistance in humans: effects of bronchoconstriction. J Appl Physiol 1997; 82:1531– 1541. 27. Fredberg JJ, Stamenovic D. On the imperfect elasticity of lung tissue. J Appl Physiol 1989; 67:2408–2419. 28. Bull HB. Protein structure and elasticity. In: Remington JW, ed. Tissue Elasticity. Washington: Waverly Press, 1957:33–42. 29. Mijailovich SM, Stamenovic D, Brown R, Leith DE, Fredberg JJ. Dynamic moduli of rabbit lung tissue and pigeon ligamentum propatagiale undergoing uniaxial cyclic loading. J Appl Physiol 1994; 76:773–782. 30. Mijailovich SM, Stamenovic D, Fredberg JJ. Toward a kinetic theory of connective tissue micromechanics. J Appl Physiol 1993; 74:665–681. 31. Suki B, Barabasi AL, Lutchen KR. Lung tissue viscoelasticity: a mathematical framework and its molecular basis. J Appl Physiol 1994; 76:2749–2759. 32. Gandley RE, McLaughlin MK, Koob TJ, Little SA, McGuffee LJ. Contribution of chondroitin-dermatan sulphate-containing proteoglycans to the function of rat mesenteric arteries. Am J Physiol 1997; 273:H952–H960. 33. Scott JE, Thomlinson AM. The structure of interfibrillar proteoglycan bridges (shape modules) in extracellular matrix of fibrous connective tissues and their stability in various chemical environments. J Anat 1998; 192:391–405. 34. Sata M, Takahashi K, Sato S, Tomoike H. Structural and functional characteristics of peripheral pulmonary parenchyma in golden hamsters. J Appl Physiol 1995; 78: 239–246. 35. Martin CJ, Sugihara T. Simulation of tissue properties in irreversible diffuse obstructive pulmonary syndromes Enzyme digestion. J Clin Invest 1973; 52:1918–1924. 36. Gleisner JM, Martin CJ. Lung tissue tension and glycosaminoglycans. Respir Physiol 1986; 66:247–258. 37. Shimura S, Martin CJ, Boatman ES, Dhand R. A role for interstitial matrix in tissue tension of alveolar wall. Respir Physiol 1985; 62:293–303. 38. Snider GL, Celli BR, Goldstein RH, O’Brien JJ, Lucey EC. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 1978; 117:289–297. 39. Starcher BC, Kuhn C, Overton JE. Increased elastin and collagen content in the lungs of hamsters receiving an intratracheal injection of bleomycin. Am Rev Respir Dis 1978; 117:299–305. 40. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycans in granulomatous lung diseases. Eur Respir J 1997; 10:2731–2737. 41. Ebihara T, Venkatesan N, Tanaka R, Ludwig MS. Changes in extracellular matrix


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5 Hyaluronan and Its Receptors in Lung Health and Disease

RASHMIN C. SAVANI

HORACE M. DELISSER

Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A.

University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Cell interactions with constituents of the extracellular matrix (ECM) are fundamental to a variety of processes ranging from embryonic development to host responses to infection and injury, to tumor growth and metastasis. While much attention has been given to cell interactions with protein members of the ECM, there is increasing recognition that receptor-mediated cell interactions with hyaluronan (hyaluronic acid; HA), a glycosaminoglycan constituent of the ECM, may also be of considerable importance. Specifically, a growing body of literature has provided compelling evidence for the role of HA and its major receptors (CD44 and RHAMM) in cell proliferative and motility responses required for wound healing, inflammatory cell recruitment and activation, and angiogenesis. This is particularly relevant to the lung, where HA has been implicated in lung development and may be important in inflammatory responses that contribute to the pathogenesis of acute lung injury, neonatal lung diseases, airway mucosal defense, and even emphysema. As our understanding of the role of HA and its receptors in lung health progresses, new and potentially very exciting approaches to treating important human lung diseases, based on inhibition of HA-dependent interactions, may emerge. 73

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HA, an important constituent of the extracellular matrix, is an evolutionary ancient molecule found throughout phylogeny from bacteria to humans (1,2). Meyer and Palmer first described HA from the vitreous of bovine eyes in 1934 (3) and, after 20 years of analysis, resolved its structure as a polymer of repeating disaccharides of d-glucuronic acid and d-N-acetyl glucosamine linked together by alternating beta-1,4- and beta-1,3-glycosidic bonds (4) (Fig. 1). In physiological solutions, HA is negatively charged, assumes a twisting ribbon structure with hydrophilic and hydrophobic faces that results in an expanded random coil structure, and occupies a very large domain (2,5). This dynamic structure is thought to result in “pores” that allow a large capacity to entrap water. Most tissues in the body contain HA with 10,000 or more disaccharides reaching molecular masses of ⬃4 million daltons. This high-molecular-weight HA (HMW HA) or native HA (nHA) is the predominant constituent of certain tissues such as cartilage and the vitreous of the eye and can reach concentrations of 25–50 mg/g wet weight. Indeed, human umbilical cord contains 4 mg/mL and rooster comb 7.5 mg/mL of HA (6). Specific interactions both with itself and with other matrix macromolecules and proteins result in organization of the ECM to provide viscoelastic properties that play an important role as a scaffold to maintain tissue structure (2,5). The metabolism of HA is very active and it has been estimated that almost one-third of the total HA in humans is metabolically removed and replaced

Figure 1 Disaccharide unit structure of hyaluronan. HA is made up of repeating disaccharide units of d-glucuronic acid and d-N-acetylglucosamine linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. The beta configuration allows the formation of a highly stable structure that in solution assumes a twisting ribbon conformation with hydrophobic and hydrophilic surfaces.

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during an average day (7,8). Furthermore, HA turnover has been associated with embryogenesis (9), tumorigenesis (10), transformation by oncogenes (11), and responses to injury (12,13). In addition to its role in maintenance of tissue structure, HA has been implicated in the regulation of cell locomotion, proliferation, and differentiation (14). Differential effects are noted between HMW HA and low-molecular-weight (LMW HA, or oligosaccharide-HA, oHA, 10,000–600,000 daltons) in these effects. For instance, HMW HA inhibits leukocyte chemotaxis, phagocytosis (15), and elastase release (16), but LMW HA regulates monocyte activation into macrophages (17). Further, LMW-HA increases cytokine gene expression in macrophages and fibroblasts (18–20). Some controversy exists in the literature about these effects, and inconsistencies may be the result of varying purity, concentration, and molecular weights of HA used in the various studies. For instance, the proinflammatory effects of HA may actually be from contaminating DNA (21). A number of growth factors and cytokines have been reported to increase HA synthesis in a wide variety of cell types, with fibroblasts being the predominant cell type examined. While exhaustive review of all reports is beyond the scope of this chapter, some key observations are possible. For instance, several groups have shown that transforming growth factor-beta (TGF-β) is a strong stimulator of HA synthesis (22–31). However, stimulation of HA synthesis is context dependent with respect to specific TGF-β isoforms, cell density, and the nature of the substratum (32), as well as cell phenotype (23,33) and the presence of other growth factors (34). Platelet-derived growth factor (PDGF), both the AA and BB forms, also stimulates HA synthesis in a dose-dependent manner (22,34– 38). Signaling mechanisms implicated in growth factor stimulation of HA synthesis include growth factor–receptor interactions, protein tyrosine phosphorylation (39,40), and protein kinase C (41,42) and cytokine regulation of HA binding to cell-associated receptors (43). Inhibition of HA synthesis has been reported for corticosteroids (44), drugs such as suramin (45) and vesnarinone (46), and nonsteroidal anti-inflammatory agents such as indomethacin and mefenamic acid (47). Hyaluronan is synthesized by hyaluronan synthases (HASs), single proteins that utilize both sugar substrates to form HA. HAS genes have been identified throughout phylogeny in viral, prokaryotic, and eukaryotic organisms. While glycosaminoglycan chains attached to proteoglycan protein cores are synthesized in the Golgi network, HA is synthesized at the inner surface of the plasma membrane (48). The growing HA polymer is then extruded through the membrane to the outside of the cell as it is being synthesized. Several groups independently cloned three eukaryotic HAS genes that have been designated HAS 1, 2, and 3. Each of these proteins has a large central domain and two or three transmembrane domains at both amino and carboxy ends, with the central domain containing the catalytic site for the enzymes (49). The eukaryotic HASs are ⬃40% larger than the bacterial proteins, but the regulatory functions of the additional sequences

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remain to be clarified. Although each mammalian HAS is able to synthesize HA independently, their enzymatic properties differ. Thus, intrinsic activity of each HAS is different, with HAS3 more active than HAS2, which is more active than HAS1. Furthermore, while HAS1 and 2 form HMW HA (2 ⫻ 10 6 daltons), HAS3 forms LMW HA (2–3 ⫻ 10 5 daltons) (50). A number of cytokines and growth factors, including TNF-α, interleukin-1β, basic fibroblast growth factor, insulinlike growth factor-1, epidermal growth factor, and platelet-derived growth factor-BB increase HAS gene expression (51–55), whereas glucocorticoid treatment suppresses HAS2 expression (55,56). Interestingly, antisense inhibition of HAS2 in chondrocytes results in decreased matrix assembly and proteoglycan retention (57), while targeted disruption of HAS2 in mice results in severe cardiac and vascular abnormalities with embryonic lethality (58). The reader is referred to recent reviews (59–63) and Chapter 6 by Hamilton et al. for more detailed information about this intriguing set of molecules. More recently, increased intracellular HA has been associated with cell migration and proliferation (64). For example, in keratinocytes, increased intracellular HA was seen following in vitro wounding and with stimulation by epidermal growth factor (53,65). Some internalized HA is destined for degradation (65); however, HA has also been localized to the mitotic spindle as well as the cleavage furrow of mitotic cells (66). Intracellular HA is, at least in certain cell types, internalized via CD44 (65), but the possibility remains that other receptors may be involved or that HA synthases may be directed internally to synthesize HA. The role(s) of intracellular HA in cell types that respond to lung injury are completely unknown. The activities of HA on cell behavior are regulated, at least in part, by receptor and binding protein interaction. A growing list of proteins that interact with HA have been described and are collectively called the hyaladherins.

III. Hyaladherins This family of proteins consists of extracellular and cell-associated proteins that bind to HA with high affinity (Fig. 2). While the function of such binding for many of these proteins has not been fully determined, interaction of HA with cellular proteins regulates cell adhesion, motility, and proliferation, whereas extracellular interactions are thought to contribute to structure and maintenance of tissue architecture (67–70). Cellular HA-binding proteins consist of two classes of molecules. CD44, a prototypic type I transmembrane receptor, is predominantly present on the cell surface and binds to HA via a complex tertiary structure defined as the link module (71,72). Nontransmembrane proteins, of which RHAMM (Receptor for HA-Mediated Motility) is a prototype, are found in multiple locations including the cell surface, cytoplasm, and nucleus (10,13,73). These proteins characteristically lack a link module and do not contain transmembrane


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Figure 2 Hyaladherins. RHAMM, receptor for HA-mediated motility; HBP, hepatocyte-binding protein; IHABP, intracellular HA-binding protein.

or signal sequences. HA binding to these proteins is a result of interaction with simple domains containing basic amino acids that conform to the motif [BX 7 B], where B represents basic residues (usually arginine or lysine) and X represents nonacidic residues (74). Enhanced HA binding is achieved with intervening basic residues (usually position 5 in the motif ) and additional positive charge at either end of the sequence (74). A discussion of each hyaladherin is beyond the scope of this review, which will focus on CD44 and RHAMM, molecules that have been characterized the most with respect to responses to lung injury. A. CD44

Several independent descriptions of an 85-kDa cell surface molecule of SV3T3 cells, T-lymphocytes, granulocytes, thymocytes, chondrocytes, and macrophages preceded final identification of CD44 as a widely expressed molecule that functions as a major receptor for HA (75,76). CD44 is a multifunctional transmembrane receptor involved in cell-cell and cell-ECM interactions that contribute to anchoring of HA-rich pericellular matrices, internalization of HA, cell locomotion, lymph node homing, lymphocyte activation, adhesion, presentation of chemokines and growth factors, and transmission of regulatory signals intracellularly. The reader is directed to recent reviews that explore these functions in more depth (68,71,72,77,78). The CD44 gene consists of 20 exons and is located on the short arm of human chromosome 11 (79). The standard form of CD44, CD44S, most prominent on hematopoietic cells, consists of exons 1–5, 16–18, and 20. Exons 6–15 represent exons that give rise to the variant forms of CD44 arising as a result of alternative mRNA splicing (78). Exon 19 is alternatively spliced in place of exon 20 in certain forms of CD44, giving rise to a shorter cytoplasmic tail (77). Thus,

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CD44 consists of four functional domains. The distal extracellular domain (exons 1–5 and 16 in CD44S) is largely responsible for HA binding (80,81) whereas the membrane-proximal domain is the primary site for alternative splicing that results in variant isoforms of the receptor (82). The transmembrane domain has been shown to regulate HA binding as well as interaction with the cytoskeleton (83). The cytoplasmic domain consists of two possible tails that interact with the cytoskeleton, and the longer tail has serine-threonine phosphorylation sites and specific protein-binding sites for interaction with the cytoskeleton, thereby carrying the potential for intracellular signaling (84). The open reading frame of the CD44S mRNA is 1482 bp and encodes a 37-kDa protein. Posttranslational modification of this primary polypeptide by N- and O-linked oligosaccharides (85), heparan sulfate (86), and chondroitin sulfate (87) results in the final 85kDa protein identified on immunoblots. All told, with variant forms of CD44 and extensive posttranslational modification, there are at least 20 different isoforms of CD44 that range from 80 to 250 kDa (71,78). Specific variant forms of CD44 have been associated with tumor invasion and metastasis. For instance, CD44v6, expressed in metastatic rather than nonmetastatic pancreatic carcinoma cells, has been associated with the metastatic potential in vivo (88,89). The HA-binding ability of CD44 resides in the distal extracellular domain. Mutational analysis of this area reveals specific basic amino acids (in particular, arginine 41) and residues 150–162, another cluster of arginines (Arg41, Tyr42, Arg78, and Tyr79), critical for HA binding (81). While most cells express CD44 that constitutively binds HA, several reports have demonstrated CD44 that is not able to bind HA or that can be induced to bind HA using antibodies (68). These observations suggest that at least in some situations, receptor cooperativity is required to effect HA binding. CD44 expression is regulated at the transcriptional level by a number of cytokines such as IL-1 (90), TGFβ (91), BMP-7 (92), and EGF (93). Increased expression correlates with increased ability to bind HA and, in some cases, to internalize and degrade HA (65). Two cis-acting response elements have thus far been characterized in the CD44 gene—a 120-bp EGF-responsive element (93), and an AP-1 site (94). Signaling via CD44 involves protein tyrosine and serine-threonine phosphorylation, binding to the cytoskeleton via ankyrin or ERM (ezrin, radixin, moesin, and merlin), and direct interaction with activated Lck and Fyn kinases within lipid rafts. Ligation of CD44 by HA also results in NFkB activation and production of IL-1β, TNFα, and IGF-I in macrophages and increased expression of IL-2 in lymphocytes. Chapter 6 in this volume and a recent review (13) provide more details of CD44-mediated signaling. With respect to responses to injury, CD44 is clearly expressed in tissues undergoing repair after wounding (95,96), and Stamenkovic’s group has presented direct evidence that supports CD44 regulation of the motility of a human melanoma cell line (97). In addition, McCarthy’s group has shown that the chon-


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droitin sulfate chains associated with CD44 are critical for TGFβ1-stimulated locomotion and invasion by melanoma cells (98,99). On the other hand, the invasiveness of cytotoxic T-cell hybridomas was independent of CD44 regulation (100), and the cellular localization of CD44 in locomoting lymphocytes and thymocytes was not in cell protrusions and ruffling (101). Furthermore, CD44 is downregulated in neutrophils migrating across endothelial cell monolayers (102). Interestingly, mice with a targeted disruption of CD44 are viable, show no abnormalities in any tissue architecture, and are fertile (103). However, treatment of these mice with granulocyte colony-stimulating factor resulted in a defect in egress of hematopoietic progenitor cells from the bone marrow (103). In addition, granuloma formation to Cryptosporidium antigen (a lymphocyte and macrophage response) was increased in mutant mice, suggesting that macrophage responses are intact in these mice (103). Further, SV40-transformed fibroblasts from CD44⫺/⫺ mice were highly tumorigenic in nude mice, an effect that could be abrogated by stable transfection of standard CD44 into these cells (103). B. RHAMM (CD168)

RHAMM (Receptor for HA-Mediated Motility) was first isolated as a 56- to 58kDa protein from the supernatant media of nonconfluent embryonic chick heart fibroblasts and shown to regulate their ruffling and migration (104). A number of critical functions, including the motility of thymocytes, lymphocytes, hematopoietic progenitor cells, malignant B lymphocytes, fibroblasts, smooth muscle cells, endothelial cells, and macrophages, as well as regulation of MAP kinasemediated proliferative responses and cellular transformation, have been ascribed to it (13,69,73). Study of this intriguing molecule has been the subject of recent controversy with two diametrically opposing views on whether RHAMM is expressed on the cell surface (105). Cell surface localization of this receptor has been demonstrated by subcellular localization, surface labeling, antibody blocking, and FACS analysis (29,104,106–111). However, studies in adherent cells transfected with full-length RHAMM cDNA showed an exclusively intracellular protein that interacts with the cytoskeleton (112,113), and other studies of RHAMM expression have failed to demonstrate surface expression (114,115). These differences are likely the result of either incomplete knowledge of the regulation and posttranslational modification of this protein; its potential interaction with chaperones, transporters, or accessory signaling molecules; and/or variations in the cell types and conditions of cell culture used in the various published studies. The gene for RHAMM contains 18 exons and is located on human chromosome 5q33.2 and on mouse chromosome 11 (19.0 cM) (116–118). Human and mouse genes for RHAMM are 85% homologous. While still incompletely understood, various isoforms of RHAMM arise from alternate splicing of this gene (119). Using sequence-specific antibodies, the longest RHAMM transcript, en-

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coding an 85-kDa protein in humans, and two other shorter transcripts, one lacking exon 4 and the other lacking exon 13, have been demonstrated in multiple myeloma (120). Other, smaller proteins that have been identified as RHAMM remain to be more extensively studied. Various domains within RHAMM have been proposed. However, the best characterized are the two HA-binding domains near the carboxy terminus of the protein (121). Site-directed mutagenesis of these HA-binding domains indicates that the minimum binding requirement for HA is represented by B(X 7 )B, where B represents the basic amino acids arginine or lysine and X represents any nonacidic amino acid (74). Identification of this HA-binding motif has allowed the creation of synthetic peptides with varying affinities for HA have been shown to competitively inhibit cell locomotion both in vitro (74) and in vivo (109,122). Other domains of RHAMM that potentially mediate protein-protein interactions and putative sites for posttranslational modifications, including N-glycosylation, myristoylation, and phosphorylation, remain to be more extensively characterized. Regulation of RHAMM expression includes both increases in gene expression and changes in subcellular localization. For example, transforming growth factor-beta 1 (TGFβ1) increases the expression of RHAMM in a doseand time-dependent manner (29), and TGFβ1-stimulated fibroblast migration can be blocked both by RHAMM antibody and by HA-binding peptides, thereby implicating this glycosaminoglycan and its receptor in TGFβ1-stimulated cell motility (29). Further, the mechanism of TGFβ1 regulation of RHAMM appears, at least in part, to be due to stabilization of its message via the binding of protein(s) to the 3′-untranslated region of RHAMM mRNA (123). Alternatively, HA ligation of basal surface-localized RHAMM in thymocytes results in the redistribution of intracellular RHAMM to the surface, where it participates in stimulation of cell motility (119). Signaling via RHAMM has recently been reviewed (13) and is also discussed further in Chapter 6. Binding of HA to membrane RHAMM results in a transient burst of protein tyrosine phosphorylation and focal adhesion turnover (124), activation of src (125), and regulation of the erk kinase cascade through ras (126). How these pathways are activated and their potential interactions or hierarchy remain to be clarified.

IV. HA and Its Receptors in Wound Healing Studies of wound repair may give insight into potential mechanisms of the contribution of HA in lung injury. This has been studied best in the skin. The role of HA and its receptors in wound healing has been reviewed extensively (13,127– 129). The remarkable observation that fetal wounds heal without fibrosis has resulted in extensive investigation as to the differences between fetal and adult


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wound repair. Fetal wound healing occurs in a sterile environment and is associated with more rapid reepithelialization and decreased inflammatory and angiogenic responses (130). A critical difference between the two wounding responses is an increased HA content of fetal skin (131). While both fetal and adult wounds increase HA content upon injury, fetal wound HA concentrations are sustained whereas adult wounds subsequently decrease wound HA content in association with the development of scarring (132). Interestingly, if made large enough, fetal wounds can be made to heal with scar formation (133). In this situation, scarring fetal wounds are associated with increased expression of CD44 and RHAMM and decreased wound HA content (134). However, in contrast to our findings, one study comparing quiescent fetal versus adult cultured fibroblasts reported increased CD44 expression in fetal versus adult cells (135). These differences are likely due to the differences in species examined (rabbits vs. sheep) and examination of cells rather than skin biopsies. Further, hyaluronidase is found in the cytoplasm of wounded fibroblasts and is responsible for HA degradation (136). Collectively, these observations suggest that receptor-mediated internalization and breakdown of HA are likely critical to the scarring phenotype. In healthy skin, HA and CD44 are expressed on keratinocytes, hair follicle cells, exocrine sweat glands, and dendritic cells in the dermis (134,137,138). Epidermal keratinocytes actively synthesize and catabolize HA (8). Consequently, skin HA has a high rate of turnover (139). Keratinocyte CD44 is the receptor thought to be largely responsible for HA uptake in these cells and has specific binding requirements for HA (65,140). In wounded skin, both HA and CD44 show increased expression around keratinocytes (141), a situation that is associated with increased keratinocyte proliferation (142). Interestingly, transgenic expression of antisense CD44 in keratinocytes results in accumulation of HA in the skin and decreased proliferative responses of keratinocytes to epidermal growth factor (143). Conversely, the concentration of HA in cell medium modulates HA receptor expression, suggesting a dynamic, homeostatic system to regulate extracellular HA content (13,144). RHAMM expression is increased in keratinocytes and infiltrating macrophages after full length excisional wounding of the skin (122). Treatment of these wounds with HA-binding peptide, an intervention that is predicted to interfere with HA interactions with all receptors and binding proteins, results in decreased wound contraction and fibroplasia (122). In independent studies, Mummert et al. have confirmed the efficacy of HAbinding peptides in limiting inflammation in hapten-induced dermatitis (145).

V.

HA and Its Receptors in Inflammation

The mechanisms by which inflammatory cells accumulate at sites of injury have received considerable attention. Circulating monocytes undergo activation

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(146,147) and become adherent at sites of endothelial activation (148,149), a process that localizes the activated cells close to the site of injury. Migration of these cells through the endothelium and subsequently in the affected tissue results in their accumulation in injured areas (150). The process is highly ordered and requires the involvement of several distinct molecules including selectins and integrins (151). While the precise role that HA plays in these processes is not clear, critical components of this pathway are regulated by HA. Thus, HA is involved in monocyte activation (17–19), leukocyte adhesion to endothelium (152,153), and macrophage motility and chemotaxis (109,122). HA regulation of processes relevant to inflammation appears to be both dose and molecular size dependent. HA at doses of 1 mg/mL or greater has been reported to inhibit inflammatory cell chemotaxis (15), phagocytosis (15,154), elastase release (16), and respiratory burst activity (154). HA also acts as an anti-inflammatory and antifibrotic agent in rheumatoid arthritis and osteoarthritis (155), and in repair of tympanic membrane perforations (156). In addition, HA accelerates cutaneous wound healing (157) and reduces adhesion formation after intra-abdominal surgery (158). On the other hand, monocyte maturation into macrophages as measured by production of insulinlike growth factor-1 is partly dependent on LMW-HA (17), and LMW-HA stimulates the expression of a number of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) (18,19). Also, HA is greatly increased during inflammatory conditions such as myocardial infarction (159), arthritis (160), and transplant rejection (161), whereas removal of HA by early treatment of myocardial infarction with hyaluronidase results in reduced myocardial fibrosis and infarct size (162). The roles of CD44 and RHAMM in inflammatory cell functions have been studied using blocking antibodies and knockout mice. Thus, CD44 has been implicated as the major receptor that mediates HA-stimulated macrophage maturation and cytokine gene expression (17–19). However, these interesting effects of CD44-HA interaction have recently been questioned, as macrophages obtained from CD44 knockout mice still demonstrate increased chemokine gene expression when exposed to LMW HA (163). The possibility that the HA used in these studies was contaminated—e.g., with other stimulatory proteins, LPS, or DNA— still remains. These controversies will have to be clarified by the use of cells derived from CD44-null mice and defined, pure, and concentration/molecular size–specific HA formulations. Also, using a blocking antibody approach, RHAMM has been shown to be important for the migration of leukocytes, including neutrophils, lymphocytes, and macrophages (122). Detailed characterization of the relative contributions of these receptors to inflammatory cell functions in defined circumstances has yet to be accomplished.

Hyaluronan and Its Receptors V.

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HA and Its Receptors in Vasculogenesis and Angiogenesis

Vasculogenesis, a process occurring during early development, is defined as the de novo formation of blood vessels from precursor cells called angioblasts. Angiogenesis, on the other hand, is the process of the formation of new blood vessels from an existing vasculature. Angiogenesis, an integral part of the response to tissue injury, is a dynamic process that results in the formation of granulation tissue rich in capillaries, and precedes final repair. Although a number of cell types are involved in the formation of new vessels, the central player is unquestionably the endothelial cell (164,165). These cells are normally quiescent but in the context of an angiogenic stimulus can be mobilized to form a new vasculature. The process begins with proteolytic degradation of the basement membrane with subsequent endothelial cell invasion of and migration into the perivascular extracellular matrix. This invasion is accompanied by proliferation of endothelial cells at the leading edge of what becomes a migrating cord of cells. In time, endothelial cells cease proliferating, change shape, and tightly associate with another to form lumen-containing tubes. These “sprouting” tubes eventually fuse to establish a patent circulation in the newly vascularized region. Reestablishment of the basement membrane and recruitment of periendothelial support cells (pericytes) contribute to the remodeling and maturation of these newly formed vessels. In understanding the process of angiogenesis, a paradigm has emerged that the regulation of angiogenesis involves the programmed temporal and spatial expression and activity of (1) soluble angiogenic and angiostatic factors and (2) endothelial cell-cell and cell-matrix interactions (164,165). HA has been implicated in both vasculogenesis and angiogenesis. Its actions, however, are molecular size and concentration dependent. Thus, in chick embryo limb buds, HMW HA localizes to avascular areas, transplantation of HArich mesoderm from normally avascular to vascular areas causes avascularity, and exogenous administration of HMW HA also results in a lack of vascular development (166). HMW HA also either has no effect or inhibits endothelial cell migration, proliferation, and tube formation (167,168). On the other hand, (LMW) HA fragments (3–25 oligomers) are potent and cell-specific stimulators of endothelial cell proliferation (167,168), tube formation (169), and angiogenesis in chick chorioallantoic membranes (168). Stimulation by HA oligomers is concentration dependent, with maximum stimulation at 2 µg/mL (170). Further, in vitro studies have shown a synergistic stimulation of endothelial cell invasion into a three-dimensional collagen gel by coadministration of HA oligosaccharides and VEGF, but not with bFGF (171). It should be noted that one report demonstrates that both HMW HA and

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chondroitin sulfate individually act in a synergistic manner with phorbol ester to promote endothelial cell tube formation (172). However, the molecular size of HA was not determined in these studies. In vivo studies also support the angiogenic nature of LMW HA. Application of HA oligosaccharides to rat skin results in an increased number of blood vessels per mm skin length (173), and accelerates wound healing in a delayed revascularization model (174). Interestingly, quiescent endothelial cells do not synthesize HA whereas activated cells do (169,175), thereby providing the means for more complex regulation of the HA content in any particular microenvironment. A number of observations have implicated HA receptors in endothelial cell functions and angiogenesis. For instance, West and Kumar (176) showed saturable binding of radiolabeled HA to endothelial cells. Rahmanian et al. (169), using both radiolabeled HA binding studies and as live-cell immunofluoresence with anti-CD44 and antibrain hyaladherin antibodies, demonstrated cell surface HA receptors on a brain endothelial cell line (169). Both groups estimated between 2000 and 3000 binding sites per cell. A role for CD44 in endothelial cell function was supported by Trochon et al. (177), who demonstrated 30% inhibition of proliferation, migration, and tube formation in vitro using Mab J173, an antiCD44 antibody that inhibits the adhesion of primitive myeloid cells to HA-coated plates (178). Recently, Lokeshwar et al. (242) demonstrated expression of CD44 and RHAMM in primary human endothelial cells. In these studies, RHAMM-HA interactions were primarily responsible for tyrosine phosphorylation of p125 FAK, paxillin, and activation of p42/44 erk. Further, LMW HA (10–15 oligosaccharide units) rather than HMW HA was more effective in stimulating proliferative responses. Using specific function-blocking antibodies, we have recently demonstrated differential contributions of CD44 and RHAMM to endothelial cell functions (111). Thus, endothelial cell adhesion to HA and proliferation were blocked by anti-CD44 antibody, while anti-RHAMM antibody blocked endothelial cell migration. While both antibodies blocked in vitro tube formation, only antiRHAMM antibody was able to block in vivo angiogenesis in response to bFGF (111). Further definition of the regulation of angiogenesis by HA and its receptors will aid in understanding their contribution to repair processes after injury. VII. HA and Its Receptors in Lung Health and Disease Numerous reports of the involvement of HA and HA receptors in lung biology exist in the literature. These range from their roles in lung development to homeostatic regulation of airway biology and finally to responses to lung injury. While it is impossible to provide an exhaustive discussion of all reports to date, there are several key areas of lung biology affected by HA that have been addressed to date.


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A. Development

Lung development commences in the embryonic phase with the lung bud appearing as an outpouch of the foregut endoderm with subsequent dichotomous divisions that results in conducting airways, vasculogenesis, and formation of accompanying neurovascular sheaths (179). In the pseudoglandular phase, a cuboidal epithelium with thick interstitial septae are seen. Progression to the canalicular phase consists of a thinning to terminal bronchioles and the appearance of more recognizable lung cells such as alveolar type I and II epithelial cells (179). Primary septation and vascularization to develop precursors of future alveoli occur in the saccular phase. Secondary septation and crest formation define the alveolar phase of lung development with great expansion of the air exchange area, maturation of the type II cell, and full establishment of a capillary blood supply (179). The extracellular matrix profoundly regulates the development and differentiation of the lung (180). Mechanisms of such regulation include binding and slow release of growth factors, specific receptor activation, or direct regulation of gene expression (181). In rabbits, HA concentrations decrease during lung development, with the lowest concentrations found at term (182). Similarly, a strong negative association between gestational age and lung HA content exists in human infants (ref. 182 and Savani and Ballard, unpublished). The interstitium consists of the largest HA content during the embryonic phase, with subsequent decreases with increasing gestation. One potential mechanism suggested for the decrease in lung HA during gestation is the progressive increase in lung macrophages that internalize HA via a CD44-dependent pathway with subsequent degradation (183). However, in monkeys, de novo HA synthesis, as measured by [ 3 H]-glucosamine incorporation into minced lung parenchyma, is maximal at term and falls off rapidly thereafter (184). Antenatal corticosteroid treatment, aimed at maturation of the type II cell and synthesis of surfactant components, results in decreases in lung HA concentrations (44,182). The endogenous increase in corticosteroid concentration with gestation is therefore another potential mechanism for the observed decrease in lung HA content. However, one study of antenatal administration of one to four doses of beta-methasone to pregnant ewes (that included mechanical ventilation for 40 min after preterm birth) showed no differences in lung HA concentrations (185). B. Lung Injury

An increased recovery of HA in bronchoalveolar lavage (BAL) has been found in various disease states involving lung injury such as sarcoidosis (186), occupational lung disorders (187), and ARDS (188). The most extensive studies of HA and its receptors in lung injury have examined the rodent model of acute

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lung injury induced by intratracheal instillation of the antitumor antibiotic, bleomycin. Pulmonary inflammation and fibrosis can be induced reproducibility in rodents by intratracheal bleomycin (189). A large number of changes in gene expression after intratracheal bleomycin, both positive and negative, have been reported using gene microarray analysis, and show distinct patterns of gene expression in relation to inflammation and fibrosis (190). The most consistent findings after bleomycin injury are the accumulation of macrophages and the abnormal deposition of collagen in the interstitium of the lung (191). In the acute phase, macrophages infiltrate injured areas, which is a prerequisite for the subsequent chemotaxis of fibroblasts to the same areas (192). The dramatic increase in absolute numbers of fibroblasts correlates well with the accumulation of collagen in the injured lung (192). Bleomycin selectively increases type I procollagen-α1, elastin, and fibronectin mRNA (193). However, these effects are mimicked in explant culture for only 5–7 days (194). Furthermore, bleomycin reduces the uptake of labeled proline into lung slices in culture and inhibits the hydroxylation of proline into collagen (195). These findings suggest that bleomycin-induced increase in collagen synthesis in vivo occurs via an indirect mechanism. It has been suggested that an extrafibroblastic source produces compound(s) that then maintain(s) the high levels of mRNA for type I procollagen-α1 and fibronectin (194). An extensive number of growth factors and cytokines have been implicated in bleomycin-induced pulmonary inflammation and fibrosis (196–198). Of these, transforming growth factor-beta-1 (TGFβ1) has received particular attention (199,200). TGFβ1 is a multifunctional regulator of cell growth and differentiation (201). It is produced as a proprotein that is posttranslationally processed into an active peptide associated with latency-associated proteins (LAP). This in turn is bound by a number of latent TGFβ-binding proteins (LTBP), thereby affording several levels of control. Activation of TGFβ is complex, occurs at the surface of cells, and requires a number of coactivating proteins and proteolytic enzymes that process the protein to its active form. These mechanisms are amply discussed in recent reviews (202–204). Interestingly, in tumor cells, CD44, through interaction with HA, associates with MMP-9 and is required for activation of TGFβ (205). TGFβ1 stimulates fibrosis, angiogenesis, and collagen synthesis in vivo (206), exerts transcriptional control of collagen genes (207), and is also a strong chemoattractant to monocytes (208), lymphocytes (209), and fibroblasts (210). In the bleomycin model, a three- to fourfold increase in the transcription of the TGFβ1 gene occurs in the acute phase of injury and is maximal at 7 days (211). TGFβ1 is localized in the cytoplasm of bronchiolar epithelial cells and the subepithelial extracellular matrix within 2 h of injury (212). Peak staining coincides with the accumulation of macrophages at sites of injury. After 7 days, TGFβ1


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is associated with the extracellular matrix in areas of tissue repair, coinciding with maximum fibroblast collagen synthesis. Several lines of evidence point to a critical role for this growth factor in the pathogenesis of bleomycin-induced pulmonary fibrosis. Decreased fibrosis is observed when TGFβ is blocked from receptor interaction using anti-TGFβ-blocking antibodies (213), decorin [a TGFβ-binding protein (214)], or soluble TGFβ receptor (215). Further, blocking of TGFβ activation, for instance, by using peptides mimicking the thrombospondin-CD36-binding site (216), also results in decreased fibrosis. In an elegant series of experiments by Munger et al. (217), mice with targeted disruption of the type II cell-specific integrin subunit-β6 failed to activate latent TGFβ and did not develop fibrosis after intratracheal bleomycin. Interestingly, these mice showed increased inflammation, suggesting that TGF-β is anti-inflammatory and profibrotic, at least in this model. HA has also been shown to increase dramatically in the alveolar interstitium (218,219) as well as in bronchoalveolar lavage (BAL) (109,220) following intratracheal instillation of bleomycin in rats. Histologically, in the lung interstitium, HA is most prominent at 4 days after injury (109) and gradually declines over the next 3 weeks (218). In bronchoalveolar lavage, the increase in HA is maximal at 4–7 days, and is normal again by 3 weeks after injury (109,220). Furthermore, the increased recovery of HA temporally correlates with an influx of inflammatory cells (109,220), and we have colocalized HA with macrophages in the lungs of bleomycin-injured rats (Fig. 3). In bleomycin-induced lung injury, the molecular weight of HA obtained by BAL is 200–700 kDa (221,222), whereas exogenously administered HA is considerably larger at ⬎10 6 daltons. In our own studies, inflammatory cells isolated by lavage from bleomycininjured animals are more motile than those from control animals, and that HAbinding peptide is able to completely inhibit this increased motility in vitro (109). Further, systemic administration of HA-binding peptide to animals prior to injury

Figure 3 Localization of HA after intratracheal bleomycin. (a) Control section of uninjured rat lung probed with biotinylated HABP (bHABP) that had been preabsorbed with excess HA showing a lack of staining and confirming the specificity of the probe. (b) Uninjured rat lung probed with bHABP showing staining of the subepithelial matrix around bronchiolar smooth muscle (dark stain). (c) Rat lung section 4 days after bleomycin showing accumulation of HA-positive staining macrophages (arrows).

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resulted in decreased macrophage accumulation and fibrosis (109), suggesting that HA is an early component of the response to acute lung injury. In independent studies using a phage display strategy, Mummert et al. recently identified a 12mer peptide that binds and blocks the function of HA in a model of haptenstimulated allergic contact dermatitis in mice (145). Collectively, these studies suggest that HA is upstream of and critical for inflammation and that competitive inhibitors of HA interaction with its receptors and binding proteins are potential therapeutic agents in attempts to limit the inflammatory responses to injury. Teder et al. (222) have proposed mechanisms for the increased production of HA after bleomycin injury in rats. They showed that fibroblasts exposed to BAL from injured animals increased their production of HA, a response that was largely abrogated by blocking antibodies to TGFβ1 (222). Further, alveolar macrophages obtained 5 days after bleomycin injury bound less [ 3 H]-hyaluronan than those from saline-treated controls, suggesting lower HA receptor expression in macrophages after injury. This lower expression of HA receptors was also thought to contribute to elevated HA, since less HA would be internalized by these cells (222). In an extension of these studies, Teder and Heldin (114) confirmed decreased HA binding in alveolar macrophages at 5 days after bleomycin injury, a 30% increase in CD44 expression, no expression of RHAMM, and no differences in either macrophage lysosomal hyaluronidase activity or liver endothelial clearance of HA. They concluded that decreased HA-binding capacity of alveolar macrophages was responsible for the elevation of HA after bleomycin injury. However, several investigators have shown that growth factors such as TGFβ1 and PDGF-BB increase both HA production and HA receptor expression (22,29,99). Changes in the expression of certain CD44 isoforms after bleomycininduced lung injury in rats have also been reported (223). While the authors found no correlation between the presence of CD44 isoforms and the extent of pulmonary injury, reactive changes in epithelial and nonepithelial cells were found (223). The expression of CD44S was increased in alveolar macrophages and in the interstitium of the lung in areas of thickened alveolar wall, while the expression of CD44v6, thought to be the epithelial form, was decreased in type II pneumocytes (223). Further, we have noted an increased surface expression of RHAMM in lavage-derived cells from bleomycin-injured animals (Fig. 4). The reasons for these discrepancies remain to be resolved, but likely involve differences in the methods of processing and/or analyses used by the two groups. Teder and Noble have reported the effects of bleomycin injury in CD44 knockout mice (224). Their data show that bleomycin injury in these CD44-null mice results in nonresolving inflammation, a failure to clear HA from the lung, and increased mortality by 10–20 days after injury. The authors speculate that CD44 is required for clearance of HA and abrogation of the inflammatory signal. Since treatment with HA-binding peptides decreases inflammation after bleomycin injury (109), it is clear that HA is also involved in the recruitment of inflam-


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Figure 4 Increased cell surface expression of RHAMM after bleomycin injury. Cells isolated by bronchoalveolar lavage 4 days after either bleomycin or saline treatment were studied by flow cytometery using anti-RHAMM antibody (R36) with normal IgG as a control. Control cells, obtained from animals given intratracheal saline, showed very low cell surface expression of RHAMM, whereas those from bleomycin-treated animals showed increased cell surface expression of RHAMM. (From Ref. 13.)

matory cells to the lung. Independent effects of HA on fibroblasts via CD44 and RHAMM are also possible. We propose a model for the involvement of HA in acute lung injury (Fig. 5). Further study and clarification of these findings are likely to reveal interesting and independent contributions of these two HA receptors to the response to acute lung injury. C. Neonatal Lung Diseases

Pulmonary surfactant, a complex mixture of phospholipids, neutral lipids, and proteins, serves to reduce surface tension at the air-liquid interface in alveoli, thereby preventing alveolar collapse, and plays a critical role in innate host defense against bacterial challenge (225). Deficiency or dysfunction of surfactant results in respiratory distress syndrome (RDS) that consists of respiratory failure, atelectasis with intrapulmonary arteriovenous shunt, and hypoxemia. Preterm infants, born with surfactant deficiency, are at increased risk of pulmonary infection

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Figure 5 Involvement of HA in acute lung injury. We propose a model in which the ratio of LMW vs. HMW HA is increased after acute lung injury. This LMW HA activates circulating monocytes and facilitates their recruitment to the lung from the pulmonary microcirculation. Further HA-mediated maturation of macrophages results in increased inflammatory molecule gene expression, including cytokines and iNOS, as well as increased macrophage motility. The level of subsequent fibroblast activation determines the balance of resolution with healing and the development of fibrosis.

and subsequent development of bronchopulmonary dysplasia (BPD), a fibrotic disease with evidence of an arrest in lung development and decreased alveolization (226). Most infants that subsequently develop BPD show an acute inflammatory response 7–10 days after birth (227). Increased lung HA content has been reported in preterm infants with RDS (182). Indeed, preterm monkeys with RDS show increased lung HA concentrations and alveolar HA staining with increased severity of lung disease (228). Increase in HA appears to be, at least in part, due to increased HA synthesis (184). Term neonatal rats exposed to increased inspired concentrations of oxygen for 5–7 days show elevated lung HA content and edema (229). On the other hand, neonatal piglets with group B streptococcal (GBS) pneumonia have lower lung HA concentrations than controls (230). Limited human studies have examined HA concentrations in infants with respiratory failure. Cochran et al. (231) reported preliminary findings of a correlation between serum HA concentrations and severity of respiratory disease but, in the 16 infants examined, failed to demonstrate any relationship with gestational age or birth weight. Johnsson et al. (182) have examined HA content and staining in 117 infants that


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died between 0 and 228 days of life after term and preterm birth. Lung HA concentration was most strongly associated with gestational age at birth, again decreasing with more advanced gestation. Further, more detailed examination of lung HA changes in larger cohorts of infants may reveal associations with inflammation, expression of growth factors, severity of lung disease, and BPD. D. Airway Mucosal Host Defense

Upper-airway secretions are continuously cleared by mucociliary mechanisms involving the ciliated epithelium. Innate mucosal defenses depend on the immediate availability of relevant proteins to react to environmental changes and mediate protective responses. Exposure of the upper airways to noxious substances elicits bronchoconstriction to limit lower-airway exposure and increased ciliary beat frequency to increase clearance of the foreign material. Tissue kallikrein (TK), a key mediator of bronchoconstriction in asthmatic individuals (232), is normally bound by HA and held on the surface of tracheal epithelial cells by the interaction of HA to RHAMM (110). TK binding by HA also inhibits its activity in a reversible manner (233). Further, tracheal cells in culture show increased ciliary beating in association with increased RHAMM and HA content with time in culture, and exogenous HA stimulates ciliary beat frequency (234). In a manner similar to that of ciliary beating in sperm (235), anti-RHAMM antibody blocks HAmediated ciliary beating in tracheal epithelial cells (110). Noxious stimuli, therefore, can result in breakdown of HA and result in the release of active TK and LMW HA that stimulate bronchoconstriction and increased ciliary beat frequency, respectively (110). These findings define a unique role for HA in mucosal host defenses in which HA functions as the holder of an apical enzyme pool “ready for use” and protected from ciliary clearance, and as a stimulator of clearance upon challenge. Interestingly, superoxide and peroxynitrite have both been shown to break down HMW HA (⬃1–2 ⫻ 10 6 daltons) into LMW HA (⬍500– 700 kDA) (236). It is possible that allergen exposure results in activation of hyaluronidases and/or production of these radicals that results in HA fragmentation. E. Emphysema

Emphysema, a common disease associated with chronic cigarette smoking, results in alveolar destruction and respiratory failure that ultimately leads to right heart failure and cor pulmonale. Intratracheal instillation of elastase in rodents is an established model of emphysema and mimics the disruption of alveoli seen in the human disease. HA has been studied as a therapeutic intervention for elastase-induced emphysema produced by intratracheal neutrophil, macrophage, or pancreatic elastases in hamsters (237). Alveolar integrity, determined by mean linear intercept, was protected by intratracheal administration of an ⬃100-kDaMW HA given either 1 h before or 2 h after injury (238). Conversely, administra-

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tion of hyaluronidase followed by elastase worsened the emphysematous changes (239). Neutrophil accumulation after injury was similar between the HA-treated and control groups, suggesting that HA protection from damage was likely not due to an anti-inflammatory effect (240). Using fluorescein-labeled HA, aerosolized HA was found associated with interstitial, pleural, and vascular elastic fibers, and HA protected elastolysis of a cell-free radiolabeled ECM (241). These studies suggest that HA provides a protective coat for these fibers. Collectively, the work of Cantor and associates (237–241) suggests either a protective role for endogenous HA and/or implicates the formation of LMW HA as a mediator of damage. Taken together with the studies of airway mucosal defense, it is clear that HA plays a significant role in airway and alveolar integrity and homeostasis. VIII. Summary and Conclusions In summary, HA, a ubiquitous glycosaminoglycan component of the ECM, has unique physical and biochemical properties that allow a multitude of functions that depend on molecular size and concentration. In addition to structural integrity and maintenance of airway and alveolar homeostasis, the interaction of HA with hyaladherins appears to regulate key responses to tissue injury including inflammation, angiogenesis, and fibrosis. The roles played by HA in the lung, both in health and in disease, are increasingly being clarified and hold promise for unique insights into the physiology and pathophysiology of lung function and responses to injury. These will likely lead to the development of novel therapeutic approaches that will promote homeostasis and limit the adverse effects of lung injury. References 1. Laurent TC, Fraser JRE. Hyaluronan. FASEB J 1992; 6:2397–2404. 2. Hascall VC, Laurent TC. Hyaluronan: structure and physical properties. Glycoforum Group. 1997; http:/ /www.glycoforum.gr.jp/science/hyaluronan. 3. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J Biol Chem 1934; 107:629–634. 4. Weissman B, Meyer K. The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. J Am Chem Soc 1954; 76:1753–1757. 5. Toole BP. Hyaluronan. In: Izozzo RV, ed. Proteoglycans: Structure, Biology and Molecular Interactions. New York. Marcel Dekker, 2000:61–92. 6. Laurent TC. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. Wenner-Gren International Series, Vol 72. London: Portland Press, 1998. 7. Morales TI, Hascall VC. Correlated metabolism of proteoglycans and hyaluronic acid in bovine cartilage organ cultures. J Biol Chem 1988; 263:3632–3638.


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6 Hyaluronan and Hyaladherin Signaling in the Lung

SARA R. HAMILTON, FU-SHENG WANG, and EVA A. TURLEY London Regional Cancer Centre University of Western Ontario London, Ontario, Canada

I.

Introduction

In the adult, lungs move and change volume continuously in order to provide adequate respiratory exchange. Since lung tissue is exposed to repeated environmental insults owing to its constant intake of air, vigilant repair of damaged alveoli and tight regulation of response to injury processes must occur. Chronic inflammation that accompanies diseases such as asthma, fibrosis, and emphysema impair the ability of the lung to perform this function, and this is caused, at least in part, by remodeling of the extracellular matrix.

Abbreviations: Alveolar macrophage: AM; basic fibroblast growth factor: bFGF; betaglucuronic acid: β-GlcUA; bronchoalveolar lavage fluid: BAL; epidermal growth factor: EGF; extracellular regulated kinase: erk; ezrin/radixin/moesin: ERM; fluorescent activated cell sorter: FACS; focal adhesion kinase: FAK; glycophosphoinositide: GPI; hyaluronan: HA; hyaluronan synthase: HAS; insulin like growth factor 1: IGF-1; interferongamma: IFN-γ; interleukin-1 beta: IL-1β; interleukin-10: IL-10; N-acetyl-glucosamine: GlcNAc; inducible nitric oxide synthase: iNOS; nuclear factor-kappaB: NF-κB; plateletderived growth factor: PDGF; sarcoma virus kinase: src; transforming growth factor beta: TGFβ; tumor necrosis factor alpha: TNFα; phosphinositide 3 kinase: PI3 kinase.

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Maintenance of a normal extracellular matrix milieu is essential for normal lung function (1). The extracellular matrix of tissues including the lung is composed of proteins such as fibronectin and collagens; polysaccharides such as HA, chondroitin, and heparan sulfate; and polysaccharide-binding proteins (2–4). Polysaccharides, in particular HA, perform many important functions that maintain homeostasis in the lung. These include regulation of neovasculogenesis/angiogenesis (5–7), participation in maintaining the integrity and function of the surfactant layer at the cellular interface in alveoli (8), and regulation of inflammatory and response-to-injury (5–7,9–12) processes. For example, HA modifies the activity of kallikrien involved in allergic response (9) and the susceptibility of elastin fibrils to degradation into fragments (10–12) that attract leukocytes (13) and control the migration and activation of resident monocytes (14,15). In addition, HA regulates the function of mesothelial cells lining the pleura, which participate in the constant repair of lung tissue (16–18). Injury to the lung tissue modifies the above examples of the normal functions of HA by altering both the expression and location of HA binding proteins called hyaladherins and the size, synthesis, metabolism, and localization of HA. For example, the fragmentation of HA by hyaluronidases (19) or oxidation (20) results in an enhanced ability of HA to activate signaling cascades (21–24) and transcription factors that regulate pro-inflammatory gene products (25,26). For instance, activation of these signaling cascades promote cell migration, proliferation, and production of proteins (27–34) that attract circulating leukocytes into lung tissue and that initiate the remodeling of extracellular matrix within the injured lung tissue. In this chapter, we will review HA and the hyaladherins that are relevant to lung response-to-injury processes.

II. Hyaluronan HA is a simple polymer composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and β-glucuronic acid (β-GlcUA) (35,36). HA can attain molecular weights in excess of a million daltons and its size can be tissue specific. For example, it has been reported that HA is 2000–7000 kDa in normal synovial fluid and cartilage but 220 kDa in lung tissue. More often, the molecular weight of HA is heterodisperse and can range from 40 to 600 kDa within one tissue (8). HA is ubiquitous in the extracellular matrix of most tissues and is also present in lymph and blood. It accumulates to the highest concentration in joints, eye, and skin (35,36). In addition to its occurrence in the extracellular matrix, HA associates with the surface of many cell types and has also been reported to occur in subcellular compartments including the cytoskeleton and cell nucleus (See “Science of Hyaluronan Today” at http://www.glycoforum.gr.jp).

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Three distinct HA synthases (HAS1, HAS2, and HAS3) have been cloned as separate gene products that polymerize HA from uridine diphosphosugar precursors (37–39) (Fig. 1). They are the first class of glycosyltransferases identified in which a single protein catalyzes the transfer of two distinct monosaccharides. These enzymes uniquely occur at the plasma membrane as integral proteins and they, and possibly as-yet-uncharacterized accessory proteins, direct the HA chain that forms on the cytoplasmic face to the outside of the cell. HAS enzymes contain multiple membrane spanning sequences, and the carboxyl terminal 25% of Streptococcus pyrogenes HAS sequence has been proposed to contain reentrant loops or amphipathic helices that may form pores through which HA is extruded out of the cell (40). A similar mechanism may permit HA to exit mammalian cells (Fig. 1). All three mammalian HAS enzymes are able to synthesize highmolecular-weight HA chains both in vitro and in vivo. HA synthesis is regulated by a multitude of cytokines and growth factors, including TNFα, interleukin-1β (41,42), PDGF (43,44), EGF (45), bFGF and IGF-1 (46), but expression of HAS enzymes is differentially sensitive to these

Figure 1 HA synthases synthesize HA at the plasma membrane. The HA polymer can be synthesized by any one of three synthases—HAS1, HAS2, or HAS3. HAS proteins are homologous but are encoded as separate gene products. HAS1–3 proteins are located at the plasma membrane and contain multiple domains that pass into the membrane. The activated (UDP) sugars are added at the cytoplasmic face and the growing hyaluronan chain passes out of the cell, possibly through a channel created by the synthases.

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factors. For instance, HA production is stimulated in lung fibroblasts by N-acetylcysteine (47) and by PDGF (43,44); the latter response is blocked by hypoxia (44). However, PDGF only promotes expression of HAS2 in lung mesothelial cells and has no effect on HAS1 or 3 expression (48). Similarly, EGF promotes only HAS2 expression in keratinocytes (45). HAS2 expression in lung cells, but not HAS1 or HAS3 expression, is inhibited by both TGFβ and hydrocortisone (48). HAS enzymes are also likely to be differentially regulated in vivo. For instance, genetic deletion of HAS2 results in embryonic lethality due to malformations in heart tissue (49). HAS1 and 3, still present in these genetically altered mice, are clearly not required for HAS2 regulated development. A simple explanation is that HAS1 and HAS3 are regulated separately from HAS2 and are not expressed during heart formation. In vitro analyses suggest that HAS1–3 also differ in their enzyme characteristics. To date these include the rates at which HA chains can be elongated and the apparent K m s for UDP-GlcNAc and UDPGlcUA (50). During normal lung function, HA is present primarily around bronchioles and blood vessels and in the bronchoalveolar lavage fluid (BAL), suggesting a location in the superficial layers at the lung :air interface (8). In vitro, HA can be produced by mesothelial cells from the pleural cavity, by lung fibroblasts and by type II pneumocytes/epithelial cells (8). HA and hyaladherin expression are increased in both lung tissue and in infiltrating leukocytes during pulmonary diseases including fibrosis, asthma, chemical-mediated injury, and neoplasia (51–53). HA performs many functions in the body as a consequence of both its unique hydrodynamic properties and its interactions with a growing list of extracellular and cellular hyaladherins. Interactions between HA and extracellular hyaladherins are essential for the hydrodynamic properties of HA while interactions of HA with cellular hyaladherins result in cell signaling. These functions of HA are affected by its molecular size and its concentration (e.g., 21,24). When high-molecular-weight HA (⬎1 ⫻ 10 6 daltons) is fragmented into oligomers (⬍2 ⫻ 10 5 daltons), it loses its unique hydrodynamic properties. Conversely, the ability of HA to bind to hyaladherins is often also modified (8) and, importantly, its signaling capabilities are often enhanced (e.g., 24). Thus, HA has both a structural and a signaling property depending on its molecular weight. The hydrodynamic properties of HA permit it to form hydrophilic gels whose properties are important to lung function (8). The hydration, viscosity, and self-aggregation properties of HA contribute to the formation of these hydrophilic gels, while the presence of extracellular hyaladherins such as versican stabilizes the gels. A central function of HA in the formation of gels is therefore likely that of a networking molecule (e.g., as in Fig. 2). In lung alveoli, HA gels provide a seamless connection that links the endothelial and epithelial cell surfaces to the surfactant layer (Fig. 2). Even in tissue culture, HA synthesized by


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Figure 2 Functions of HA in normal lung physiology. Possible important functions of high-molecular-weight HA in the normal lung are participation in maintaining the integrity of the surfactant layer and providing a smooth coat that covers the underlying uneven surface of endothelial and type I and II epithelial cells.

cell monolayers is retained as a coat, or miniature gel, that covers individual cells (Fig. 3) (54,55). Although the precise function(s) of such HA coats are not clear, HA-rich gels present at the air/alveolar interface have been proposed to provide a “smooth” surface covering the cellular layer and, through interactions with the phospholipids associated with surfactant proteins, to stabilize the surfactant layer (Fig. 2) (8). High-molecular-weight HA (⬎1 ⫻ 10 6 daltons) has sluggish signaling capabilities, and, in high concentrations, inhibits processes required for a

Figure 3 High-molecular-weight HA can coat cells in vitro. A coat of HA is often observed in cells maintained in culture, particularly in locomoting and dividing cells. This coat may act as a mobile reservoir for growth factors and provide exquisite hydrodynamic regulation for the cell. HA coats appear to be attached to the cell surface via binding to CD44.

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Figure 4 Signaling pathways regulated by HA and cellular hyaladherins. Hyaladherins such as RHAMM and CD44 regulate and coordinate signaling pathways through HA and growth factors. CD44 is an integral plasma membrane protein, and its cytoplasmic domain has been shown to bind to many signaling complexes, including c-src, Tiam1, and RhoA. The cytoplasmic domain of CD44 also binds to actin-binding proteins, such as the ERM proteins and ankyrin. This hyaladherin therefore likely couples signaling pathways to the cytoskeleton. In contrast to CD44, RHAMM does not contain a membrane-spanning sequence and is present on the cell surface as a nonintegral protein. It is also present in several cytoplasmic compartments and in the cell nucleus. Cell surface RHAMM (CO 168) is required for HA-regulated and growth factor–mediated signaling pathways, including activation of src and the map kinase erk. Intracellular RHAMM associates with both the microtubule and actin cytoskeletons and binds to signaling complexes including the map kinase, erk. Whether the functions of cell surface RHAMM (CO 168) are coordinated with those of intracellular RHAMM so that it can act as a “modular” receptor is not yet clear.


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response to tissue injury. For instance, high-molecular-weight HA can also inhibit angiogenesis (25) and block degradation of elastin into fragments that are chemotactic to circulating leukocytes (10–13). High-molecular-weight HA in the lung interstitium also maintains the water homeostasis in the tissue by both attracting water by osmotic forces and resisting water flow (56,57). HA gels can be degraded by hyaluronidases, by oxidation, and/or by synthesis of smaller (⬍10 6 daltons) HA fragments (19). HA fragments produced by these processes are able to stimulate cellular signaling and to initiate cellular responses to tissue damage (58). For instance, HA fragments (⬍2 ⫻ 10 5 daltons) promote the activation of protein tyrosine kinase cascades that result in signaling through the ras-erk and PI3 kinase cascades in endothelial cells and fibroblasts. Activation of these and other signaling cascades by HA fragments requires the presence of at least two cellular hyaladherins, RHAMM and CD44 (58), and results in transcription of genes involved in the response-to-injury process (Fig. 4). Signaling directed by HA fragments promotes cell motility and proliferation and appears to play a key role in regulating the functions of lung macrophages and endothelial cells, the latter particularly during angiogenesis (21–34). For example, HA fragments can activate NF-κB/IκB, AP-1, and other transcription factors in macrophages and endothelial cells (25,26). As a result, HA promotes the production of chemokines and cytokines, such as IL-10 and TNFα, and of enzymes associated with an inflammatory response, including nitric oxide synthase, metalloproteinases, and tPA, uPA, and PIA-1, the latter particularly in response to cytokines such as TNFα (24–27). These disparate functions of native vs. fragmented and of high vs. low concentrations are mediated by hyaladherins, and the availability of HA to bind to extracellular vs. cellular hyaladherins is likely to be key to the precise response of a cell to environmental HA.

III. Hyaladherins Extracellular hyaladherins are required for the important hydrodynamic functions and properties of HA in the lung as noted above, and these proteins also play important structural roles in lung architecture (35,36). For instance, both link protein and aggrecan are present in tracheal cartilage and contribute to the structure of this tissue (59). Extracellular hyaladherins, such as versican (60,61), occur in the surfactant layer in alveoli (8) and likely play a role in retaining HA in this layer. Cells within the alveolus (62,63) express CD44. In addition to its ability to signal, this hyaladherin maintains HA coats on cell monolayers in vitro (54,64,65) (Fig. 3). It is therefore reasonable to propose that CD44-HA interactions anchor the HA/surfactant layer to the cells within the alveoli (8). In addition to CD44, lung epithelial cell lines also express RHAMM in vitro (66). Additional

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novel cellular hyaladherins are also expressed in lung tissue. One of these is an endothelial-specific hyaladherin (67), and one is expressed in the lymphatic tissue (68). Both CD44 and RHAMM have been shown to be important in regulating signaling in endothelial cells and fibroblasts in vitro, and these two receptors clearly play important roles in lung response-to-injury processes (56). The nature of these two receptors and the signaling pathways that they are known to regulate are summarized below. A. CD44

The CD44 gene contains 20 exons and is subject to alternative splicing of exons 6–15. These 10 splicing exons are also known as variant exons v1–v10 and are capable of being individually spliced, thus generating multiple variant forms. CD44 standard transcript (CD44s) lacks the 10 variant exons and has a predicted MW of 85 kDa. The larger CD44 variant transcripts (CD44v) encode proteins with molecular weights of ⬎85 kDa, and have been estimated to amount to 1– 10% of all CD44 isoforms. Variant forms are commonly expressed in hemopoetic, endothelial, and mesodermal tissues, and many of these variant forms are expressed following tissue injury (69). The epithelial form encodes CD44s plus three additional exons (v8–v10) and is expressed primarily by epithelial cells. In addition to alternative splicing, heterogeneity in CD44 proteins is created by cell type–specific addition of glycosaminoglycans and N- and O-linked carbohydrates, which can account for more than half the mass of the proteins, for example, the CD44s protein. All CD44 forms contain a “link module” sequence that is responsible for binding to HA. CD44/HA interactions affect cell adhesion, motility, proliferation, and some, but not all, events associated with angiogenesis (70,71). Intracellular domains of CD44 interact with the actin cytoskeleton and signaling molecules, directly linking extracellular HA to the cell cytoskeleton and signaling complexes. The cytoplasmic domain of CD44 can bind both to ERM (ezrin/radixin/ moesin) proteins in a phosphatidylinositol 4,5-bisphosphate-dependent and -independent manner, and to the actin-binding protein ankyrin (72–74). Although HA/CD44 binding appears to be unaffected by ERM-CD44 interactions, the association of CD44 with ankyrin is required for efficient binding of HA to CD44, and for HA-CD44-mediated cell adhesion to occur (75). CD44-ankyrin interactions have therefore been suggested to be required for cytoskeleton activation during HA-mediated signaling. CD44 interacts directly with at least two tyrosine kinase-based signaling regulators, her 2/neu and pp60csrc (76,77). Importantly, CD44-HA interactions can activate src, and this activation results in the protein tyrosine phosphorylation of cortactin, a cortical actin-binding protein, whose overexpression stimulates the


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motility of fibroblasts (78,79). Tyrosine phosphorylation of cortactin is required for its trafficking to the cortex of the plasma membrane and for its motilityenhancing capability (78). In addition to its direct interaction with her 2/neu and src, CD44 can associate with other nonreceptor protein tyrosine kinases in lipidrich cell membrane “rafts” (80). CD44 is also involved in activating the Rholike GTPases (81) and rac 1 signaling pathways (82). These pathways coordinate remodeling of the actin cytoskeleton during cell process formation and are required for cell motility and vesicle trafficking (83). Likely as a consequence of its ability to promote signaling cascades, CD44 interactions with HA (2 ⫻ 10 5 daltons) result in the activation of NF-κB/IκB transcription factors and expression of pro-inflammatory enzymes including plasminogen activator, nitric oxide synthase, chemokines, cytokines, and metalloproteinases (35,36). In summary, the paradigm by which CD44 signals is similar to that of growth factor receptors insofar as both classes of receptors are transmembrane proteins and their extracellular ligands bind to the extracellular portion of the receptor, stimulating the accumulation of signaling complexes and actin-binding proteins on the cytoplasmic portion of the receptor (84). B. RHAMM

RHAMM is unlike CD44 in that it is not an integral plasma membrane protein and does not contain a signal peptide for export through the Golgi/ER, yet it can occur on the cell surface as well as inside the cell along the cytoskeleton and in the cell nucleus (85). RHAMM likely occurs on the cell surface as a peripheral or GPI-linked protein, although this has not been unequivocally established (85). The mechanisms by which RHAMM signals clearly do not fall into the CD44/ integral growth factor receptor paradigm (84). Even though the mechanisms by which RHAMM affects signaling may be frustratingly enigmatic, it is clearly required for a signaling response to HA. For instance, even when cells express CD44, the presence of RHAMM is required for activation of protein tyrosine kinase cascades by small HA polymers (24). This role for cell surface RHAMM in activating protein tyrosine kinase cascades and signaling through the raserk kinase cascade has also been demonstrated using blocking antibodies (21,22,24,71,86–88). Although the majority of RHAMM protein is likely intracellular and associated with the cell cytoskeleton or the cell nucleus, cell surface RHAMM regulates signaling through HA (21,24,71) and is required for the activation of erk kinase by several stimuli (89,90). In fact, a clear role or roles for intracellular RHAMM forms in signaling have not yet been reported. Interestingly, HA itself has been reported to occur within nonlysosomal compartments including caveoli, the mitotic spindle, and cell nucleus (91,92; http:/ / www.glycoforum.gr.jp/). Whether such intracellular HA binds to intracellular

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RHAMM forms and whether these putative interactions play a role in HA-mediated cell signaling is not yet understood. RHAMM occurs on the surface of a variety of cell types including leukocytes, fibroblasts, endothelial cells, and smooth muscle cells (50,93), as measured by FACS (e.g., 88), subcellular fractionation of membranes (e.g., 71), and the ability of antibodies to block the signaling and motility functions noted above (21,22,24,71,86–88). Surface RHAMM on leukocytes has recently been designated CD168 by the International Leukocyte Workshop (Leukocyte typing VI, 1998, Garland Press; http://www.ncbi.cim.nih-gov/prow). In substrate-attached fibroblasts and smooth muscle cells, expression of cell surface RHAMM is variable and the factors that promote a cell surface localization are not well defined, although TGF-β increases cell surface RHAMM expression (94) and cell-cell contact may decrease it. Several groups (95,96) have noted an absence of cell surface RHAMM, and this discrepancy with other studies suggests that export of RHAMM be subject to strict regulation. Unlike CD44, RHAMM does not contain a link module, and RHAMMHA interactions are mediated by small sequences rich in basic amino acids (97). The HA/RHAMM interaction regulates protein tyrosine kinase cascades, at least in part through src (97), focal adhesion kinase (FAK) (21,24), and receptor protein tyrosine kinases such as the PDGF receptor (95). HA/RHAMM interactions are required for signaling through ras and can result in activation of the map kinase erk (22,95). In addition, cell surface RHAMM is required for PDGF-mediated and stretch-induced activation of erk kinase (95,96), but to date these effects have not been directly demonstrated to require HA binding to RHAMM. Unlike CD44, HA-RHAMM interactions only transiently activate src (98); furthermore, focal adhesion kinase is first phosphorylated then de-phosphorylated (21), an event that is coincident with the turnover of focal adhesions. These results suggest that HA-RHAMM interactions may also regulate the activity of phosphatase(s) and may thus provide a mechanism for limiting the extent and timing of protein tyrosine phosphorylation. The signaling regulated by RHAMM/HA interactions is required for angiogenesis (71), random cell motility (e.g., 21), and progression through G 2 M of the cell cycle (99). The role of intracellular RHAMM forms in signaling has not been extensively investigated, but RHAMM coimmunoprecipitates with erk kinase in the presence of PDGF (95) and NGF (100), suggesting that intracellular forms associate with this map kinase. Furthermore, although the secondary structure of RHAMM is a series of discontinuous coiled coils reminiscent of structural proteins such as myosin, analysis of its primary sequence by MOTIF SCANNER (http://cansite.bidmc.harvard.edu) reveals Abl, Amphiysin, and crk SH3-binding sites; Abl, Nck, Src, and PI3 kinase SH2-binding sites; two erk docking sites; several nuclear localization signals; and multiple kinase phosphoacceptor sites including ones for erk, ATM, CDK2, cdc2K, cdK5, GSK3B, PDK1, PKA, PKC,


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and AtkK. These motifs suggest that, in addition to erk, intracellular RHAMM forms have the potential for interacting with disparate signaling complexes. This ability, combined with both its secondary structure and its association with the cytoskeleton (101,102), predicts that intracellular RHAMM forms may act as adapter proteins, linking signaling complexes to the cytoskeleton much like paxillin, vinculin, and WASP proteins (103,104). Since CD44 and RHAMM commonly regulate cell motility, proliferation, and angiogenesis, as well as similar if not identical signaling cascades, it is reasonable to predict that these two hyaladherins coordinate the signaling pathways necessary for at least some of these cell functions. For instance, the ability of HA fragments to activate protein tyrosine kinase cascades and erk kinase occurs in the presence of both RHAMM and CD44 (24). However, several studies suggest that at least some functions of RHAMM and CD44 are distinct. For instance, anti-RHAMM, but not anti-CD44, antibodies block neovascularization, yet both receptors are involved in endothelial cell tubulogenesis in vitro (71). Further, CD44, but not RHAMM, is involved in the proliferation of melanoma cells (105), and CD44 mediates adhesion of multiple myeloma cells to HA while RHAMM, but not CD44, is required for their motility on HA substrata (106).

IV. Hyaluronan and Lung Diseases A. HA in Clinical Fibrosis

Clinical studies show that a correlation exists between increased levels of HA in BAL and various interstitial lung diseases (8,51,107,108). For example, an increased recovery of HA in BAL and lung interstitium has been described in adult respiratory distress syndrome (109,110), occupational lung disorders (111), sarcoidosis (112,113), and idiopathic pulmonary fibrosis (114,115), suggesting that HA may serve as an indicator of the activity of interstitial lung disease (8,107,108). However, while HA levels in BAL may be a sensitive marker of lung injury, the underlying molecular mechanisms of its accumulation are still not well understood (8,107,108). Furthermore, the functional role that HA plays in lung injury repair is only beginning to be defined. Repair of the lung following injury involves rapid restoration of tissue integrity and function through complex interactions between humoral factors, cells, and their extracellular matrix (1,8). This repair process occurs in a sequential, yet overlapping, process involving coagulation, inflammation, granulation tissue formation, and reestablishment of normal parenchymal–stroma cell interactions (1,8,116,117). However, in interstitial lung disease the persistence of chronic inflammation promotes fibroproliferation and deposition of extracellular matrix, resulting in deregulated, exaggerated, and unrelenting tissue repair that disrupts normal lung function (1,9,116–121). The early clinical stages of fibrosis are char-

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acterized by a transient accumulation of HA in the lung interstitium that is associated with enhanced accumulation of water, influx of inflammatory cells, and remodeling of the extracellular matrix leading to a net deposition of matrix in the interstitium of lung tissue, and impairment of lung function (3,107,115). B. A Rodent Model of Bleomycin-Induced Lung Fibrosis

In order to dissect the sequel of human lung fibrosis and to characterize the roles of various molecules linked to clinical lung fibrosis, for instance, HA, several animal models have been studied. One of these is the bleomycin-induced lung injury/fibrosis in rats (108). This is an established and well-studied animal model that resembles progressive clinical pulmonary lung fibrosis in key characteristics (108,122,123). Indeed, bleomycin, which is used as an antineoplastic agent, causes similar fibrosis in some, but not all, treated humans (124–126). In this model, lung injury appears to be due to the production and activation of TGFβ (123,127–132) and results from a persistent inflammatory infiltrate, consisting largely of macrophages, as well as chronic extracellular matrix remodeling characterized by increased deposition of fibronectin, collagen, and HA, that leads to the gradual development of pulmonary fibrosis (1,8,107,108,121–123). The histology and pathophysiologic changes observed in rodents after bleomycin treatment include the death of alveoli epithelial cells, initial influx of neutrophils followed by macrophages into the injured area, and enhanced deposition of the above extracellular matrix components within the interstitium and alveolar space (1,52,116,123,132–135). HA dramatically accumulates in the alveolar interstitium and BAL acutely after bleomycin insertion into the lung (107,136,137). These elevated concentrations of HA, as in clinical lung fibrosis, are temporally correlated with edema and the influx of inflammatory cells into the lung interstitium (52,107,115,132). Therefore, a possible consequence of abnormally high accumulation of HA within the alveolar interstitial tissue is immobilization of water, which would contribute to interstitial lung edema (58,107). AMs appear to be key mediators of bleomycin-induced fibrosis, and thus the mechanism by which macrophages accumulate at injury sites has received considerable attention (51,107,138). The mechanism(s) by which HA accumulates within the lung following bleomycin insertion are not yet well characterized. Since a major source of HA in lung tissue is likely lung fibroblasts (8,108,114,139,140), to clarify the role of both lung fibroblasts and alveolar cells activated by pro-inflammatory growth factors in HA production following bleomycin insertion, temporal HA accumulation in both the BAL and the supernatant medium of cultured lung fibroblasts, isolated at various times after bleomycin exposure, were compared (108). Levels of HA in the fibroblast-conditioned medium were significantly higher than those from saline-injected controls 1 day after bleomycin insertion (108). HA produc-


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tion by the lung fibroblasts peaked 3 days after exposure to bleomycin and gradually declined to near control levels by day 14 (51,108). These results suggest that interstitial lung fibroblasts activated in the presence of bleomycin produced the majority of HA that accumulated in the lung during the very early stages of pulmonary fibrosis (108). In contrast, HA levels were significantly increased in the BAL only after 3 days following bleomycin insertion into the lung (108). These HA levels peaked at day 7, later than HA produced by lung fibroblasts in vitro, and returned to control levels by day 28 (108). These combined results suggest either that HA produced by lung fibroblasts moved from the interstitium to reach the alveolar space or that HA is produced by alveolar cells, for example, type II epithelial cells (108,141). HA accumulation within injured vs. normal lung can also be modified by alterations in the uptake and degradation of HA (107). Resident AMs are the major cell type known to internalize HA for degradation in normally functioning lungs (107,142,143). Interestingly, bleomycin-treated and control AM express different amounts of active HA-binding sites during early stages of lung fibrosis (107,144). For example, the binding of [ 3 H] HA to AM from bleomycin-treated rats is decreased by eightfold 1 day following bleomycin insertion and 15-fold 8 days after injury, compared to AM taken from saline-injected lungs (107). Binding levels of AM taken from bleomycin-injured lungs returned to normal 14 days after the injury (107). Concomitant with reduced binding, both internalization and degradation of labeled HA were also decreased (107). Therefore, impaired uptake of HA by AM probably contributes to the enhanced accumulation of HA during bleomycin-induced lung fibrosis (107). AM cells have been reported to express both CD44 and RHAMM, and since both receptors are involved in internalization of HA in other cell types (89; http://www.glycoforum.gr.jp11), these cellular hyaladherins may play key roles in regulating the accumulation of HA within the injured lung tissue. CD44 in particular mediates uptake of HA and delivery of this polysaccharide to the lysosome for degradation (http://www. glycoforum.gr.jp11).

V.

A Role for HA in Regulating Leukocyte Trafficking and Macrophage Activation

HA potentially plays several roles regulating leukocyte trafficking and activation during lung fibrosis, depending on its molecular weight (35,36). As noted above, high-molecular-weight HA protects key extracellular matrix proteins, such as elastin, from degradation into fragments that are chemoattractants for circulating leukocytes (10–13). HA oligomers do not possess this property, and therefore fragmentation of HA would permit more rapid generation of chemoattractive peptides. Furthermore, HA fragments are themselves chemoattractants. HA frag-

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ments also promote the expression of chemokines and growth factors by macrophages that regulate leukocyte trafficking and activation (28–34). In a similar paradigm of disparate biological functions, high-molecular-weight HA suppresses the angiogenesis that accompanies tissue injury whereas HA oligomers promote this process (25). This dual nature of HA is key to understanding the role of HA in response to injury processes. Clinical pulmonary fibrosis is typically accompanied by chronic tissue inflammation (1,122). Similarly, in bleomycin-injured lung tissue, a significant influx of inflammatory cells, in addition to AM, occurs as early as 1 day following bleomycin insertion (108,132,144,145). The accumulation of inflammatory cells at sites of injury requires that they be activated, followed by their adhesion to vessel endothelium and subsequent extravasation into the injured tissue. HA likely plays a role in this recruitment since it is involved in monocyte activation (26,146) and leukocyte adhesion as well as extravasation (147–149), and monocyte and smooth muscle cell migration following injury (51,150,151). For instance, Savani et al. found that after treatment with bleomycin, and concomitant with an increase both in the HA and macrophage content, the motility of AM was increased ex vivo (51). Treatment of AM with an HA-binding peptide significantly reduced macrophage motility ex vivo and decreased the accumulation of AM in the injured lung tissue (51). Importantly, treatment with peptide limited bleomycin-induced collagen deposition in the lung as well. These results are consistent with a key role for AM in matrix remodeling and fibrosis (96,152–154) and provide direct evidence for a role of HA in regulating macrophage accumulation at sites of lung injury in vivo. Activated macrophages release a variety of factors that amplify the inflammatory process including chemokines, reactive oxygen and nitrogen species, proteases, cytokines, and growth factors (155). Small-molecular-weight HA (2 ⫻ 10 5 daltons) regulates the expression of some of these mediators, for instance, stimulating the expression of IL-1β and TNFα (156,157) in macrophage cell lines. High-molecular-weight HA (⬎1 ⫻ 10 6 daltons) does not have this same effect on macrophages. More recently, small HA forms have been shown to promote the expression of additional macrophage mediators, including chemokines such as macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), cytokine responsive gene-2 (crg-2), monocyte chemoattractant protein-1, and murine regulated on activation, normal T-cell expressed and secreted (RANTES). These factors have been implicated in the pathogenesis of inflammatory disorders (146,158–160). To begin to assess the mechanisms by which small HA forms regulate expression of these chemokines, the minimum HA fragment able to elicit a response was determined (146). It was found that HA fragments as small as hexamers are sufficient to induce expression of these genes in a mouse AM cell line (MH-S) (146). These results are consistent with HA acting through a cellular hyaladherin, which requires at least a hexamer


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for detectable binding to occur. Indeed, an anti-CD44 monoclonal antibody inhibited the binding of HA to these cells and significantly decreased HA-induced gene expression (146). Further, it is likely that CD44-regulated signaling pathways, such as discussed in Section III.A and diagrammed in Figure 4, activate specific transcription factors that mediate expression of these pro-inflammatory factors. An example of an HA-activated transcription factor is NF-κB/IκB, a transcriptional regulatory system shown to be an essential component of the inflammatory response (161). Noble et al. found that stimulation of macrophages with HA fragments resulted in an activation of NF-κB/DNA binding (162). HA fragments also resulted in an increase in the mRNA levels of IκB, and restoration of IκB protein levels, which were initially decreased, in a time course consistent with an autoregulatory mechanism (162). In vitro, translated murine IκB inhibited HA-induced NF-κB/DNA binding activity (162). Conversely, stimulation of the macrophages with high-molecular-weight HA and other GAGs did not result in this induction (162). NF-κB inhibitors or mutation of the proximal NF-κB binding site blocked the induction of iNOS by HA fragments (26). NF-κB mediates the expression and regulation of a large number of inflammatory genes, and so the ability of HA fragments to induce this transcriptional regulator provides further evidence that HA may be an important regulator of macrophage activation at sites of chronic inflammation and fibrosis. HA fragments have also been shown to induce the same pro-inflammatory mediators in human lung macrophages from idiopathic pulmonary fibrosis patients ex vivo (146). HA fragments can synergize with cytokines such as IFN-γ to promote expression of key mediators, such as iNOS (26), Mig, and IP-10, which regulate T-cell recruitment and angiogenesis (32), and both IFN-γ and IL-10, which inhibit HA-induced expression of MIP-1α and MIP-1β (32). These results suggest that HA participates in an intricate cobweb of positive and negative regulation of macrophage activation that permits a delicate control of the response-to-injury process. Clearly, during chronic inflammation, regulation of this balance is lost. For instance, while normal AM cells express only minimal levels of iNOS in response to HA fragments, inflammatory AM cells show a significant induction of iNOS expression (26).

VI. Summary The role of HA in normal and injured lung is still unfolding. At present, data suggest that high-molecular-weight HA in both the alveoli and the interstitium plays an important hydrodynamic role both in maintaining the integrity of the superfactant layer and water homeostasis within the interstitium. As well, highmolecular-weight HA may contribute to the regulation of inflammation by suppressing angiogenesis, monocyte activation, and degradation of extracellular ma-

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trix molecules to fragments that can act as leukocyte chemoattractants. When high-molecular-weight HA is degraded into smaller fragments, its hydrodynamic properties are lost and its previously latent ability to promote signaling is activated, resulting in macrophage activation and the production of factors that attract additional leukocytes to lung tissue (Fig. 5). HA and pro-inflammatory cytokines such as IFN-γ may work together to alter chemokine gene expression during inflammation, and, conversely, the effect of HA fragments on macrophage effector functions may be negatively regulated by cytokines such IFN-γ and IL10. These opposing effects likely provide one of many mechanisms to limit the extent of the pro-inflammatory response permitting return of lung tissue to homeostasis. Disease ensues when these regulatory mechanisms are lost. As an example, the HA-binding ability of macrophages isolated from bleomycin-treated lungs to HA differs from macrophages isolated from untreated lungs, and this will impact upon signaling and consequent generation of leukocyte mediators. For instance, some of the pathology resulting from the bleomycin injury can be prevented by

Figure 5 A model of HA function in lung response to injury and chronic inflammation. In the normal lung, high-molecular-weight HA may function in part to suppress monocyte activation as well as to sustain water homeostasis and the integrity of the surfactant layer. Following injury, fragmentation of HA is proposed to promote activation and recruitment of macrophages and other leukocytes to the lung through multiple direct (signaling) and indirect (elastin degradation) actions. HA fragments can also synergize with specific cytokines (see text) to regulate macrophage activation; conversely, combinations of cytokines can inhibit specific signaling functions of HA fragments. These regulatory loops likely permit the injured lung tissue to return to homeostasis. Persistent insult blocks return to homeostasis and likely involves loss of these regulatory loops. HA-regulated signaling contributes to the maintainance of chronic inflammation and fibrosis, since exposure to an HA-binding peptide prevents the development of fibrosis (e.g., bleomycin injury).


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hyaluronan-binding peptides that likely compete with hyaladherins for HA, thus blocking signaling by HA (51). Although much remains to be learned about the role of HA in lung disease, studies to date suggest that the development of reagents to regulate HA production, fragmentation, and/or interaction with hyaladherins may represent an effective clinical approach to controlling persistent inflammation in lung tissue. Figure 5 summarizes this model.

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nary fibrosis. A potential mechanism for the recruitment and activation of neutrophils in lung fibrosis. J Clin Invest 1991; 88(6):1802–1810. Gauldie J, Jordana M, Cox G. Cytokines and pulmonary fibrosis. Thorax 1993; 48(9):931–935. Nathan CF. Secretory products of macrophages. J Clin Invest 1987; 79(2):319– 326. Noble PW, Lake FR, Henson PM, Riches DW. Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha– dependent mechanism in murine macrophages. J Clin Invest 1993; 91(6):2368– 2377. Boyce DE, Thomas A, Hart J, Moore K, Harding K. Hyaluronic acid induces tumour necrosis factor-alpha production by human macrophages in vitro. Br J Plast Surg 1997; 50(5):362–368.

7 Hyaluronan in Malignant Mesothelioma

ANDERS L. THYLEŃ and GUNNAR MARTENSSON Sahlgrenska University Hospital Go¨teborg University Go¨teborg, Sweden

I.

Introduction

Malignant mesothelioma is an uncommon fatal tumor that can originate from mesothelial cells of the pleura, peritoneum, pericardium, or tunica vaginalis testis. The most common type is malignant pleural mesothelioma (MPM) which often, though not always, is present as a pleural effusion. A pleural tumor probably a MPM was histologically described as early as 1970 by Wagner (1). The multipotent ability of the malignant mesothelial cells results in varied histological tumor patterns, such as epithelial, sarcomatous, or mixed (2). This causes diagnostic difficulties especially in distinguishing MPM from adenocarcinomas. Access to tissue specimens combined with immunohistochemical staining is thus shown to be of great importance in the diagnosis of MPM (3). MPM being a sarcoma would display an immunophenotype pattern that differs from adenocarcinomas. The sarcomatous origin of the tumor also results in production of extracellular matrix substances such as hyaluronan. Cytological examination of pleural fluid is less sensitive than examination of tissue specimens but can be improved with the use of electron microscopy (4). An association between malignant mesothelioma and exposure to asbestos was first described by Wagner et al. in 1960 (5). All types of asbestos, with the 135

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possible exception of anthophylite, can cause malignant mesothelioma (6). The widespread use of asbestos is due to its high tensile strength, stability, and insulation from heat and electricity. Apart from asbestos other possible etiologic factors include live polio vaccine contaminated during the 1950s and 1960s with simian virus 40 (SV40) (7), exposure to erionite (fibrous zeolite) (8), irradiation (9), genetic susceptibility (10), and spontaneous disease. The number of patients with malignant mesothelioma diagnosed in a region depends on both the local use of asbestos and the use of accurate diagnostic procedures. II. Hyaluronan Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of D-glucuronic acid and N-acetyl D-glucosamine. It is a highmolecular-weight polymer and has a molecular weight that varies between 10 6 and 10 7 daltons. The HA chains are very long and can span several microns in length (11). Great water-binding properties can make the molecule very hydrated. HA is a connective tissue component and is involved in a variety of physiological processes, such as tissue repair, angiogenesis, and cell growth (12). The substance is synthesized by many cells at the cell membrane and deposited in the extracellular matrix of most connective tissues throughout the body (13). A large amount of HA is degraded locally or eliminated by lymphatic tissue (14). The remaining amount enters the circulation where it is degraded by the liver endothelial cells (15). A healthy adult has serum HA levels of 10–100 µg/L (16). III. HA in Malignant Mesothelioma Raised level of HA in pleural fluid from a patient with malignant mesothelioma was initially reported by Meyer and Chaffee (17) in 1939. HA is synthesized at the cell membrane in about two out of three cases of MPM. The ability to synthesize HA is a sign of cell differentiation and as such associated with a more highly differentiated tumor cell especially of the epithelial cell type, even if HA production is found in all histological types. IV. HA Analysis A. Histochemical Methods

Histochemical demonstration of HA in MPM has been attempted. Alcian Blue or colloidal iron staining has been used to demonstrate the presence of HA in tissue samples (18). However, this staining has a low sensitivity.

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Another diagnostic tool used on tissue samples was described by Azumi in 1992 (19). A probe that specifically binds to HA in formalin-embedded tissue was evaluated for its sensitivity and specificity as a diagnostic adjunct to other diagnostic methods. In a study group of 33 patients with MPM and 37 with adenocarcinomas, moderate to strong membrane or cytoplasmic staining was observed in ⬎30% of the MPM tumor cells. The biotinylated probe specific for HA was shown to have a sensitivity of 79% and a specificity of 92%. The staining pattern described was seen in all major histologic subtypes of MPM. B. Other Methods

Other methods with varying specificity and sensitivity have been used to analyze HA in pleural fluid. One older quantitative/semiquantitative method determined HA by studying the electrophoretic migration pattern of plasma proteins in the fluid before and after the addition of hyaluronidase (20), and had a sensitivity of 30% to detect MPM (21). Two modern methods, a radioassay (RA) and a highperformance liquid chromatographic (HPLC) method, have significantly higher sensitivity toward MPM. The RA method (22,23) utilizes the interaction between HA and the HA-binding region of cartilage aggrecans and can be used on both serum and pleural fluid. Two different HPLC methods with equivalent results (24,25) have been used on pleural and peritoneal fluid. The HPLC method described by Hjerpe (25) identifies a HA-derived disaccharide. Both the RA and HPLC methods are very sensitive, but the HA-binding reagent used in the RA method can also interact with other carbohydrates, such as in the bacterial capsule, thereby giving false-positive results in infectious effusions. When simultaneously performed on 21 patients, the HPLC method recognized 70% and the RA 64% recognized of MPM patients using a HA cutoff level of 100 mg/L pleural fluid (26). In a retrospective study of 100 cases of MPM, the HPLC method recognized 62% of the cases (27). The HPLC method has been evaluated with regard to both specificity and sensitivity in a study of 1610 patients (28)—1039 patients with pleural fluid and 571 with peritoneal fluid of whom 43 had pleural and 7 peritoneal malignant mesothelioma. With a HA cutoff level of 225 mg/L the specificity for malignant mesothelioma was 100% and the sensitivity was 56%. The HPLC method described by Roboz showed a pleural fluid HA content above 250 mg/L in 13 of 14 MPM patients while no such levels were noted among controls (24). Studies of HA production in cell cultures of MPM may show conflicting results as this capability could be lost during generations of cell culturing (29). V.

Alterations of HA in Serum

Frebourg reported significantly increased levels of HA in serum among patients with MPM in comparison to patients with pleural effusion due to other causes

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or healthy adults (30). Dahl studied serum HA in seven groups of patients with untreated and well-defined malignancies (MPM, sarcomas, lymphomas, breast carcinomas, brain tumors, lung cancers, and a group of various malignancies) (31). Three control groups were included: patients with radically operated malignant tumors; benign pulmonary disease; and benign tumors. The concentration of HA was significantly raised in the MPM group as compared to healthy adults. An elevated content of HA was also seen in a few patients seriously ill owing to other malignancies. This finding is probably explained by decreased clearance of HA (22,23). VI. Immunohistochemical Characterization of HA-Producing Malignant Pleural Mesothelioma Immunohistochemical differences were studied in 33 patients with biopsies of proven MPM, all without reactivity for monoclonal carcinoembryonic antigen (CEA) (32). MPM should not show CEA reactivity, and a finding of CEA reactivity would contradict the diagnosis. Among the 33 patients, 23 had a pleural fluid HA level larger than 100 mg/L and the remaining 10 had levels lower than 100 mg/L. Immunohistochemical reactivity is presented in Table 1 and categorized into those showing more or less than 50% of all cells with reactivity for a certain antibody. The antibodies studied were vimentin, epithelial membrane antigen (EMA), and antibodies to cytokeratin (CAM 5.2). Vimentin reacts above all with the sarcomatoid subtype or sarcomatoid part of the mixed type of MPM. EMA

Table 1 Immunohistochemical Reactivity to Vimentin, CAM 5.2, and EMA Versus HA-Producing or Non-HA-Producing Malignant Mesotheliomas Pleural fluid HA level

Vimentin ⬍50% immunopositive ⬎50% immunopositive CAM 5.2 ⬍50% immunopositive ⬎50% immunopositive EMA ⬍50% immunopositive ⬎50% immunopositive

⬍100 mg/L (N ⫽ 10)

⬎100 mg/L (N ⫽ 23)

cells cells

3 7

16 7

cells cells

4 6

2 21

cells cells

7 3

6 17


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reacts mainly with the epithelial and mixed subtypes, whereas CAM 5.2 reacts with all three subtypes of MPM. The results show that regardless of histological subtypes, MPM cases with HA levels ⬎100 mg/L showed a significantly greater reactivity toward EMA (P ⬍ .05), a greater reactivity toward CAM 5.2 (P ⫽ .05), and a lower reactivity against vimentin (P ⫽ .06). VII. HA as a Prognostic Factor in MPM Previously epithelial histology (21,33–36) and good performance status (35,36) have been shown to be favourable prognostic factors in many studies. The prognostic significance of pleural fluid HA levels ⬎100 mg/L as compared to HA levels ⬍100 mg/L was evaluated in a study of 100 cases of biopsy-verified MPM (24). The median age of these patients was 64 years with a median survival of 11.5 months. A significant unfavorable prognosis was noted in patients with pleural fluid HA ⬍100 mg/L as compared to HA ⬎100 mg/L in a univariate analysis. In a multivariate analysis pleural fluid HA ⬎225 mg/L as compared to HA ⬍225 mg/L had a favorable prognostic impact. An explanation to this finding could be that the ability of HA production with regard to tumor biology should be seen as a sign of higher differentiation. VIII. Serum HA Versus Tumor Volume A healthy adult shows serum HA levels ⬍100 µg/L (16). In a report by Dahl (37) a positive correlation was noted between changes in serum HA levels and changes in tumor volume. However, this finding was not consistent, as two patients with tumor progression did not show rising serum HA levels. An explanation for this could be that all cases were not characterized as HA-producing or non-HA-producing MPM by pleural fluid analyses. In a prospective study of 19 MPM patients (38), all with previous pleural fluid HA analysis, repeated serum HA samples and simultaneous serially performed CT scans were performed. The HA analyses were done with the RA method. Tumor volume was calculated on the CT scans by means of a digital planimeter. This study showed a correlation between the content of HA in pleural fluid and the initial serum HA level. Furthermore, an increase in tumor burden was followed by rising levels of serum HA in HA-synthesizing MPMs. This correlation was not found in non-HA-producing MPMs. IX. Conclusions In ⬃65% of all cases, MPM is associated with an elevated content of HA in pleural fluid. HA may serve as a marker in the management of the disease. Reli-

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able methods for analysis of HA are available. The sensitivity of pleural fluid HA analyses as a means of diagnosing MPM is ⬃60–65% with a specificity of 100%. HA-synthesizing MPMs differ immunohistochemically from non-HAsynthesizing MPMs. One such difference relates to survival; others might be of importance for response to various treatments. References 1. Wagner E. Das tuberkelahnliche Lymphadenom. Arch Heilk 1870; 11:495– 525. 2. Klemperer P, Rabin CB. Primary neoplasms of the pleura. A report of five cases. Arch Pathol 1931; 11:385–412. 3. Boutin C, Rey F. Thoracoscopy in pleural malignant mesothelioma: a prospective study of 188 consecutive patients. Part 1: diagnosis. Cancer 1993; 72:389–393. 4. Martensson G. Diagnosing malignant pleural mesothelioma. Eur Respir J 1990; 3: 985–986. 5. Wagner JC. Diffuse pleural mesothelioma and asbestos exposure in the North-West Cape Province. Br J Ind Med 1960; 17:260–271. 6. Meurman LO, Kivilouto R, Hakama M. Mortality and morbidity among the working population of anthophyllite asbestos miners in Finland. Br J Ind Med 1974; 31:105. 7. Pepper C, Jasani B, Navabi H, Wynford-Thomas D, Gibbs AR. Simian virus 40 large T antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue. Thorax 1996; 51:1074–1076. ¨ zesmi M, et al. An outbreak of pleural mesothelioma and 8. Baris YI, Sahin AA, O ¨ rgru¨p in Anatolia. Thorax 1978; chronic fibrosing pleurisy in the village of Karain/U 33:181–185. 9. Cavazza A, Travis LB, Travis WD, Wolfe JT III, Foo ML, Gillespie DJ, Weldner N, Colby TV. Post-irradiation malignant mesothelioma. Cancer 1996; 77:1379–1385. 10. Ma˚rtensson G, Larsson S, Zettergren L. Malignant mesothelioma in two pairs of siblings: is there a hereditary predisposing factor? Eur J Respir Dis 1984; 65:179– 184. 11. Waxler B, Eisentein R, Battifora H. Electrophoresis of tissue glycosaminoglycans as an aid in the diagnosis of mesotheliomas. Cancer 1979; 4:221–227. 12. Comper WD, Laurent TC. Physiological function of connective tissue polysaccharides. Physiol Rev 1978; 58:255–315. 13. Prehm P. Hyaluronate is synthesized at plasma membranes. Biochem J 1984; 220: 597–600. 14. Laurent UBG, Reed RK. Turnover of hyaluronan in the tissues. Adv Drug Rev 1991; 7:237–256. 15. Smedsro¨d B, Pertoft H, Eriksson S, Fraser JRE, Laurent TC. Studies in vivo on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells. Biochem J 1984; 223:617–626. 16. Tengblad A, Laurent UBG, Lilja K, Cahill RNP, Engstro¨m-Laurent A, Fraser JRE,


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35. Boutin C, Rey F. Thoracoscopy in pleural malignant mesothelioma: a prospective study of 188 consecutive patients. Part 2. Prognosis and staging. Cancer 1993; 72: 394–404. 36. Hemdon JE II, Green MR, Chahinian AP, Corson JM, Suzuki Y, Vogelzang NJ. Factors predictive of survival among 337 patients with mesothelioma treated between 1984–1994 by the Cancer and Leukemia Group B. Chest 1998; 113:723– 731. 37. Dahl IM, Solheim OP, Erikstein B, Mu¨ller E. A longitudinal study of the hyaluronan level in the serum of patients with malignant mesothelioma under treatment. Cancer 1989; 64:68–73. 38. Thyleń A, Wallin J, Ma˚rtensson G. Hyaluronan in serum as an indicator of progressive disease in hyaluronan-producing malignant mesothelioma. Cancer 1999; 86: 2000–2005.

8 Matrix Proteoglycans in Development of Pulmonary Edema

DANIELA NEGRINI, ALBERTO PASSI, and GIANCARLO DE LUCA

GIUSEPPE MISEROCCHI

University of Insubria Varese, Italy

University of Milano-Bicocca Monza, Italy

I.

The Pulmonary Interstitial Space

The alveolar wall consists of a layer of epithelial cells, their basement membrane, the capillary basement membrane that supports the capillary endothelial cells, and a thin layer of interstitial space lying between the capillary endothelium and the alveolar epithelium. The structure of the lung extracellular matrix (ECM) is unique as it fulfills two main functions. On the one hand it provides a strong and expandable framework supporting the fragile alveolar epithelial-capillary intersection; on the other hand, it provides a low resistive pathway for guaranteeing an effective exchange of the respiratory gasses. Alveolar gas diffusion is optimized through a very large surface area (⬃70 m 2 ) (1) and by the structure of the alveolar-capillary membrane, whose extreme thinness (⬃0.5 µm) essentially depends upon the hydration state of the ECM interposed between the capillary endothelium and alveolar epithelium. The size of the epithelial and endothelial cells accounts for most of the thickness of the alveolar-capillary membrane, the interstitial space being only a thin stratum interposed between the two cell layers. In many places, the two

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Figure 1 Schematic drawing of the structure of alveolar interstitial matrix.

basement membranes are even physically fused to reduce as much as possible the distance for gases to diffuse from the alveolar space to red blood cells. Where the two basement membranes are not fused, the interstitium includes cells (fibroblasts, macrophages, etc.), a macromolecular ECM, and a free liquid phase (Fig. 1). Both the macromolecular fibrous component and the fluid phase of the ECM form a continuum that pervades the entire lung (1), functioning as a three-dimensional mechanical scaffold; the former is ensured by a fibrous mesh consisting mainly of collagen types I and III, which provide tensile strength, and elastin, conveying elastic recoil. The three-dimensional fiber mesh is filled with other macromolecules, mainly hyaluronan (HA) and proteoglycans (PGs), which are the major components of the nonfibrillar compartment of the interstitium. II. Lung Proteoglycans and Hyaluronan PGs include families of multidomain core proteins, which are genetically unrelated and contain one or more covalently linked glycosaminoglycan (GAG) chains, like chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (2,3). The protein component of PGs is responsible for different molecular constructions and functions. So far, ⬎20 genetically different species of core proteins have been identified. With the exception of HA, GAGs are sulfated polysaccharides made of repeating disaccharides (typically 40–100), which consist of uronic acid (or galactose) and hexosamines. GAG sulfation in CS chains is usually regular, one sulfate per disaccharide throughout the chain, while in HS chains, sulfation is somewhat irregular, resulting in intensely sulfated and sparsely sulfated regions on a single GAG chain. In addition to GAGs, PGs normally have other

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carbohydrate units including O-linked and N-linked oligosaccharides, as found in other glycosylated proteins. The most abundant GAG in lung interstitium (0.4 mg/g) is HA, the only GAG without sulfate residues secreted in the extracellular matrix. The HA content of ECM changes during one’s lifetime, dropping from 40% to 4% of the dry lung weight on development from fetal to adult stage. HA is an extraordinary simple but large molecule, reaching 4 ⫻ 10 6 daltons, due to repetition of up to 10,000 disaccharide moieties. These disaccharides are constituted by an uronic acid residue covalently linked to an N-acetyl-glucosamine (β-1,4-GlcUA-β-1,3GlcNAc). In physiological conditions, HA carboxylic groups are ionized and the molecule assumes the shape of a random coil, filling an enormous space, as predicted by the rigid chemical bonds in the HA molecule, by intramolecular hydrogen bonds and by solvent interactions. These structural features are the basis of the HA function as “water content regulator” in tissues. The HA interactions with solvent also explain its action as a molecular sieve, regulating the extracellular traffic of molecules. In tissuerepairing situations, as in former-smoker lungs or after radiation exposure, HA increased in early phase of tissue recovery (4). HA also has a protective function in infective lung disease and in proteolytic activity of granulocyte enzymes, where HA molecules are utilized as protective agents (5). Therefore, pulmonary lesions are often linked to HA metabolism, involving both synthetic and catabolic pathways. In repair processes and wound healing, HA molecules are produced early and can stimulate collagen type I and III synthesis (6). Different PG populations are present in the lung parenchyma. CS-containing PG (CS-PG, versican) is a large molecule with an apparent molecular mass of ⬎1000 kDa, widely expressed in many tissues. It is substituted with 12– 16 chondroitin sulfate chains, many fewer than with aggrecan, and with a number of N- and O-linked oligosaccharides. Its core protein of ⬃500 kDa may form aggregates with HA in the interstitial matrix. Morphological studies of both developing and aging lung parenchyma indicate that versican is present in the alveolar interstitial space surrounding lung fibroblasts and blood vessels in regions not occupied by the major fibrous protein collagen and elastin (3). In both the walls of airways and pulmonary arteries, versican seems to be associated with smooth muscle cells. Versican binds to fibronectin and various types of collagen and inhibits cell adhesion to the matrix (7). It may be involved in differentiation of developing mesenchymal cells and is thought to play a specific role in influencing matrix synthesis by cells, thus favoring wound healing (3). Although CS-PGs represent a minor share of the dry lung weight, they are essential for structural matrix integrity and pulmonary interstitial tissue compliance. PG organization in the ECM is based on noncovalent linkages with other macromolecules, involving mainly low-energy ionic and/or hydrophobic interactions. The polyanionic nature of GAGs is the main determinant of the physical

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properties of PG molecules, allowing them to resist compressive forces and to simultaneously maintain the hydration of the tissue. HS containing PGs (HS-PGs) include perlecan in epithelial basement membrane and syndecan at cells surface (3,8,9). Lung HS-PGs were shown to be localized primarily in the vascular basement membranes (8–10), even though HS-PG-like syndecans and glypicans are more densely arranged along the alveolar than the capillary surface. Three members of the syndecan family (syndecan1/syndecan, syndecan-2/fibroglycan, and syndecan-4/ryudocan [amphyglycan]) are transmembrane HS-PGs specific to the basolateral surfaces of vascular endothelial cells (11). Glypican-1, another cell surface HS-PG, is present on vascular endothelial cells where, like syndecan, it is believed to play an important role in regulating the biological activity of fibroblast growth factors (12). HS-PGs like perlecan, agrin, and bamacan are typical components of the basement membrane. The core protein of human perlecan, 467 kDa, is composed of five domains, suggesting that the core protein carries multifunctional properties, although there is little actual evidence for them. Its HS chains are heterogeneous in size; however, each HS chain averaged 380 kDa in molecular mass and 87 nm in length when examined by electron microscopy (13). In addition to HS chains, perlecan bears a total 20 kDa of N- and O-linked oligosaccharide chains, which are suggested to function in the secretion of perlecan (13). Agrin induces clustering of acetylcholine receptors and acetylcholine esterase at the postsynaptic membrane of neuromuscular junctions; however, it has been shown to be distributed in basement membranes of other cell lines, like in the renal glomerular cell lining (14). Bamacan, a CS-PG of the basement membrane, is localized in various kinds of basement membranes except in the glomerular capillary loop. The core protein of bamacan, 138 kDa, consists of five domains: the amino-terminal globular head; the second rod domain attached with a CS chain and a N-linked oligosaccharide chain; the third globular domain; the fourth rod domain containing two N-linked oligosaccharide chains; and the fifth globular tail with two CS chains. The function of bamacan has not been well elucidated (15). HS-PGs are emerging as important homeostatic mediators at the cell surface and in the pericellular environment. A diverse group of proteins bind HSPGs, including glycoproteins that mediate cell matrix adhesion, growth factors, enzymes and inhibitors, viral coat proteins, and regulatory proteins in the nucleus (7). In addition, HS-PGs interact with fibroblast growth factors (FGFs), thus contributing to the regulation of cell migration, proliferation, and gene expression, and in many developmental and pathological processes including wound healing and angiogenesis. Binding of the HS-PG betaglycan with FGF increases its affinity to cell receptors, protects FGF from proteolysis, and promotes epithelial regrowth on denuded basal lamina (3). HS-PGs in basal lamina of the kidney influence charge-dependent selective filtration properties (16); hence, it is possible


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that HS-PG may also influence the regulation of fluid balance across the alveolarcapillary membrane (3,8). Large amounts of lumican, a keratan sulfate-containing PG expressed in several connective tissues including cornea, muscle, intestine, and cartilage, were immunologically detected in human adult lung (17). Small DS-containing PGs (DS-PG, decorin) are associated with collagen fibrils, playing a role in the final organization of collagen in all tissues (3). Moreover, decorin specifically interacts with TGF-β molecules, and a role in modulating TGF-β activity has been reported in various tissues, including the lung (18). These data clearly indicate that the function of GAGs and PGs in the lung is not limited to the maintenance of the mechanical and fluid-dynamic properties of the organ. These molecules also play a pivotal role in tissue development and recovery after injury, interacting with inflammatory cells, proteases, and growth factors (2,3). ECM is thought to transmit essential information to pulmonary cells, regulating their proliferation, differentiation, and organization. As an example, acute lung injury is associated with an increase in synthesis of HA, CS, and DS, suggesting that PGs influence migration of cells into the damaged area, cellular response to injury, and production of other extracellular components such as collagen or elastin (2,3,8). The structural integrity of pulmonary interstitium largely depends on the balance between the regulation of synthesis and degradation of ECM components. The turnover and remodeling of ECM involves a family of structurally related zinc-dependent endopeptidases, known as matrix metalloproteinases (MMPs), that are active at neutral pH and can collectively degrade almost all components of ECM (19,20). Human MMP genes encompass interstitial collagenases, stromelysins, putative metalloproteases, and gelatinases. In particular, the MMP subfamily of gelatinases (gelatinase A, MMP-2 and gelatinase B, MMP-9) degrade denatured collagen such as gelatin, type IV collagen, fibronectin, and elastin (19,20), and it was recently found that MMP-2 and MMP-9 are able to cleave large CS-PGs isolated from rabbit lung (21). The activity of gelatinases was shown to be involved in pathological process including pulmonary emphysema (22,23), interstitial lung disease (24), and hyperoxia-induced acute lung injury (25,26).

III. Pulmonary Interstitial Pressure In the interstitium, water is partly bound to polyanionic macromolecules like HA or PGs and partly freely moving into the porous mesh of extracellular fibrous macromolecules. These two fluid components are in equilibrium with each other, and both contribute to determine the hydration state of the pulmonary interstitium. Under steady-state conditions, total tissue fluid volume depends on: (1) the net

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rate of fluid filtration across the capillary endothelial layer; (2) the rate of fluid absorption into the draining route provided, in most normal tissues including the lung, by the initial lymphatic system; (3) the polyanionic macromolecules included in ECM; and (4) the local mechanical tissue compliance. The pressure of the free fluid phase is defined as pulmonary interstitial pressure (Pip ); its value depends on the total tissue hydration, as well as on other mechanical factors such as the tissue stress related to lung volume and the alveolar surface tension phenomenon (27,28). In addition, regional differences in Pip ought to be expected on the basis of: (1) the interdependence phenomenon, by which the stress acting on the outer surface of rigid structures (like bronchi and vessels) is greater than at the pleural surface of the lung parenchyma; (2) the gravity-dependent distribution of regional lung expansion; and (3) the complex lung–chest wall mechanical interaction (28). Pip has been measured in anesthetized supine rabbits with lungs physiologically expanded in the intact pleural space through the micropuncture technique (27,28). A glass micropipette, driven by a micromanipulator under stereomicroscopic view, was advanced through the intact parietal pleura exposed after removal of the external and internal intercostal muscles. This experimental approach allowed the recording of Pip in the periadventitial interstitial space of in situ lungs without disrupting the functional integrity of the lung–chest wall coupling. At a given lung volume, Pip is not uniform throughout the whole lung; indeed, as depicted in Figure 2, in supine rabbits Pip decreases as a function of lung height (27) by about 0.7 cmH 2 O/cm of vertical height from the lowermost point of the lung. The relationships referring to hydraulic pressure measured in the pleural cavity (Pliq ) (29,30) and in the extrapleural interstitial space (Pepl ) (30,31) are also reported for comparison. It is evident that, at any lung height, Pip is significantly more subatmospheric than the pressure recorded in the other two adjacent compartments, indicating that the pulmonary parenchyma is a more “dehydrated” compartment than the extrapleural and the pleural ones. Figure 3 presents the dependence of Pip , Pliq , and Pepl from pleural surface pressure (Ppl ) that reflects the degree of regional lung expansion, the more negative pleural surface pressure the larger regional lung volumes. Both Pliq and Pip become more subatmospheric on decreasing Ppl for unit change in the latter (Fig. 3). On mechanical ground, the greater drop observed in Pliq compared to Ppl may be interpreted as a tighter fitting of the opposing pleura during lung expansion, generating deformation pressures at the points of contact between the sliding surfaces (28). Such reasoning may also be applied to pulmonary interstitial space, where increasing lung volume may determine complex mechanical deformations between macromolecules of the tight fibrous matrix. The fact that at a given Ppl , Pip is more negative than Pliq indicates that lung expansion determines greater deformation pressures in the pulmonary interstitium compared to pleural space.


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Figure 2 Hydraulic pressures recorded by micropuncture in the extrapleural parietal interstitium (Pepl ), in the costal pleural space (Pliq ), and in the perimicrovascular pulmonary interstitium (Pip ) are plotted as a function of lung height. Data were collected in supine rabbits at a lung volume corresponding to the functional residual capacity.

At variance with what is observed in the intact lung, in most isolated lung preparations Pip is less negative or essentially equal to Ppl . This indicates that Pip is not only dependent on the mechanical stress applied to the pulmonary interstitium, but it is instead primarily set by the mechanisms controlling the interstitial fluid turnover. IV. Microvascular Fluid Exchanges Filtration of fluid (J v ) between the visceral capillary (c) and the pulmonary interstitium (i) across the pulmonary endothelium considered as an homogeneously porous membrane may be calculated on the basis of the thermodynamic modification of the Starling law as: J v ⫽ L p ⋅ S ⋅ ∆Pnet

(1)

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Figure 3 Pepl , Pliq , and Pip plotted as a function of pleural surface pressure (Ppl), the latter becoming more subatmospheric on increasing lung volumes. Data collected in supine rabbits at right atrium level.

where L p is the hydraulic filtration coefficient of the membrane, S its surface area, and L p ⋅ S ⫽ K f is the membrane filtration coefficient. The net pressure gradient across the membrane, ∆Pnet , is given by: ∆Pnet ⫽ [(Pc ⫺ Pi ) ⫺ σ(π c ⫺ π i )]

(2)

where P and π represent the hydraulic and colloidosmotic pressures of plasma in the pulmonary capillary (subscript c) and of pulmonary interstitial fluid (subscript i), respectively. The reflection coefficient of the endothelium for total proteins (σ) is a correction factor depending on the ratio between the protein molecular radius and endothelial pore size. On the basis of Eq. (2) one may calculate ∆Pnet at any lung height and at a given lung expansion in various experimental conditions, beginning with the physiological one. Direct measurements of the hydraulic pressure in the arteriolar and venular superficial microvascular network, performed through micropuncture in the intact normal lung (32), allow one to extrapolate an average functional capillary pressure (Pc ) of ⬃10 cmH 2 O. As shown in Figures 2 and 3 at a lung volume corresponding to FRC and at the level of the right atrium (⬃3 cm from the lowermost lung height in rabbits), Pip is ⬃ ⫺10 cmH 2 O. π i is a critical parameter in determining transendothelial fluid flux and a key factor in setting pulmonary tissue hydration either in control conditions or during edema development. The presence of collagen and other large macromolecules like HA and PGs limits the interstitial space accessible to solutes dispersed


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in the interstitial fluid, in particular to plasma proteins. Indeed, PGs establish water-filled compartments, only part of which are available to other ECM components, thus constituting a selective sieve of various pore size and charge density. The result of this phenomenon, called “volume exclusion,” is that the protein concentration of the free interstitial fluid is higher than that expected on the basis of the extravascular plasma protein quantity and interstitial fluid volume. In the normal lung, plasma proteins are excluded from 66% of total interstitial volume (33), a value much higher than that observed in muscle and skin (33,34), suggesting that the composition and structural organization of pulmonary ECM differ substantially from more compliant tissues. In normal rabbit lung, protein concentration measured with the implanted-wick technique averages 2.7 g/dL (35) yielding a π i value of ⬃12.5 cmH 2 O. As shown in Figure 4A, by considering a plasma colloid-osmotic pressure of ⬃30 cmH 2 O and assuming an average σ value of 0.8, the ∆Pnet value calculated according to Eq. (2) across the capillary endothelium is ⬃8 cmH 2 O. Thus, in normal conditions, because of the microvascular hydraulic

Figure 4 Schematic drawing of the pulmonary capillary and the surrounding interstitium including the corresponding hydraulic (P) and colloidosmotic (π) pressure values at right atrium level. Bold numbers indicate the filtration transendothelial pressure gradient (∆Pnet ) calculated from Eq. (2) for an average reflection coefficient of the endothelium for total proteins (σ) of 0.8. Black arrows depicts qualitatively the direction and entity of transendothelial fluid fluxes. In mild edema ∆Pnet and corresponding fluid fluxes are nil. White arrows indicate pulmonary lymphatic flow direction. In severe edema ∆Pnet is about one-fourth of control but filtration flux is higher, reflecting an increase in microvascular permeability. (A) Physiological conditions; (B) mild hydraulic interstitial edema; (C) severe hydraulic edema.

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pressure profile, pulmonary capillaries provide net filtration into the perivascular interstitial space even at their venular end. Hence, even though the hydraulic pressure in the pulmonary capillaries is lower than in the systemic ones, in the normal lung the pulmonary circulation does not provide a route for drainage of interstitial fluid. As in all other tissue, except in the brain and in the renal medulla, where the lymphatics are absent, such a role is played by the lymphatic system. The latter provides the most efficient mechanism for fluid drainage and the only mechanism for macromolecules removal from the lung tissue under physiological conditions, acting as an efficient feedback system to prevent pulmonary edema. Interstitial fluid volume is maintained at a minimum, as demonstrated by the fact that Pip is very negative (⫺10 cmH 2 O) (27,28). This pressure is set by the initial lymphatics and forces liquid to filter out of the pulmonary capillaries across the endothelial barrier, whose permeability to water is very low (36). Hence, under physiological conditions, the hydration state of the lung parenchyma is minimized (wet weight to dry weight ratio ⫽ ⬃4.8 g/g dry tissue weight) by the low microvascular filtration permeability and by the powerful draining action of the pulmonary lymphatics. No direct recording of the net lymphatic absorption pressures and flows has been attained yet in the physiologically expanded lung. However, the features of the lymphatic system have been extensively studied in the adjacent pleural cavity. It has been shown that initial pleural lymphatics are able to sustain pressures of ⬃ ⫺20 cmH 2 O (37), even lower than pulmonary interstitial pressure. This evidence suggests that the pulmonary lymphatic system might also be able to generate an interstitial to lymphatic pressure gradient favoring removal of fluid and solute from the pulmonary parenchyma. In several studies performed on the pleural space (see 38 for a review), it has been pointed out how the lymphatic system is well suited to drain small amounts of water in conditions close to the physiological ones, thus generating a subatmospheric tissue fluid pressure and keeping the interstitial space dry. As shown by data obtained in the rabbit, a similar model of maintenance of tissue fluid balance may be applied to the normal lung. The choice of the rabbit as an experimental animal model was motivated by the possibility offered by this species to approach the lung with the least invasive micropuncture technique, allowing one to obtain most of the parameters appearing in Eq. (2). This model may be extended to the other mammals whose parameters are similar to those reported for rabbits. In the face of an increased microvascular filtration rate, the lymphatic flow may adapt to the increased tissue hydration by augmenting its egress flow by up to 10–15 times, a flow rate above which lymphatics saturate (38). Above the saturation threshold, notwithstanding a maximized lymphatic drainage, an imbalance is achieved between capillary filtration and subsequent fluid removal and edema develops.

Matrix Proteoglycans V.

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Pip in the Transition Toward Hydraulic and Lesional Edema

Pulmonary edema is associated with various types of pathologies and may be defined as an abnormal, diffuse extravascular accumulation of fluid in the pulmonary tissue and air spaces due to changes in the hydraulic and colloidosmotic pressures appearing in Eq. (2) (in case of hydraulic edema) or to increased capillary permeability to water and solutes (in case of lesional edema). In order to study the early transition phase from the normal physiological condition to full edema development, experimental models of various type of pulmonary edema have been used: 1. Hydrostatic edema, induced by slow [0.5 mL/(kg ⋅ min)] infusion of saline solution into the jugular vein, maintained for up to 3 h (39,40). 2. Permeability edema caused by endothelial lesion induced by intravenous addition of pancreatic elastase [200 µg (41)]. 3. Acute hypoxia, induced in anesthetized rabbits spontaneously breathing a gas mixture of 12% O 2 in N 2 (42). Figure 5 shows the Pip pattern during development of either hydraulic (saline infusion) or lesional edema (elastase treatment). The abscissa expresses the amount of extravascular water as indexed by the wet weight to dry wet weight ratio of the lung (W/D ratio). In both models of edema, Pip increased from the physiological negative pressure of about ⫺10 cmH 2 O (data recorded at heart level) to positive values subsequently dropping towards zero as edema progressed. Saline infusion caused plasma expansion, a mild increase in cardiac output, and a decrease in total pulmonary vascular resistance, coupled with a significant increase in percentagewise resistance in the precapillary segment (43). Hence, by using the saline load chosen for this slow-developing experimental hydraulic edema, the functional capillary pressure remained essentially unaltered, compared to physiological conditions, at about 10 cmH 2 O (43). Plasma protein concentration progressively decreased as a result of saline load (35). Elastase leads to an increased fluid filtration into the parenchyma by causing an increase in permeability to water caused by the damage to the endothelial wall; indeed, elastase is an omnivorous proteolytic enzyme with broad affinity for a variety of soluble and insoluble protein substrates, including ECM components. At variance with what is observed during saline loading, elastase treatment results in an increase in the interstitial protein concentration with respect to plasma (35). Data presented in Figure 4B show the pressures involved in transcapillary fluid flux at the occurrence of mild interstitial edema during saline loading. The significant increase of Pip to positive values (Fig. 5) is the major factor that tends to reduce or even nullify the pressure gradient sustaining filtration across the pulmonary endothelium. In this sense the increase in Pip prevents further fluid

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Figure 5 Pulmonary interstitial pressure (Pip ) measured at heart level in intact rabbit lung plotted as a function of the wet weight to dry weight lung ratio (wet/dry ratio) during development of hydraulic (dashed line) or lesional (continuous line) edema induced by IV injection of elastase. In both models edema development is associated with an increase in Pip from the negative control value to positive; Pip subsequently drops to zero as edema progresses.

filtration and accumulation, acting like a safety factor against the attainment of a more severe stage of edema. The extent of the increase in Pip during edema development depends on the mechanical features of the extracellular matrix or, more specifically, on its mechanical compliance. It is evident that, as edema progresses, Pip changes according to a biphasic pattern: the slope of the curves shown in Figure 5 has the dimension of a pressure change (∆Pip ) divided by a fluid volume change ∆(W/ D); hence, it represents the reciprocal of the mechanical compliance of the pulmonary tissue matrix. The compliance is very low [⬃0.5 mL ⋅ mmHg⫺1 ⋅ 100 g wet wt⫺1 (39)] in the intact normal lung and for hydration states close to the physiological ones. The lower the compliance, the more positive ∆Pip will become for a given increase in tissue water content, thus opposing further filtration into the


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interstitium. From this mechanical standpoint, the pulmonary parenchyma is much more protected against fluid accumulation leading to interstitial edema than are other organs, such as intercostal muscles, whose mechanical compliance is up to 20 times higher (39). From the similarity of the curves shown in Figure 5, the “mechanical tissue factor” represented by the low tissue compliance seems to play an important role both in development of hydraulic and lesional edema induced by elastase. The compliance increases abruptly when tissue hydration increases over a W/D ratio greater than ⬃ 5.5 g/g dry lung weight (Fig. 5), resulting in a large increase in tissue fluid content in face of a small Pip variation: the matrix loses its physiological protective role to counteract fluid filtration, leading to rapid deterioration towards severe pulmonary edema and alveolar flooding. Hence, the pressure behavior presented in Figure 5 indicates that the evolution from mild interstitial to severe pulmonary edema is strictly associated with a change in the structural and mechanical properties of ECM and endothelial basement membrane. Indeed, severe edema eventually develops in spite of a transendothelial ∆Pnet which is reduced with respect to control condition (Fig. 4C), suggesting that an increase of the endothelial wall permeability is achieved at this stage of edema development.

VI. Lung Proteoglycans During Developing of Interstitial Edema A biochemical analysis of the composition, structure, and interaction of PGs with other macromolecular structure shows that pulmonary PGs play a key role in controlling fluid balance. Analysis of lung PG composition and structure was performed in control rabbit lung and after induction of hydraulic or lesional edema. Immediately after death and exhaustive bleeding of the animals, lungs were removed, washed with PBS containing a cocktail of protease inhibitors (40,41), and cut into small slices, separating the pulmonary parenchyma from the terminal bronchioles. Tissue specimens were then treated with increasing concentrations of GuHCl, which breaks intermolecular noncovalent bonds and allows PG extraction (44). The total recovery of PGs after sequential extraction of lung specimen with GuHCl increased with the extent of edema development by 11% and 62% in hydraulic edema (40) and by 13% and 25% in lesional edema (41), respectively, reflecting an easier extraction of PGs from the matrix and thus suggesting a progressive loosening of PG intermolecular bonds with other ECM components. The molecular size distribution of 125 I-PGs extracted from lung tissue was analyzed by gel filtration chromatography performed under dissociative conditions. The elution pattern of the radiolabeled material isolated from control lungs identified

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Figure 6 Gel filtration chromatography performed under dissociative conditions on proteoglycans (PGs) isolated from 0.4 GuHCl lung extracts in control (left panel), hydraulic edema attained through saline infusion (middle panel), and lesional edema induced through pancreatic elastase (right panel). Peak 1: CS-PG; peak 2: HS-PG; peak 3: small PGs and degradation products.

three distinct peaks (Fig. 6, control), corresponding respectively to (1) versican (peak 1, CS-PG), as suggested by its sensitivity to chondritinase ABC treatment, which digests galactosamine-containing GAGs such as CS; (2) perlecan (peak 2, HS-PG), as indicated by its sensitivity to combined treatment with heparinase and heparitinase, which digest glucosamine-containing HS chains; and (3) PG degradation products of small molecular size, including CS and HS (peak 3) (45). The composition of the different peaks was confirmed by capillary electrophoresis analysis of unsaturated disaccharides released from GAG chains by the specific enzymatic treatments. The elution pattern was modified in lung extracts from rabbits receiving saline load (Fig. 6, hydraulic edema) or elastase treatment (Fig. 6, lesional edema). The W/D ratio of the edematous lungs was that corresponding to the mild edema phase. Hydraulic edema was associated with an almost complete disappearance of the band-containing large versican, with a corresponding increase of the relative content of peak 2 and 3: such an elution profile would indicate significant fragmentation of the versican component and/or partial destruction of the smaller HS-PGs. Elastase treated lungs (Fig. 6, lesional edema) also showed a reduced versican band, though not as evident as in case of hydraulic edema; the HS component was reduced with respect to control, accompanied by a relevant increase in the smaller molecule-containing band, interpretable as a significant fragmentation of HS-PGs. PG fragmentation observed in hydraulic and lesional edema had a relevant effect on the interaction of PGs with other ECM components. Indeed, the binding properties of total 125 I-PGs isolated from 0.4 GuHCl extracts of lung affected by


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hydraulic or lesional edema to other matrix macromolecules like collagen type I, type IV and HA (Fig. 7A) were all reduced compared to control, indicating the loss of structural organization of the ECM macromolecular components. The derangement from the native ECM architecture was associated with the degree of edema severity, as indicated by the progressive loss of PG binding properties to other ECM components observed in mild and severe lesional edema compared to control (Fig. 7B). The observed loss of macromolecular assembly was likely responsible for the alteration of tissue compliance during edema development, as suggested by the fact that Pip drops to atmospheric values and remains essentially unaltered as edema progresses (Fig. 5). An increased tissue compliance may well be the mechanical expression of the loss of ECM native architecture in both the experi-

Figure 7 (A) Proteogycan binding to collagen type I, collagen type IV, and hyaluronic acid in control and severe hydraulic or lesional edema. (B) Proteogycan binding to some ECM macromolecules at various degree of lesional edema severity. 125 I-proteoglycans were isolated from 0.4 GuHCl lung extracts.

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mental models of lung edema, and is probably largely dependent upon a PG breakdown. The development of pulmonary lesional edema following intravascular addition of elastase was directly related to the proteolytic activity of this enzyme, which digests not only elastin but also PG core proteins, collagen, fibronectin, and laminin (19). Long exposure to high plasma concentration of elastase is known to cause pulmonary emphysema, due to progressive reduction of total elastin, with minimal effect on lung collagen, but actually followed by new connective-tissue synthesis (46). In rats, elastase-induced emphysema was shown to be associated with a decrease in HS-PG in the lung parenchyma with a parallel increase in pulmonary GAG content and a decreased urine content of GAGs (46). Enzymes without elastolytic properties like pancreatic trypsin or collagenases cause acute lung injury, but the lung heals completely without development of emphysema (46). This suggests that pancreatic elastase triggers instead a differentiated activation of the connective tissue metabolism, resulting in a long-term imbalance among the ECM lung components and in a long-term functional tissue impairment. It cannot be excluded that elastase may also proteolytically activate other proteases, present in the tissue as zymogen forms, thus amplifying the degradation of PG core proteins and other protein components of ECM. The proteases may also affect protein components of complement and coagulation systems, as well as other protein cascades (19). Protease synthesis is mainly accounted for by inflammatory cells, neutrophils or macrophages, T-lymphocytes eosinophils, basophils, and mast cells. Fibroblasts may also synthesize proteases which are released directly into the interstitial space. Moreover, it was recently shown that reactive oxygen species released by macrophage-derived cells may trigger the activation of latent proforms of metalloproteinases in the vascular wall, thus modulating ECM degradation in areas of high oxidative stress through protease activation. On this basis, upregulation of tissue proteases may largely contribute to tissue damage in the development of lung edema, leading to a disturbance of the dynamic equilibrium between the synthesis and the degradation of ECM components. MMPs, and in particular the MMP subfamily of gelatinases, are markedly involved in the turnover and remodeling of lung ECM. Gelatinases are normally localized in the lung tissue, particularly in the ciliated cells, endothelial cells, pneumocytes, and smooth muscles cells (47). In both our rabbit models of lung edema, qualitative zymographic investigations showed an increase of the proteolytically activated forms of gelatinase A (MMP-2) and B (MMP-9) (45). Moreover, we recently showed that 125 I versican isolated from rabbit lung was cleaved by purified MMP-2 and MMP-9, as well as by a crude enzyme extract from rabbit lungs with hydraulic edema (21). The capability of the enzyme to cleave versican is direct proof that gelatinases may recognize the large PG family of lung interstitium as an in vivo substrate.


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Activation of endogenous proteases is probably involved in the severe disruption of the gas exchange alveolar-capillary structure found in acute lung injury; in fact, an increased expression of gelatinases and collagenases was found in another model of acute lung injury induced in rats by breathing 100% oxygen (25,26). Elastase and collagenase, as well as other proteases produced outside the lung, may reach the lung through the bloodstream in case of tissue inflammation or injury; they rarely cause lung damage in normal conditions, because of the protective role of aspecific antiproteases (such as α 2-macroglobulin) and of specific tissue inhibitors of MMPs (TIMPS) of which four are known (19,48,49). In the normal lung, an excess of antiproteases protects ECM by modulating macromolecule turnover and by neutralizing the proteases released by inflammatory cells like macrophages and neutrophils. However, when acute or chronic inflammatory process are triggered, the released proteases may overwhelm the antiprotease protection, leading to tissue damage (50). In particular, in the lesional model of edema, exogenous elastase treatment may cleave the inhibitors of endogenous proteases (51), thus reducing the antiproteolytic defence of the lung. On this basis, dilution of the plasma and tissue antiproteolytic pool and/or activation of endogenous proteases might well modulate the proteoglycan fragmentation and loss of PG binding properties to other ECM macromolecules associated with the development of hydraulic edema.

VII. Proteoglycans in Interstitial Lung Edema Induced by Hypoxia Exposure Pulmonary edema may develop after acute exposure to hypoxia, a condition of high clinical relevance because it relates to pulmonary and cardiac disease and has a profound influence on pulmonary vasculature. Hypoxia is known to increase pulmonary arterial pressure and to trigger a marked remodeling of vessel structure (52). Cellular responses to hypoxia are actually very complex. They include depletion of energy substrates from the aerobic cycle, shifting the cell metabolism toward anaerobic sources, and Ca 2⫹ inducing cytosolic acidosis. As a consequence of low intracellular pH, H⫹ ions compete with Ca 2⫹ ions to be extruded from the cell using a Ca 2⫹ /Na⫹ exchanger, thus causing an increases in intracellular Ca 2⫹ concentration. The intracellular hypercalcemia observed in hypoxia seems to be a key point in triggering a series of event leading to cells damage and death. Indeed, high intracellular concentrations of Ca 2⫹ may trigger the activation of phospholipases A 2 and C, leading to plasmalemma and cytoskeleton lesion, inhibition of the phospholipid resynthesis, and mechanical alteration of the cell membrane (53).

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Alternative synthesis of ATP through the adenylate kinase reaction favors the formation of intracellular AMP, which is partly converted in adenosine, a potent local vasodilator that, once released in the interstitium, favors an increased blood flow to the hypoxic organ. However, adenosine is also converted to inosine that, in hypoxic conditions, induces an oxidative stress through the purine nucleoside catabolic pathway (53). Another consequence of hypoxia exposure is an intravascular accumulation of polymorphonuclear neuthrophils (PMN), facilitating inflammatory response with increase in capillary permeability and release of proteases (53). Moreover, reactive oxygen species are known to cleave the major antiprotease in the lung, α 1-antitrypsin, and also activate local proteases, leading to potentiation of the effect of neutrophil proteases on tissue destruction (54). Unlike what has been observed in other tissues, such as the intercostal muscles, Pip progressively increases (from ⫺10 cmH 2 O up to ⬃⫹4 cmH 2 O in 6 h) after experimental acute exposure of anaesthetized rabbits to a hypoxic gas mixture (12% O 2 in N 2 ) (42). Interestingly, peak Pip was similar to that observed in hydraulic and lesional edema models, suggesting that this pressure represents an upper limit of ECM mechanical tolerance to interstitial edema. Similarly to what was observed in the other edema models, pulmonary PG extractability in-

Figure 8 (A) Hexuronate recovery for total proteoglycan (PG) extraction and CS-PG, HS-PG and smaller peptidoglycan fragments (PDGL) obtained from pulmonary tissue samples in control normoxia and at varying times of hypoxic exposure. PG extraction was performed by 0.4 M GuHCl treatment of lung specimen (38). Normoxia corresponds to time zero. (B) Hexuronate recovery from lung specimen plotted as a function of Pip during development of pulmonary interstitial edema induced by exposure to hypoxia. Control normoxia corresponds to Pip of about ⫺10 cmH 2 O.


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creases with the increasing time of hypoxic exposure (Fig. 8A), indicating a weakening of non-covalent bonds linking PGs to other ECM components. Gel filtration chromatography indicated that after hypoxia exposure the relative contents of CS-PG and HS-PG were markedly lowered with respect to normoxia, indicating a fragmentation of both PG families (42). As a result, the recovery of peptidoglycans resulting from PG fragmentation progressively increased with time of hypoxia exposure and degree of severity of interstitial edema, as shown in Figure 8A. The increase in PG extractability and degradation induced by hypoxia clearly correlates with the extent of Pip increase during development of hypoxic edema (Fig. 8B). A possible source of increased fluid filtration into the pulmonary interstitium during hypoxia might be the sequential degradation of HS-PG, which seems particularly susceptible to sustained low PO 2 (Fig. 8B). Perlecan fragmentation, contributing to the selective barrier properties of endothelial and epithelial basement membrane, might therefore account for an increase in microvascular permeability and filtration rate leading to edema. The fragmentation process of PGs, accompanied by the loss of their interactive properties with other ECM macromolecular components, likely depends on the activation of tissue MMPs, in particular MMP-9, as indicated by gelatin zymography analysis performed on purified enzyme extracts from normoxic and

Figure 9 Photodensitometric representation of gelatin zymography performed on purified lung extracts. (A) Peak at 72 kDa, corresponding to matrix pro-metalloprotease 2 (proMMP-2); (B) peak at 92 kDa, corresponding to matrix pro-metalloprotease 9 (proMMP-9); (C, D) multiple MMP-9 forms; (b) proteolitically activated form of MMP-9. Left panel: control normoxia; right panel: 6 h of hypoxic exposure.

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hypoxic lungs (Fig. 9, left and right panels, respectively). Indeed, increased amounts of latent MMP-2 and MMP-9, as well as of the proteolitically activated form of MMP-9, were found in purified enzyme extracts from lung exposed to hypoxia (42). These data suggest that upregulation of MMPs may largely contribute to the damage of pulmonary interstitial PGs during hypoxic exposure, leading to the disturbance of the dynamic equilibrium between degradation and synthesis of PGs. VIII. An Example of “Spontaneous Recovery” from Lung Edema: the Newborn Lung At birth the lung follows a process almost inverse to what is experienced during edema development: namely, the lung turns within a relatively short period of time from a liquid-filled to an air-filled dry organ, the latter condition being essential for respiratory gas exchange. This process is accomplished through interdependent mechanisms: (1) active absorption of alveolar fluid through epithelial cells in the lung interstitium (55,56); (2) lung expansion due to an increased outward chest wall recoil (57); and (3) removal of fluid from the interstitial space. From the standpoint of fluid balance, alveolar edema at birth is cleared by drainage of fluid into the parenchyma, thus passing through the stage of interstitial edema. In fact, as shown in Figure 10, in lungs of vaginally and cesarean-delivered newborn term rabbits (58), Pip rises in the first postnatal hours as a result of alveolar fluid reabsorption, attaining a value comparable to that observed during

Figure 10 Time course of Pip in lungs of term vaginally and cesarean-delivered rabbits and in ventilated and atelectatic regions of premature rabbits.


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development of interstitial edema in adult rabbits (57). Thereafter, Pip decreases, as a result of interstitial fluid drainage to progressively attain the adult steady state value at ⬃2 months of age. In the ventilated region of preterm newborns, Pip increased to similar values, but decreased at a slower rate. Interstitial fluid reabsorption into the pulmonary capillaries appears to be critically dependent on the marked increase in Pip occurring from 30 min to 2 h after birth. In the adult, the attainment of a positive peak Pip during edema development depends on the low matrix compliance; as discussed above, ECM PGs seem to play a major role in organizing the matrix, thus determining tissue matrix compliance. It is reasonable to expect that a similar role might be played by PGs in the developing lung: the similarity between peak Pip reached in postnatal phase newborn and in edema in adult suggests that at birth the matrix is already well developed to resist volume loading while fluid is absorbed from the alveoli into the interstitium. This view is supported by morphological evidence in developing lung, showing that a large quantity of CS-PG is present in the alveolar interstitial space surrounding lung fibroblasts (6). HS-PGs also are thought to be deposited early in the developing lung: fetal lung synthesizes mainly HS-PGs, whereas total PG content progressively decreases with maturation and aging (3). A significant deposition of syndecan, a cell surface HS-PG, has been observed by epithelial and mesenchymal cells during lung branching morphogenesis (59,60). This class of HS-PGs is thought to be involved in recognition and binding of growth factor receptors in the cell membrane, and in cell-to-cell and cell-to-matrix adhesion, suggesting their importance in matrix organization (3). PGs are also essential in determining early morphogenesis, and also collagen and elastin fibrillogenesis (10). Finally, HA deposition in the pulmonary interstitium is also very efficient before birth, then diminishes after delivery (perinatal period) in line with reduction of lung water content (60). Conversely to what was observed in vaginally and cesarean delivered term newborn rabbits or in ventilated preterm rabbit lungs, in unventilated lung regions of premature rabbits Pip does not increase to positive values (Fig. 10). In these atelectatic regions, lung maturation is incomplete and the alveolar epithelium still secretes fluid rather than absorbing it. Furthermore, a deficiency in protein deposition of the interstitial matrix and surfactant synthesis characterizes the immature lung (61,62). In the adult rabbit, recovery from lung edema follows a similar pattern compared to that described for vaginally delivered term newborn rabbits; namely, Pip progressively decreases from the positive peak value attained during edema development toward normally subatmospheric values (63). This behavior likely depends on a progressive removal of accumulated interstitial fluid toward the pulmonary lymphatics, as well as vascular and interstitial remodeling, restoring the original normal ECM structure. Indeed, an increase in pulmonary metallopro-

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tease content in the recovery phase following hydraulic edema can be regarded as index of increased macromolecular turnover rate leading to tissue remodeling (53). This finding is in line with the observation that apoptosis occurs in cells of the adventitia of the vascular wall during recovery from chronic hypoxia, suggesting that ECM and cells may be removed from the recovering lung, thereby returning the vessel wall to normal structure (64). IX. Conclusions Lung ECM PGs are important molecules controlling microvascular permeability and tissue compliance. Pulmonary microvascular permeability is normally very low, maintaining fluid filtration at a minimum value. Tissue compliance is also a critical parameter in setting tissue fluid content, representing a powerful tissue safety factor in case of an increase in microvascular filtration. Both features render the lung very resistant to edema. Experiments show that pulmonary edema develops when PGs of the basement membrane and of the interfibrillar substance are fragmented by tissue proteases to an extent causing an increase in microvascular permeability and tissue compliance. Recovery from lung edema requires matrix remodeling and deposition of new PGs to restore the mechanical properties of the matrix and the microvascular wall sieve. Both functional features are present the newborn lung, which may be considered as a physiological case of edema healing. References 1. Weiber ER, Bachofen H. The fiber scaffold of lung parenchyma. In: Crystal RG, West JB, Weibel ER, Barnes PJ, eds. The Lung, Scientific Foundations, 2nd ed. Philadelphia: Lippincott, 1997:1139–1146. 2. Hardingham T, Fosang AJ. Proteoglycans: many forms and many functions. FASEB J 1992; 6:861–870. 3. Roberts CR, Wight TN, Hascall VC. Proteoglycans. In: Crystal RG, West JB, Weibel ER, Barnes PJ, eds. The Lung: Scientific Foundations, 2nd ed. Philadelphia: Lippincott-Raven, 1997:757–767. 4. Skold CM, Blaschke E, Eklund A. Transient increases in albumin and hyaluronan in bronchoalveolar lavage fluid after quitting smoking: possible signs of reparative mechanisms. Respir Med 1996; 90:523–529. 5. Cantor JO, Shteyngart B, Cerreta JM, Lui M, Armand G, Turino GM. The effect of hyaluronan on elastic fibers injury in vitro and elastase-induced airspace enlargement in vivo. Exp Biol Med 2000; 225:65–71. 6. Li Y, Rahmanian M, Widstrom C, Lepperdinger G, Frost GI, Heldin P. Irradiation induced expression of hyaluronan (HA) synthase 2 and hyaluronidase 2 genes in rat lung tissue accompanies active turnover of HA and induction of types I


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9 The Role of Small Proteoglycans in the Formation of Fibrosis

GUNILLA WESTERGREN-THORSSON, ELLEN TUFVESSON, ¨M ERIK EKLUND, and ANDERS MALMSTRO Lund University Lund, Sweden

I.

Introduction

Small proteoglycans play key roles in fundamental biological contexts such as providing tensile strength to interstitial connective tissues, regulating the mechanical properties of blood vessels, and the status of mineralized tissue. In addition, they exert profound effects on cellular signaling and modulate the activities of several cytokines. The small proteoglycans belong to the small leucine-rich repeat protein family (SLRRP) and are conspicuous components of the interstitial and peribronchial tissue of the lung. They are of importance during remodeling of the lung in physiological as well as pathophysiological conditions owing to their effects on matrix maintenance and on cell and cytokine activities. Therefore they are deeply involved in disease processes such as inflammation associated with fibrosis, emphysema, or allergy. The following presentation will be limited to the relationship between the structure and function of the proteoglycans of the SLRRP family. It will focus on lung functions under both physiological and pathological conditions. Several excellent reviews concerning these proteoglycans are available (1–3). 169

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In the lung several small proteoglycans belonging to the SLRRP group have been demonstrated including decorin, biglycan, and lumican (4,5). These macromolecules can be divided into three groups and comprise at least 12 different members. The basis for this classification is the arrangement of the leucine-rich repeats (LRR) and the presence of distinct cysteine clusters. These SLRRP proteins are differently modified with dermatan/chondroitin sulfate (DS/CS), keratan sulfate, N-linked oligosaccharides, or tyrosine sulfation. Moreover the promoter regions of the various SLRRPs are quite distinctive (6). These components are therefore highly heterogeneous regarding structure, function, and metabolic regulation. The core proteins belong to a large group of proteins with varying numbers of leucine-rich repeats motifs (Fig. 1), where the leucine residues often are located in conserved positions. There are three groups with different leucine-rich repeat and gene organizations. The C-terminal regions are generally short whereas the

Figure 1 Structure and function of small proteoglycans exemplified by decorin.

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N-terminal region is longer and considerably more variable regarding both sequence and substitution (2). A. Group 1

This group has a core of 40 kDa containing 10 repeat motifs and comprises the proteoglycans decorin and biglycan, and a glycoprotein, asporin (7). The core proteins are highly homologous, and biglycan and decorin have two and three sites for N-linked oligosaccharides (Fig. 1) in the LRR region (8,9). Biglycan shows the highest expression in the spleen, bone, lung, and liver whereas decorin is most prominent in the skin, uterus, heart, bone, lung, and liver (10). Biglycan is mostly localized pericellularly and around blood vessels whereas decorin is found in the fibrous matrix (10). Biglycan is mostly substituted with two glycosaminoglycan (GAG) chains and decorin with one. The galactosaminoglycan chains consist of repeating disaccharide units of N-acetyl d-galactosamine and an uronic acid, either d-glucuronic (d-GlcA) or l-iduronic acid (l-IdoA). If the uronic acids only consist of d-GlcA, the polymer is referred to as chondroitin, but if any l-IdoA is present, the polymer is called dermatan. These backbone polysaccharides are then substituted with sulfate groups at various positions. Depending on the tissues, the structures of the polysaccharide chains differ on both biglycan and decorin. In bone decorin is substituted with chondroitin-4-sulfate, but in most other tissues both proteoglycans are substituted with DS with varying proportions of l-IdoA residues. Thus in nasal cartilage a minute amount of l-IdoA is found, in articular cartilage an intermediate proportion is noted, whereas in lung, skin, and uterus a high proportion of lIdoA acid is present (11). l-IdoA residues are distributed in blocks of varying size interspersed with d-GlcA containing blocks mostly of shorter size (Fig. 1). The sulfation is also variable with L-IdoA regions containing 4-sulfated N-acetylgalactosamine residues (Fig. 1) and some 2-sulfated L-IdoA residues (12). The latter endow the molecules the potential to bind cytokines and blood coagulation factors. B. Group 2

This group of SLRRP consists of fibromodulin, lumican, keratocan, osteoadherin, and PRELP. All in this group have 10 leucine-rich repeats and have characteristically a cysteine-rich region just N-terminal to the LRR region. Fibromodulin and lumican show high sequence homology. Fibromodulin contains sulfated tyrosine and, depending on the tissue and age, keratan sulfate. It is found in the interstitial tissue in lung (13). Lumican is substituted with keratan sulfate in cornea but with a nonsulfated lactosaminoglycan in other tissues (14). In lung, lumican is one of

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the prominent proteoglycan species (5). PRELP has also been demonstrated in lung tissue (15). C. Group 3

This group of SLRRP contains only six leucine-rich repeats. The members of this group are epiphycan, mimecan, and opticin; they have not yet been demonstrated in lung tissue.

III. Impact of Small Proteoglycans on Matrix Assembly The proteins of the SLRRP family influence tissue properties/functions by regulating collagen network formation and cytokine activities. Thus a change of tissue composition not only changes the properties but also generates a tissue with different responses to various stimuli. These macromolecules exert their effects by interaction through the protein core as well as its substitutent. Thus the highly variable polysaccharide chains exert a number of effects depending on the presence of specific polysaccharide motifs. Decorin and biglycan, being substituted with mainly DS, exert a swelling pressure due to the osmotic pressure generated by their highly negatively charged side chains. Biglycan knockout mice do not display any major changes in the lung. The only major effect is the induction of an osteoporosislike phenotype (16). The protein core of biglycan is, however, known to interact with transforming growth factor-β (TGFβ) and collagen VI (17,18). Decorin knockouts also display small effects. They show abnormal collagen fibrils leading to skin fragility (19). This may be due to the fact that decorin, named from its property to “decorate” collagen, has the potential to regulate the collagen fibril diameter; i.e., there is a relation between the collagen fibril surface and the amount of decorin (Fig. 1), and the more decorin present, the smaller the fibrils. The molecular background is that decorin has a horseshoe conformation which fits precisely over the collagen triple helix. In decorin the fourth and fifth leucine-rich repeats are involved (20) in collagen binding, and the collagen binding motif is located ⬃25 nm from the C-terminus (21). Furthermore, lumican accelerates the initial fibril formation, but, together with decorin, there is a synergistic retardation (22). The DS on decorin has an important function in regulating the distance between the collagen fibrils through specific interaction between the DS chains (23) (see below). Decorin also interacts with collagen types VI and XIV (18,24). When decorin interaction is compared to those of lumican and fibromodulin, it becomes clear that the latter two interact with a different site on the collagen fibril (25).


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Fibromodulin and lumican are both expressed in lungs. Their functions are not completely known, but knockouts of both these proteoglycans separately show disrupted collagen fibrils in a similar manner to decorin (26,27). It appears that these proteoglycans can compensate for each other, making the knockout effect of only one of the proteoglycans less noticeable. No direct effect on lung function has therefore been noticed. The side chains of biglycan and decorin encompass considerable structural variability and are of importance for the formation of a well-regulated extracellular matrix (ECM) network. In addition to the impact of DS self-interaction on the organization of the collagen fibrils, direct interaction between dermatan sulfate and collagen types I and XIV also occurs (24). Furthermore, DS interacts with tenascin-X (28), MAGP-1, and fibrillin-1 (29) to contribute to the ECM network. The latter could also from a ternary complex with DS on decorin. All these interactions are of fundamental importance for the properties of the fibrous connective tissue. The fibroblasts are central in controlling ECM, and to do this several adhesion molecules are important as well as the cytoskeletal network. Fibronectin has a great impact on ECM organization with a multitude of interactions from integrins on the cell surface to individual ECM proteins and polysaccharides. Also, decorin and fibronectin show specific interaction, thereby blocking fibroblast adhesion (30). CS/DS also influences this interplay by interacting with α-dystrophin, thereby influencing the organization of the cytoskeleton with the extracellular matrix (31).

IV. Core Proteins of Small Proteoglycans: Interaction with Cytokines and Their Receptors Increased TGFβ production is an important feature of several fibrotic disorders. Four of the SLRRP members—decorin, fibromodulin, lumican, and biglycan— bind TGFβ with high- and low-affinity sites of 10⫺9 and 10⫺7 M, respectively (17). This is of major importance in the control of the remodeling process, as TGFβ activity can be abrogated by decorin. Furthermore, decorin may function as a store for TGFβ to be released after proteolytic attack on the protein core (Fig. 1). Moreover, treatment of animals with decorin or transfection of cDNA for decorin results in inhibition of fibrosis both in kidney and lung (32–34). A finding of great potential is the function of SLRRPs to control cell proliferation. Thus, decorin has been demonstrated to retard the growth of a wide variety of tumor cells (35,36). The production of decorin also correlates inversely with the proliferation of lung fibroblasts (Westergren-Thorsson et al., unpublished). The mechanism for these effects is that decorin interacts with and thereby modulates the activity of epidermal growth factor (EGF) receptor, thus inhibiting

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the cell cycle via inhibition of cyclin-dependent kinases by P21 (35). This property has been postulated as a mechanism to prevent tumor growth, as shown in cells from breast carcinoma and lung adenocarcinoma (37,38). V.

Polysaccharide Substitution of Small Proteoglycans

The bioactivity of the glycosaminoglycan side chains is also of great importance in the functions of decorin and biglycan. In most cases these chains differ in structure among tissues, but have a greater similarity in decorin and biglycan from the same tissue (Tiedemann and Tufvesson, preliminary result) (39). Several roles of the side chains have been documented. In the coagulation system, DS with sulfated L-IdoA residues specifically interacts with the thrombin inhibitor heparin cofactor II. (40) (Fig. 1). This is known to be of physiological importance in placenta (41) and in the atherosclerotic plaque (42). Moreover, DS enhances the anticoagulant activity of protein C (43). Thus it is not surprising that dermatan sulfate is being introduced as an antithrombotic agent (44). L-IdoA-2 sulfates in DS are also involved in the binding and ensuing activation of basic fibroblast factor 2 (bFGF). The activation is of considerable importance in wound fluid, where released DS is the main activator of bFGF (45). Presentation to macrophages of IFN-γ bound to DS on mast cells resulted in activation and production of nitric oxide (46). Other cytokines, such as hepatocyte growth factor/scatter factor (HGF/SF) (47) and heparin affin regulatory peptide (HARP), bind to DS thus activating the cytokines (48). DS appears to have the capacity to inhibit expression of TGFβ possibly through PKC (49) (Fig. 1). Of interest is the effect of released DS and CS on various inflammatory processes and wound healing. Proteolytic attack releases the glycans and, in addition to bFGF activation, they also increase ICAM-1 expression through stimulation by NFκB, which results in recruitment of neutrophils (50). To further increase neutrophil recruitment, both chondroitin and dermatan sulfate interact with L- and P-selectins present on the neutrophil surface (51). The side chains of decorin and biglycan as well as the intact proteoglycan are also of importance for modulation of fibroblast proliferation and migration (Fig. 1) (52–54); Tufvesson, unpublished), thereby recruiting fibroblast with specific properties. VI. Metabolism of the Small Proteoglycans The metabolisms of decorin and biglycan are regulated differently. The effect of cytokines on ECM remodeling differs depending on the target cells, especially when comparing chondrocytes and fibroblasts. In this review emphasis is put on the effects on fibroblasts. The main regulator of biglycan is TGFβ, which upregu-


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lates both the mRNA and protein expression up to 10-fold (55). Furthermore, TGFβ cooperates with other cytokines such as platelet-derived growth factor BB (PDGF-BB) and EGF to produce synergistic effects on proteoglycan expression (56). The biglycan promoter encompasses several cis-acting elements such as AP-2, interleukin-6 (IL-6) responsive element, NFκB, and surprisingly also a TGFβ-negative element. In line with this finding, it has not been possible to activate the promoter by TGFβ, indicating the presence of an undefined postranscriptional mechanism (57). Also TNFα but not IL-1 stimulates the production of biglycan, but not as potently as TGFβ (58). In preliminary results, Eklund has noticed that IFNγ in combination with TGFβ does not inhibit the TGFβ-induced production of biglycan, whereas it does suppress the TGFβ-induced production of versican. Decorin production does not respond to TGFβ treatment. In fact, in several reports a direct downregulation has been noted (55,56). The decorin promoter has no TGFβ response element. However, two TNFα response elements and a AP-1 binding site are present, which results in an antagonistic effect between TNFα and IL-1 (59,60). Thus, TNFα downregulates decorin (58,60), whereas conflicting reports regarding the effect of IL-1 have been noted (59). Furthermore, IL-10 upregulates decorin (61). Dexamethasone, being an important drug to control inflammation, exerts a differential regulation by stimulating decorin and inhibiting biglycan production (62). In addition, budesonid affects biglycan in the same way but has no stimulating effect on decorin in human skin fibroblasts (Sa¨rnstrand, unpublished). Finally, retenoids are also of interest as they upregulate decorin and downregulate biglycan production (62). The lumican promoter contains a binding site for the transcription initiation factor Sp3, but no other cytokine response elements has been found (63). However, in breast carcinoma the production is differentially regulated compared to decorin (64) and in keratocytes bFGF upregulates lumican (65). The regulation of fibromodulin is largely unknown at present. Recently it has been documented that TGFβ not only affects the core protein production of biglycan and decorin but also affects the copolymeric structure of their DS chains, resulting in up to a 50% decrease in L-IdoA residues (55). The mechanism behind this change in polysaccharide structure is a downregulation of the chondroitin C-5 epimerase activity (Tiedemann, unpublished) An important way to regulate the actual amount of proteoglycan in the tissue is to control rate of endocytosis followed by degradation. A specific endocytosis receptor for decorin of 51 kDa has been demonstrated. Heparan sulfate proteoglycans diminish the binding capacity of this receptor (66). The effect on the endocytosis receptor may explain the inconsistent result where mRNA levels of decorin are decreased whereas the amount of decorin is increased. (56) Mechanical load has an impact on proteoglycan synthesis in fibrous connective tissue around vascular smooth muscle cells (67), in tendon (68), and in

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fetal lung cells (69). The major effect is a clear increase of versican and biglycan whereas decorin is unaffected or down regulated.

VII. Different Pathological Disorders Involving the Small Proteoglycans The proteoglycans discussed in this review are often mediators of remodeling processes involving connective tissue. They are crucial components in basic processes such as wound healing and defective variants thereof (e.g., artherosclerotic plaque, fibrosis, and emphysema). In states of tissue inflammation and remodeling, typical proteoglycan expression patterns appear. Many parasites are known to use extracellular molecules as receptors for cell entry. Decorin-binding proteins have been detected in the Lyme disease bacteria B. burgdorferi. In fact, animals lacking decorin can no longer be infected with this microorganism (70). Furthermore, chondroitin 4-sulfate seems to be the receptor for P. falciparum–infected red blood cells in the intervillous space of placenta. The selective accumulation of these cells leads to poor fetal outcome and severe health complications in the mother (71). A change of the relative distribution of decorin and collagen in some disorders results in a changed collagen organization. This actually occurs in incontinent women, who have collagen fibrils of changed diameter most likely generating the incontinence problem (72).

VIII. Normal Physiology of the Lung Proteoglycans play important roles in the normal function of the lung. Their main function is to form a foundation for the alveoli and the air duct system. Important regions in the tissue are the basement membranes containing mainly the heparan sulfate proteoglycans perlecan and agrin (73) and the interstitial connective tissue, where the main proteoglycan is decorin (74). In smaller amounts, however, it is also possible to find the SLLRPs biglycan, lumican (5), fibromodulin, and PRELP (75) in the interstitial tissue as well as the large proteoglycan versican. The main producers of these proteoglycans are the fibroblasts, but type II alveolar cells are also active (76). An important role of the proteoglycans in the tissue is to contribute to the network of ECM and to function as activators or inhibitors for various cytokines as well as function as reservoirs for storage of cytokines. The presence of cytoactive motifs both in the core protein and in the GAG chain is most likely of great importance in the defense and remodeling of the tissue during inflammatory conditions. Further studies must, however, be performed to verify the presence and possible functions of all proteoglycans in the lung tissue.


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The breathing process results in a mechanical effect on all cell types in the lung. No information on the impact of this has been recorded in the lung itself. However, mechanical influence on lung fibroblasts in vitro generates proteoglycans with longer side chains (68). Furthermore cyclic mechanical stimulation of vascular smooth muscle cells increases the production of versican and biglycan whereas that of decorin remains unchanged (67). Corresponding experiments are yet to be performed on smooth muscle cells and biopsies from the lung. This may be of special importance as the mechanical impact may be changed during certain pathophysiological disorders. The importance of the proteoglycans in normal lung function is underlined by the fact that treatment with xylosides (initiators of GAG synthesis) or elastase, which both deteriorate proteoglycan patterns of the lung, strongly changes lung architecture and function (77,78). These and other inflammatory processes involving proteoglycans, which affect lung function, will be discussed thoroughly hereafter. IX. Small Proteoglycans and Lung Pathology In chronic lung diseases the expression profile of small proteoglycans is clearly changed compared to the normal situation. It has been suggested that both inflammatory and fibrotic processes of the lung, at least in part, are mediated by specific up- and downregulation of the expression and degradation of these molecules. The most well known disorders involving proteoglycans are asthma, various forms of fibrosis, and chronic obstructive pulmonary disease (COPD). If proteoglycan metabolism in these diseases could be specifically controlled, novel therapeutic ways to treat them could be at hand. Therefore, a deeper knowledge of proteoglycan regulation in the lung is of paramount interest. A. Peribronchial Fibrosis

An important feature in the asthmatic trait is the formation of peribronchial fibrosis, which emerges early in the disease process. The changes in the extracellular matrix may contribute to abnormal airway function by altering airway mechanics and modulating inflammatory and structural cell function. Indeed, both our group and others have found significant correlation between airway hyperresponsiveness and the degree of bronchial subepithelial fibrosis in patients with asthma (79,80). With immunohistochemical characterization and by culturing fibroblasts from asthmatic patients, it has been clearly demonstrated that production of the small proteoglycan biglycan and lumican are increased, whereas decorin is not affected (Westergren-Thorsson et. al., submitted; 81) (Fig. 2). An increased production of the larger proteoglycans perlecan and versican have also been noticed. The increased production of these proteoglycans is sig-

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Figure 2 Mediators, markers, and potential treatment of remodeling ECM of the lung.

nificantly correlated to airway hyperresponsiveness to methacoline. As it is possible to retain the disease phenotype of the fibroblasts in cell culture, these data point to a mechanism where specific active fibroblast clones may have been recruited to the engaged tissue and indicate that these cells are stable even under culture conditions. The role of the peribronchial fibrosis in the development of asthma attacks is not known. Detailed characterization of cytokine interactions and the presence of cytoactive fragments of the core proteins and polysaccharide side chains of the proteoglycans therefore need to be further investigated. B. Interstitial Lung Fibrosis

Fibrosis occurs in the interstitium of the lung in a variety of disorders such as idiopathic pulmonary fibrosis (IPF), systemic sclerosis, and also possibly COPD. In these disorders a profound remodeling of the extracellular matrix takes place, where the fibroblast is regarded to play a very active role. The detailed proteoglycan changes connected with the various disorders have not been characterized in detail. However, in early lesions in adult respiratory distress syndrome (ARDS), bronchiolitis obliterans organizing pneumonia and IPF a clear accumulation of versican occurs. In addition, myofibroblasts, which produce procollagen type I and decorin, are present (82). Also, in sarcoidosis the same type of lesions with


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myofibroblasts and versican production has been recorded, suggesting a role for versican in the early repair process (83). Proteoglycan production has been more thoroughly studied in several animal models to elucidate the mechanism behind the formation of fibrotic lesions. In these models fibrosis has been induced by silica, bleomycin, or adenoviruses containing cDNAs for TGFβ, IL-1, and biglycan (Fig. 2). In silica-induced fibrosis, an increased synthesis of collagen type I has been observed and after 27 days of instillation a temporal and spatial overlap with increased production of TGFβ has been noticed, implicating this cytokine as a mediator in the formation of the fibrotic lesion (84). However, little is known concerning the presence of small proteoglycans in this model. In bleomycin-induced fibrosis, a marked increase of versican is noticeable at 7 days after instillation (85). Also, collagen production with clear increases of mRNA for collagen type I occurs in the initial phase. The active cells in this context are the myofibroblasts, and the main activation cytokine is TGFβ. Myofibroblasts are often found in a versican-rich extracellular matrix, suggesting that versican itself may influence the progression of the fibrosis (82). A major increase of biglycan mRNA and its expression as proteoglycan occurs at day 7–14 subsequent to the increase of TGFβ (Fig. 2). During the same time period a decreased production of the decorin has been noticed both as protein and as mRNA (74). Fibromodulin accumulation increases together with biglycan around day 14 (85). These severe changes of the extracellular matrix correlate well to a loss of resistance and elasticity of the afflicted tissue (86). Lumican occurs in substantial amounts in the lung (5), but little is known concerning lumican expression in fibrotic disorders. The increase in biglycan and fibromodulin together with the corresponding decrease in decorin is certainly of importance for the remodeling process, as it changes the collagen fibrillation process (21,27). Furthermore, biglycan may have other specific functions of importance for the early remodeling, such as recruitment of fibroblasts (Tufvesson, preliminary data; 54). The mechanism for upregulation of biglycan is unclear as there are no TGFβ-responsive elements in its promoter, but rather posttranscriptional mechanisms may be responsible for both mRNA as well as core protein formation of biglycan (57). Later in the process after bleomycin instillation, a clear upregulation of decorin takes place together with a further increase of collagen (Fig. 2). As decorin specifically interacts with collagen and thereby regulates fibril formation, it is crucial for the generation of the fibrotic tissue. In this tissue decorin is localized along the collagen fibrils, and furthermore TGFβ colocalizes with decorin (87). In the bleomycin model, the amount of collagen/decorin stabilizes only to decrease after 1–2 years. The bleomycin model lends itself well to experiments aimed at elucidating mechanisms behind the deranged metabolism of the extracellular matrix. Instillation of decorin macromolecule or by using adenovirus containing cDNA for de-

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corin abrogates the formation of fibrosis (33,34,88), which most likely can be explained by the binding of TGFβ to the decorin core protein. Instillation of adenovirus containing cDNA for active TGFβ leads to a heavy longstanding fibrosis (89). In preliminary results together with Gauldie and Sime, we have shown that after instillation of this construct, biglycan and decorin are regulated in the same way as after instillation with bleomycin; i.e., increase in the production of biglycan simultaneously as the production of decorin decreases. Only later do biglycan decrease and decorin increase in agreement with the bleomycin model. Instillation of adenovirus containing cDNA for biglycan or decorin leads to an unexpected differential effect on lung morphology. The biglycan construct leads to a transient fibrosis of the lung with increased collagen expression and accumulation of myofibroblasts (90). However, no effect on lung morphology or matrix production could be noticed after instillation with adenoviruses containing cDNA for decorin. These results may be explained by the fact that biglycan can act as a profibrotic factor as it is situated close to the cells and may deliver its bound TGFβ more easily to the TGFβ receptors. It is possible that biglycan per se can affect the fibrotic process. In preliminary results by Tufvesson it has been observed that biglycan can upregulate the expression of ICAM-1 and stimulate the migration of fibroblasts. The hypothesis that biglycan and decorin differently modulate the TGFβmediated fibrotic response was tested by coinfection with adenovirus containing cDNA for active TGFβ and biglycan or decorin. Under these circumstances only coinfection with virus containing cDNA for decorin could abrogate the TGFβinduced fibrosis (88). It is, however, documented that biglycan and decorin interact with TGFβ to the same extent (17). The difference in abrogating the activity may be explained by interaction of the TGFβ/decorin complex with collagen fibers thereby preventing contact with the TGFβ receptors. Another way to prevent the formation of fibrosis was presented by Ziesche et al, who showed that treatment with the cytokine IFNγ in some patients efficiently interrupted the fibrosis progression (91). These data further underline the importance of TGFβ in the development of fibrosis. IFNγ abrogates the effect of the TGFβ signaling system by modulating the activity of the TGFβ responsemediating protein SMAD-3 (92,93). In cell culture systems we have shown that IFNγ inhibits the TGFβ-induced production of versican, but that the TGFβinduced increase in biglycan remains even in the presence of IFNγ. Hence, in vivo, the lack of versican might prevent recruitment of myofibroblasts and abolish the increase of collagen production. Instillation of adenovirus containing IL-1 cDNA (94) has been shown to induce fibrosis. In this system, IL-1 induces a longstanding increase in TGFβ. An increase of myofibroblasts, which may be promoted by an IL-1, induced production of versican (58). The severity of this fibrosis is similar to that induced by adenovirus containing with cDNA for TGFβ. Also, instillation of adenovirus containing cDNA for GM-CSF results in


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the formation of granulation tissue, which results in an irreversible fibrosis. As in the IL-1-induced fibrosis, TGFβ and myofibroblasts are increased (95). C. Fibrosis in Systemic Sclerosis

Patients with systemic sclerosis often have fibrotic lesions in the skin as well as in internal organs such as the lung (96). In some patients the amount of fibrotic tissue in the lung increases rapidly, which often causes the afflicted subject to die. In fibroblasts obtained from patient skin biopsies early in their disease, biglycan and versican production is upregulated. At this stage of the disease, the proteoglycan production correlates with the grade of inflammation measured as acute-phase protein (CRP) and ESR. Later in the disease, the production of decorin is upregulated (97). Measurements of proteoglycan in fibrotic lesions from lungs of patients with scleroderma or in cell culture thereof have not been studied as yet. D. Interstitial Lung Fibrosis and Emphysema

Chronic obstructive pulmonary disease (COPD) is a complex disorder attracting considerable interest due to a very marked increase in hospitalizations and deaths in recent years. Morphologically it has both features of fibrosis as well as emphysema, which indicate that the ECM is rearranged in a complex way. Collagen type I concentration measured histochemically is increased in irregular air space enlargement sections in the lung (98). Elastin, on the other hand, is generally decreased (99). Biglycan and decorin are clearly decreased in the peribronchiolar areas, whereas no changes are noted in the perivascular areas (100). A clear indication of the role of extracellular matrix is the effect of instillation of β-Dxylosides, which through inhibition of biglycan, decorin, and versican synthesis generate emphysematous lesions (78). Also, instillation of elastase-induced emphysema in rat (101). An important correlation here is that the formation of an emphysematous lesion correlates with the presence of DS and HS in the urine, indicating the importance of intact proteoglycan in the interstitial as well as in the basal membrane system (77). Tobacco smoke is regarded as the most important inducer of COPD (102). Cadmium in tobacco smoke is one of the possible agents in this process. It decreases the production of both collagen and proteoglycans, especially versican, decorin, and perlecan, but not biglycan (103). Further studies of ECM status during various phases of COPD are necessary to understand this complex disorder. X.

Conclusions and Future Perspectives

It is clear that the composition of extracellular matrix is affected differently during various phases of inflammation in the lung (Fig. 2). During the initial phase

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of inflammation, versican and biglycan are conspicuous proteoglycans and possibly also lumican. When a fibrotic lesion is established, the dominating proteoglycan is decorin together with elevated levels of collagen. Important factors that are involved in this process are IL-1, GM-CSF, TGF-β, and maybe biglycan in the initial phase of the inflammation (Fig. 2). Biglycan and decorin may directly affect the progression of inflammation or participate through their binding to TGFβ. Versican can also affect the inflammatory process via the initiation of myofibroblast formation and its capacity to recruit neutrophils. The GAG chains of the proteoglycan most likely play important roles in these processes by binding, activating, inhibiting, and stimulating receptor signaling of cytokines, as well as by directly inhibiting cell proliferation. A better understanding of the structure and function of the GAGS and proteoglycan core protein that can bind TGFβ could result in the development of new therapeutic agents. Decorin may inhibit the proliferation of the fibroblast in the fibrotic lesion. Another promising tool to inhibit the formation of fibrosis has been the use of IFNγ, which has the potential to inhibit the TGFβ-stimulated synthesis of versican but not that of biglycan. IFNγ can thereby maintain the inflammatory ECM and prevent the transition into a fibrotic phase with increases of collagen and decorin. In emphysema, the main issue is to elucidate the disease mechanism. Is it a direct consequence of a release of proteases, due to toxic components tobacco smoke, resulting in a degradation of ECM, or is it a compensatory stimulation of ECM production by these toxic components or released proteases leading to an initial fibrosis, which after prolonged stimulation transforms into degradation and emphysema? The importance of proteoglycans is especially seen when xylosides are given to rat lung, where these animals develop emphysema. During the formation of emphysema the airway walls are destroyed and the production of versican and decorin decreases. The various disorders afflicting lung extracellular matrix and its constituent proteoglycans generate a heavy burden on medical care. The main disorders generating an incredible cost are asthma, fibrosis, and COPD. Despite extensive research efforts, we are still in need of new treatments. To this end, the structure, metabolism, and especially the functions of decorin, biglycan, lumican, and other members of the SLRRPs need to be better understood. Other important areas are the functions of the protein cores, specifically their interplay with the collagen/ tenascin network and the cytokine cascades, headed by TGFβ and possibly other cytokines. The structural requirements for the cytoactivity of DS/CS side chains need to be studied regarding anticoagulant activity, modulation of cytokine activities, and cell activation concerning the expression of adhesion molecules, cell proliferation, and cell migration. Inhibiting fibrosis with the protein cores of proteoglycans, both directly and after viral instillation, has shown several promising effects. Heparin derivatives such as Fragmin and DS are already in clinical use as anticoagulant and antithrombotic agents. Also decorin gene transfer–mediated suppression of TGFβ


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abrogates experimental malignant glioma growth, which opens new therapeutic possibilities in vivo (104). In fact, efforts have already been made using these molecules in the treatment of asthma (105). In conclusion, an increased knowledge of cytoactive DS/CS and heparan sulfate motifs, together with proteoglycan core motifs, can be expected to open more effective treatment for lung disorder.

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10 Versican in the Cell Biology of Pulmonary Fibrosis

CLIVE R. ROBERTS University of British Columbia Vancouver, British Columbia, Canada

I.

Introduction

Pulmonary fibrosis is the replacement of lung architecture with collagenous extracellular matrix, and is a frequent consequence of chronic inflammation. Fibrosis affects primarily the alveolar structures of the lung in the interstitial lung diseases (ILDs), a group of life-threatening disorders with ⬎150 known causes or associations. ILD may be caused by inhaled particles such as asbestos or silica, by inhaled gases such as nitrogen dioxide, by xenobiotics such as the chemotherapeutic agent bleomycin, by systemic autoimmune disease such as scleroderma, by the reaction to aeroallergens such as fungal spores or to infectious agents such as mycobacteria and viruses, or by as yet undefined environmental agents and/or predisposing genetic factors. In most individual patients the cause is impossible to determine. The disease process is classified based on a combination of histolic appearance, which includes division into nongranulomatous and granulomatous inflammatory processes, and cause, if a particular cause is known or likely. The most prevalent ILDs in the developed world are idiopathic pulmonary fibrosis (IPF), occupationally induced fibrosis caused by particle inhalation, and sarcoidosis, a multisystem idiopathic granulomatous disease (1). Tuberculosis, the sec191

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ond leading infectious cause of death in the world (2), involves a granulomatous and progressive fibrotic reaction to Mycobacterium tuberculosis, and tissue fibrosis is an important component of the pathophysiology of the disease. The cell biology of tissue remodeling in the major fibrotic lung diseases/disorders shows some similarities, and altered proteoglycan metabolism is central to each of them. In many of the fibrotic lung diseases, the progressive replacement of areas of normal lung by fibrotic tissue causes decreased lung air space volume and alterations in gas exchange and lung mechanics that eventually become perceptible, then limiting, and may ultimately lead to respiratory failure and death. This insidious progression may be subclinical for years in the most common form, idiopathic pulmonary fibrosis (IPF), and patients often present with persistent cough and dyspnea that limit physical activity, as a result of losing considerable lung function. The pathological pattern associated with IPF is termed usual interstitial pneumonia (UIP) [known as cryptogenic fibrosing alveolitis (CFA) in the United Kingdom]. UIP involves patchy nongranulomatous inflammation, distinctive areas of active remodeling where myofibroblasts proliferate and begin to deposit collagen, areas of dense collagen (fibrosis), areas of distortion including the creation of large spaces (honeycombing), and normal lung, all in proximity to one another. Figure 1A shows a typical image of normal control lung, in contrast with the section shown in Figure 1B, taken from a biopsy of UIP lung, where alveolar walls appear thickened or coalesced and there is also thickening of airway and vascular walls and apparent smooth muscle hypertrophy. A decrease in alveolar surface area and increase in area fraction (and therefore relative volume) occupied by tissue compared to air space is readily apparent in Figure 1B, compared to the normal lung (Fig. 1A). Many cases of ILD with a clear occupational association also exhibit this histological pattern, as does ILD associated with systemic sclerosis. ILDs are diagnosed and classified on the basis of radiological analysis, presence of a progressively worsening restrictive defect on lung function testing, and pattern of histological changes on lung biopsy, together with clinical history and presumed cause. Median survival after diagnosis of IPF is 3–5 years (3). The prevalence of this class of diseases is difficult to determine: estimates range from five to 10 per 100,000 population (4) to 74 per 100,000 (1), and ILDs in their many forms have been estimated to account for 15% of pulmonary specialty problems (3,4). Current anti-inflammatory therapy, with corticosteroids or cytotoxic agents, slows the rate of deterioration in lung function in only 10–30% of ILD patients but does not reverse fibrotic changes that have already occurred. Therefore, inhibiting changes in lung architecture is the key future goal in the therapy of ILD. Future therapeutic strategies will likely involve inhibition of collagen secretion (e.g., using colchicine) or assembly (e.g., using prolyl hydroxylase inhibitors),

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(A)

(B)

Figure 1 (A) Histological section of normal lung stained with Gomori’s trichromealdehyde fuchsin stain. Note the thin walls of the membranous bronchiole (Br) and alveoli (aw) in the normal lung. (B) Histological section of IPF lung stained with Gomori’s trichrome-aldehyde fuchsin stain. Note the thickened and distorted structure of alveolar walls (aw) replaced by dense collagenous fibrotic tissue, and the thick walls of a small bronchiole (Br), with apparently hypertrophied smooth muscle (sm) in IPF.

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inhibition of the expression of collagen genes (e.g., by inhibiting the production or activity of fibrogenic cytokines), or the inhibiton of fibroblast proliferation. Future epidemiological studies may point to risk factors for ILD that will facilitate preventive measures in individuals at risk, or early therapeutic intervention to modify the course of disease. An excellent example of this is the demonstration that a population of workers in the nylon flocking industry had an incidence of ILD 250 times greater than background (5), implicating a previously unrecognized hazard (fine nylon fibers) as a potentially substantial risk for ILD.

II. Proteoglycans The remodeling process in pulmonary fibrosis has been best studied in diagnostic biopsies from individuals with ILD, where diagnostic pathologists have long recognized that in the most common pathological pattern UIP, normal lung, areas of inflammation, active remodeling, and mature collagenous fibrosis may be observed in different areas of the same lung, allowing observation of different phases of remodeling in the same patient. Mechanistic studies have used animal models of alveolitis leading to collagen deposition such as bleomycin-induced alveolitis that resemble human ILD, though these models generally lack the progressive nature of the most common serious human fibrotic lung diseases. Thus a paradigm for the disease process in humans is well established (4,6). Infectious agents or inhaled toxins damage the epithelium, leading to the release from alveolar macrophages of cytokines that recruit granulocytes into the lung and activate them (7). Alternatively, epithelial injury may be initiated by allergic inflammation. Injury may be mediated by oxidants (8) or proteinases released from granulocytes or macrophages and ultrastructural damage to the basal lamina in IPF is consistent with proteolysis (9). Inflammation may become chronic, because of repeated or persistent insult or because epithelial regeneration is prevented (10). A fibrinous exudate containing inflammatory cells, fibrin, and fibronectin covers the alveolar surface (11). Fibroblasts migrate through holes in the basal lamina into this alveolar clot. Initial pulmonary inflammation involves fluid exudation onto the surface of the lung, activation of the clotting system in former air space and interstitium, and recruitment and activation of polymorphonuclear leukocytes (PMNL) and mononuclear phagocytes. The exudate forms a clot in former air space, and if the clot persists, fibroblasts colonize it, where they proliferate and synthesize a provisional matrix. This constitutes a distinctive ‘‘subepithelial fibroblast focus’’ that is recognizable on hematoxylin-eosin staining by pulmonary pathologists, and is an important diagnostic and functional aspect of the cell biology of disease. Figure 2 shows such a proliferative lesion, and it is immediately distinctive because it consists of large, highly aligned ‘‘myofibroblasts’’ in a sparse matrix.


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Figure 2 A subepithelial fibroblast focus (sff ), a characteristic fibroproliferative lesion of interstitial fibrosis composed of highly-aligned myofibroblasts in a distinctive glycosaminoglycan-rich matrix in former air space; the fibroblast focus is stained metachromatically (purple) by toluidine blue O at pH 3.0, indicating a high concentration of glycosaminoglycans. We have found that the glycosaminoglycan is predominantly chondroitin sulfate/dermatan sulfate, by selective enzyme digestion using serial sections. (From Ref. 17.)

It is in these ‘‘active lesions’’ that fibroblast proliferation is inferred to occur, because of the increased number of fibroblasts, and the fact that these are often outside the original basal lamina and therefore in former air space and thus must have arisen by some combination of fibroblast migration and proliferation (6). The myofibroblasts express the gene for type I collagen (6,12), and the active lesions are recognized to have the capacity to evolve into deposits of relatively acellular collagen. In addition to being active in the synthesis of collagen, myofibroblasts stain for alpha smooth muscle actin and are contractile (13), and their contractility, together with the local replacement of the normal architecture of the lung with collagenous matrix, causes changes in tissue mechanics. Our studies of diagnostic biopsies and postmortem tissue samples from human patients with both nongranulomatous and granulomatous forms of interstitial lung disease have allowed us to fit altered proteoglycan and glycosaminoglycan metabolism into this paradigm. We studied glycosaminoglycans, collagen, and elastin by histochemistry, and versican, decorin, biglycan, and hyaluronan using specific antibodies, in conjunction with localization of alpha smooth muscle actin, sites of type I collagen synthesis, and cell populations in tissue sections.

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Biopsies from control patients were compared with those from individuals with usual interstitial pneumonia, bronchiolitis obliterans organizing pneumonia and organizing diffuse alveolar damage associated with fibroproliferative ARDS as the most common nongranulomatous disorders (14). Control biopsies were compared with those from patients with sarcoidosis, extrinsic allergic alveolitis, and tuberculosis as representative granulomatous conditions (15). Additionally, a study using the same procedures, but a more limited number of tissue samples from individuals who had died as a result of asthma, was performed (16). We found deposition of large amounts of glycosaminoglycan, corresponding to the deposition of versican, in association with hyaluronan in the active lesions (as defined by alpha smooth muscle actin-positive, alpha 1–type I collagen-snythesizing ‘‘myofibroblasts’’) of all of the major forms of granulomatous and nongranulomatous fibrotic lung disease. An example of a typical subepithelial fibroblast focus in IPF, showing metachromatic staining characteristic of glycosaminoglycans, is shown in Figure 3. Though the architecture of the remodeling lung associated with each disease process is of course different, the presence of versican within the fibroproliferative lesions was a constant feature of the earliest stages of the remodeling lesions in all of these diseases. More subtle changes in decorin and biglycan deposition were observed in all of the diseases; notably, decorin was localized intracellularly in α1(I) procollagen-positive myofibroblasts, consistent with an involvement in

Figure 3 Immunohistochemical localization of versican (A) and tenascin C (B) in serial sections from a patient with idiopathic pulmonary fibrosis. This patient exhibited the pathologic pattern of BOOP, characterized by a presence of a large number of even-aged intraluminal lesions. The intraluminal lesions stained intensely for versican and tenascin; colocalization is readily apparent throughout the biopsy at low magnification.


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collagen synthesis (14,15). The spatial and temporal association of versican with proliferating myofibroblasts suggested a specific involvement of versican in the cell biology of tissue remodeling in all of the human chronic inflammatory lung diseases that lead to fibrosis. In all of the fibrotic disorders, the deposition of glycosaminoglycans in the active lesions was dramatic and easily demonstrable by histochemistry (Fig. 3). We have used enzymatic degradation in situ to determine that the predominant glycosaminoglycans in these fibroproliferative lesions are chondroitin sulfate/dermatan sulfate and that the fibroproliferative lesions contain very little or no detectable heparan sulfate or perlecan as assessed by this technique (17,18). Only in fatal asthma was the disposition of glycosaminoglycans more subtle, but the association between versican and alpha smooth muscle actin-positive cells was constant. In biopsies collected prospectively from patients with early IPF, we found versican in association with fibroblasts that were apparently migrating into air space, associated with hyperplastic epithelium, and in the alveolar wall thickening in the pattern of desquamative interstitial pneumonia, believed to be a very early stage of IPF. Reverse-transcriptase PCR showed increased synthesis of versican mRNA splice variants V0 and V1 in these patients (17). It is instructive to compare and contrast the pattern of versican deposition in relation to collagenous fibrosis in these diseases. In organizing diffuse alveolar damage associated with adult respiratory distress syndrome (ARDS), histochemically increased deposition of glycosaminoglycan throughout the thickened alveolar walls occurred within 7–14 days of the onset of clinical symptoms. This clearly preceded collagen deposition in patients biopsied at an early stage in the process, though collagen type I gene expression was widespread in the myofibroblasts at this stage (14). It should be noted that though none of the patients studied in our series actually died as a result of ARDS or fibrosis, 30–50% of survivors typically show signs of long-term impairment due to fibrosis (19). Biopsies showing the pattern termed bronchiolitis obliterans organizing pneumonia (BOOP) showed a multitude of even-aged intraluminal lesions that were particularly versican rich (See Fig. 3). It should be noted that this pattern is associated with the best prognosis and best response to steroid treatment, and these lesions are believed to be reversible with and possibly without corticosteroid treatment. This implies degradation of versican and apoptosis of the myofibroblasts in patients whose lesions regress. In the patchy pattern characteristic of usual interstitial pneumonia we were able to observe active versican-rich lesions, areas of mature collagenous fibrosis, which appeared glycosaminoglycan and versican poor, and areas of normal lung and honeycombed end-stage lung, all in proximity to one another. The granulomatous processes similarly showed a tight temporal and spatial association between versican and hyaluronan, and the expression of type I collagen and alpha smooth muscle actin, in the rim of active remodeling that encapsulates the central core of the granuloma (15). As in IPF, in older,

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collagen-rich granulomatous lesions, there was far less versican or hyaluronan, and far fewer alpha-smooth muscle actin/collagen I–positive fibroblasts. It was clear from our studies that in the evolution of the remodeling lesion, which is a transient entity, there is a spatial and temporal association between the myofibroblast and versican (see Fig. 4 for a schematic representation). Versican’s localization in the normal lung is consistent with a role in smooth muscle function; versican is associated with airway and vascular smooth muscle, and with the alpha smooth muscle actin–positive cells that are believed to be contractile, in the alveolar entrance rings. Versican appears to be associated

Figure 4 Schematic representation of the remodeling process in interstitial fibrosis. Fibroblast proliferation is associated with versican deposition in the fibroproliferative lesion (see Fig. 3). Alpha smooth muscle actin (α-SMA)-positive myofibroblasts in a versicanrich matrix are abundant in the organizing diffuse alveolar damage lesions of fibroproliferative ARDS, in BOOP, and in the active lesions of usual interstitial pneumonia. In both BOOP and organizing diffuse alveolar damage, the remodeling is potentially reversible, but may proceed to fibrosis, which is characterized by deposition of large amounts of relatively acellular collagen. In the transition to a collagenous matrix, the myofibroblasts disappear, presumably by apoptosis, and the glycosaminoglycan-containing parts of versican are lost or resorbed. Fibroblast proliferation, versican synthesis, and HA synthesis are regulated by PDGF. Fibroblast expression of alpha smooth muscle actin, versican, and collagen is stimulated by TGFβ.


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with elastic fibers throughout the lung (14,17). Versican’s association with this cell type is consistent with the hypothesis that the myofibroblasts whose number is increased following chronic inflammation arise from this preexistent pool of versican-positive, alpha smooth muscle actin–positive cells in airways and/or lung parenchyma/microvascular. That smooth muscle hypertrophy occurs in IPF (see Fig. 1B, for instance), even though this is underrecognized, suggests that local growth factors that promote the proliferation of cells of the smooth muscle/ myofibroblast lineage act on smooth muscle in diseases also. III. Functions of Versican in Provisional Matrix Though a number of specific functions for versican have been identified and continue to be discovered, it is becoming apparent that versican has a multitude of functions in the provisional matrix. Just as the basal lamina or cartilage matrices have a multitude of functions, so the matrix of fibroproliferative lesions in the lung may be a distinct matrix with a set of specific biological activities. Indeed, the number of biological processes that versican can influence suggests that the name versican, which is derived from ‘‘versatile proteoglycan’’ (20), is prophetic. Versican is a member of a family of large hyaluronan-binding proteoglycans of which aggrecan is the archetype, and which have been termed hyalectans or lecticans [see Iozzo, 1998 (21), for a review of classification and structure and function]. Four isoforms of a single human versican gene (22) are generated by alternative splicing of the pre-RNA (23). Two glycosaminoglycan attachment domains (GAGα and GAGβ) are differentially expressed in the four variants. V0 contains both GAGα and GAGβ; V1 has GAGβ; V2, GAGα; and V3 contains neither glycosaminoglycan attachment domain. All variants have a hyaluronanbinding domain (‘‘G1’’) near the N-terminus (24) and a carboxy-terminal domain (‘‘G3’’) composed of two epidermal growth factor–like (E), one lectinlike (L), and one complement regulatory protein–like (C) nodule. Versican is the only member of this family of proteoglycans that shows significant alternative splicing, and the significance of tissue-specific expression of different mRNA splice variants is unknown. However, ectopic expression of V2 in the vascular wall has recently been shown to have a profound effect on elastin accumulation (see Chapter 14, in this volume). A. Versican as a Structural Element

Versican V0 variant has an estimated molecular mass of ⬎1,000,000 and, as an aggregate with hyaluronan, could form a ‘‘scaffolding’’ with which other glycoproteins and cells could interact. Aggrecan and hyaluronan form a hydrated matrix in which type II collagen fibrils are embedded, and this is the major structural

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element of cartilage. Such a versican-hyaluronan aggregate would likely contribute to the local compressive modulus of tissue, as does aggrecan-hyaluronan in cartilage; however, mechanical and other properties depending on charge density would be affected by the versican splice variants involved, and their glycosylation—and could be locally variable. We have shown that degradation of human airway smooth muscle-associated matrix is associated with decreased passive tension and alterations in smooth muscle contractility that are consistent with this matrix exhibiting resistance to compression (25) and modulating smooth muscle contractility. We have discussed the likely functional consequences of pathological deposition of a versican-rich matrix in the walls of the airways in human airway disease (26,27). Although airway wall thickening, which involves deposition of proteoglycan, could narrow the airways and increase resistance to airflow, proteoglycan-mediated changes in the local matrix of the smooth muscle cells themselves could have geometric, local mechanical, or cell biological consequences that could either exacerbate airway resistance after allergen challenge or inhibit smooth muscle contraction, so the functional consequences of airway remodeling require further specific study. B. Versican-Hyaluronan Matrix

Hyaluronan and versican (14), tenascin C, thrombospondin, and SPARC (28) together with fibronectin are found in the active fibroproliferative lesions. Tenascin, thrombospondin, and SPARC are considered ‘‘antiadhesive’’ and, like versican, are expressed during morphogenesis. Tenascin and versican appear to closely colocalize in the remodeling lung; Figure 3 shows immunolocalization of versican (panel a) and tenascin (panel b) in serial sections from a lung biopsy from an individual with BOOP. Each major component of this matrix binds at least two other components (see 29–32 for discussions of the binding properties of fibronectin, versican, and tenascin). Thus, the provisional matrix can be considered a gel with multiple cell-, growth factor –, and protein-binding activities. Recent data show that a number of growth factors and cytokines bind versican, and that versican-rich matrices may concentrate these signals and modulate their biological activities, which may also contribute to the ability of provisional matrix to support mesenchymal cell proliferation. Versican has been shown to bind a range of chemokines including secondary lymphoid chemokine and stromal-derived factor (SDF)1β (33), and we have recently shown that binding of SDF1 to matrix proteoglycans does not inhibit processing by matrix metalloproteinases (34). C. Versican as a Modulator of Cell Adhesion, Migration, and Proliferation

Provisional matrix appears to facilitate migration and proliferation of myofibroblasts, and it is in this matrix in fibroproliferative lung disease that they begin


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to synthesize and assemble collagen fibers. As cells isolated from their normal matrix usually undergo apoptosis or anoikis, the provisional matrix in chronic inflammatory sites presumably contains factors that support cell survival and proliferation. Such signals may include integrins or other matrix receptors, and growth factors that specifically localize to the provisional matrix. Myofibroblasts express the fibronectin receptor integrin α5β1 (35). Fibroblasts isolated from individuals with ARDS have been shown to use the hyaluronan receptor CD44 to invade fibronectin- and hyaluronan-containing matrices in vitro (36), suggesting that hyaluronan receptors may be important as fibroblasts invade exudates in vivo. In 1996 we showed that versican is found with hyaluronan in the thickened alveolar wall matrix in ARDS (14) and in the early stages of the other fibroproliferative lesions (14,15). In biopsy samples from individuals with early, active disease we have subsequently found that fibroblasts apparently migrating into air spaces at the earliest stages of remodeling are surrounded by versican (17). As CD44 is now known to bind both versican and hyaluronan (37), it is possible that versican binding may complement or modulate in some way CD44mediated adhesion and migration. Versican is associated with cell proliferation in atherosclerosis lesions (38,39). Versican-hyaluronan-rich pericellular matrices have been shown to promote migration and proliferation of vascular smooth muscle cells (40). Versican is associated with healing skin wounds and the stromal reaction to tumors (41). Inhibition of versican expression by transfection of transformed cells with antisense RNA reduced cell proliferation and increased attachment to near-normal levels (42), consistent with a functional role for versican in enhancing cell proliferation. An engineered chimeric molecule called ‘‘miniversican’’ has been shown to modestly stimulate cell proliferation, an effect dependent on its EGF-like modules (43), and to inhibit mesenchymal chondrogenesis (44). Versican gene ablation is lethal in development, causing cardiac malformation (45). This effect is believed to be secondary to a defect in cell migration in endocardial cushion formation in heart development. Versican inhibits adhesion of cells to other matrix molecules (46) and has been considered antiadhesive; versican appears to antagonize some integrinligand interactions, and this could modulate gene expression and cellular activities. A plausible mechanism for antiadhesive activity is simple steric hindrance of the interaction of other ligands with their receptors, given the huge hydrodynamic size of this ⬎1,000,000-dalton molecule, and still larger macromolecular aggregates that it may form with hyaluronan. Such antagonism would occur in localized cell surface zones and change the affinity or number of integrins occupied by ligand. It has been shown in a series of elegant experiments that subtle changes in integrin-ligand binding properties influence cell migration speed (47); thus, subtle changes in integrin binding consequent to the interaction of a large proteoglycan at the cell surface could have similar effects. Conversely, versican has

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some positive effects on adhesion of specific cell types: versican exhibits low affinity binding to selectins and CD44, mediated by its chondroitin sulfate chains (37). Chondroitin sulfate binds to α4β1-integrin (48), but conversely versican inhibits α4β7-integrin-mediated adhesion to mucosal adressin cell adhesion molecule-1, suggesting versican is involved in local modulation of cell matrix adhesion in inflammation (33). Versican’s lectinlike domain may bind heparin sulfate and sulfated glycolipids at the cell surface (49). The diversity of effects described suggest that proteoglycan–cell surface interactions in the remodeling lung have effects that are cell and possibly proteoglycan/glycosaminoglycan specific. The interaction of another lectican or hyalectan family member, neurocan, with a cell surface glycosyltransferase inhibits both β1-integrin-mediated and Ncadherin-mediated adhesion and neurite outgrowth (50). Indeed, versican, neurocan, and brevican are believed to have important morphogenetic functions in the brain (51), consistent with a range of important regulatory functions for this class of proteoglycans that are the subject of active current research. Tenascin C colocalizes with versican in development (52) in healing wounds and the matrix of malignant tumors (53–55). Tenascin-fibronectin complexes (but not either alone) stimulate secretion of matrix-degrading metalloproteinases by fibroblasts (56). Proteinases so induced might contribute to the removal of this versican-tenascin-fibronectin ‘‘provisional’’ matrix, a process that must occur in the evolution of the lesions to form areas of collagen-rich and proteoglycan-poor fibrosis (14,15,17). Concomitantly the myofibroblasts disappear, presumably by apoptosis. The evolution of the lesions from versican rich, collagen poor to collagen rich and versican poor appears common to the remodeling process driven by chronic inflammation, whether the cause is acute severe injury as in ARDS (14), idiopathic BOOP or IPF (14), allergic injury in extrinsic allergic alveolitis (15), or encapsulation of a chronic inflammatory stimulus such as Mycobacterium tuberculosis (15). The proteinases involved in these processes are unknown, but versican is known to be cleaved by macrophage MMP7, an event that may be associated with atherosclerotic plaque lysis (57), and versican V1 has recently been shown to be cleaved by ADAMTS-1 and -4 in artery walls (58).

IV. Platelet-Derived Growth Factor and Transforming Growth Factor Beta Expression of platelet-derived growth factor (PDGF) is increased in IPF (59), and PDGF is localized to the fibroblast foci (60) and to sites of injury in experimental asbestos-induced fibrosis (61). PDGF is a stimulator of smooth muscle cell proliferation (62), hyaluronan synthesis, and versican synthesis (63). Transforming growth factor beta (TGFβ) is also found in the fibroblast foci (64,65), and is


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Figure 5 Matrix changes in fibroproliferative lesions in lung fibrosis. The major cell biological synthetic processes in lung remodeling—cell proliferation, hyaluronan deposition, versican deposition, and collagen deposition—can be controlled by PDGF and TGFβ in vitro. In contrast, the control of apoptosis and provisional matrix degradation are not understood.

synthesized by regenerating epithelial cells in IPF (64). Indeed, thrombospondin, a component of the provisional matrix that seems to underlie regenerating epithelium (27), appears to be able to activate TGFβ (66), as can integrin αVβ6 (67), which may release TGFβ from its latent complex at remodeling sites. TGFβ1 induces expression of the genes for alpha smooth muscle actin (67) and a number of matrix proteins, including hyaluronan (68), versican (63), and collagen. Mononuclear phagocytes have been shown to overproduce PDGF (59) and TGFβ (64) in human pulmonary fibrosis, and to be able to activate TGFβ1 in a process that involves endogenous plasmin (69). Thus, PDGF and TGFβ, released by macrophages locally, could induce cell proliferation, expression of alpha smooth muscle actin, and synthesis of hyaluronan and versican in target mesenchymal cells (see Fig. 5). Both TGFβ and PDGF are strongly linked to cell proliferation and accumulation of ECM in wound healing and fibrosis in a number of organs, and modulation of their biological activities is a key therapeutic goal for therapy of many diseases that involve aberrant matrix synthesis.

V.

Parallels Between Pulmonary Fibrosis and Wound Healing in Other Systems

In wound healing, a well-established sequence of events occurs (Fig. 6). Cellular injury and vascular leak are followed by platelet activation, blood clotting, and release of mediators of acute inflammation. Polymorphonuclear leukocytes (PMNL) and macrophages enter the exudate; PMNL endocytose and kill micro-

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Figure 6 The processes involved in wound healing. The relationship between wound healing in the lung and the pathological process leading to fibrosis is discussed in the text.

organisms, and macrophages remove erythrocytes, fibrin, and cellular debris. Release of plasmin, from plasminogen activator, by macrophages is important in this process. Fibroblasts proliferate and migrate into the area of the exudate and lay down a new extracellular matrix; these cells have a distinct phenotype and are known as myofibroblasts. Myofibroblasts express alpha smooth muscle actin, resemble smooth muscle cells ultrastructurally, and have been shown to contract in response to agonists that cause smooth muscle contraction (70,71). These cells not only synthesize and assemble structural elements of the matrix, but also contract the granulation tissue, a phenomenon important in closing the wound. The origin of the myofibroblasts that are central to wound healing is controversial. They may be derived from tissue fibroblasts, from expansion of a preexistent pool of alpha smooth muscle actin–positive connective tissue cells, such as pericytes, in the microvasculature, as occurs in the skin (72) and liver (73), from ‘‘myofibroblasts’’ in the subepithelial matrix of the airways of the lung (74), or, even more controversially, from a circulating pool of mesenchymal cells originating in the bone marrow (75). Though macrophages are still generally considered to direct the remodeling process, wounds in animals depleted of macrophages were shown to heal adequately, though leaving a repair cluttered with debris, many years ago (62). This indicates that myofibroblast proliferation, migration, and new matrix synthesis can occur, at least in the skin, independently of macrophage-derived factors. The signals that stimulate the proliferation of connective tissue cells and the generation of myofibroblasts are incompletely understood. However, a relatively simple mechanism exists that could be central in inducing the generation of myofibroblasts adjacent to the exudate, their proliferation, and synthesis of new matrix. Thrombin, produced from its zymogen in the coagulation cascade, is a strong inducer of fibroblast and smooth muscle cell proliferation (through its actions on proteinase-activated receptors (PAR1 and PAR2) (76), and thrombin releases the smooth muscle cell mitogen PDGF from


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matrix stores by proteolysis (77). Thrombin also induces synthesis of TGFβ by smooth muscle cells (78). Though other schemes are possible, this relatively simple mechanism links coagulation and inflammation to the production of the mitogen PDGF and the production of TGFβ, a potent inducer of both the expression of alpha smooth muscle actin and matrix accumulation. Such a mechanism depends on competent mesenchymal cells in vivo responding to thrombin as cultured smooth muscle cells do in vitro. After the granulation tissue has been replaced by collagen-rich scar tissue, the myofibroblasts disappear, and this appears to involve apoptosis (79) rather than a change to the fibroblast phenotype, since there is a decrease in cellularity at this stage. The cellular signals that control this process are as yet unknown, but are of great interest in seeking to control tissue repair and fibrosis. VI. Conclusions Why does chronic inflammation lead to pulmonary fibrosis, rather than wound healing in the lung? Lung architecture can return to normal after inflammation and fluid exudation; most cases of pneumonia resolve successfully. Pulmonary fibrosis is considered by many to be an example of wound repair that is aberrant because of some combination of the magnitude, kind, and repetitive nature of the injury, or defective removal of the initial clot. A number of aspects of the healing process in chronic lung inflammation have been identified as potentially abnormal, and these may contribute to the development of fibrosis rather than regenerative wound healing. A. Persistence of Initial Injury

The initiating stimulus is clearly likely to be persistent in forms of lung fibrosis that have a major autoimmune component, such as scleroderma, or where exposure to antigen is not discontinued, as is the case in allergic alveolitis. The pneumoconioses are initiated by toxic particles that cannot be effectively neutralized by phagocytes or other cells, and animal studies convincingly demonstrate that rapid generation of growth factors including PDGF at the site of injury is linked to fibrotic changes (61,80). Such stimulation likely persists as long as the particle remains in the lung—i.e., indefinitely. B. Magnitude of Initial Injury

The studies of Vracko (81,82), using a model in which lung inflammation in dogs was elicited by increasing doses of oleic acid, showed that a greater degree of

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damage to the subepithelial basal lamina correlated with the likelihood that complete restoration of lung architecture could not occur. He suggested that the basal lamina provides information to epithelium and mesenchymal cells that defines the original architecture of the lung, and that loss of this information (implicitly, due to matrix degradation in inflammation) results in uncontrolled repair. In support of the hypothesis that fibrosis may result from excessive proteolysis in inflammation, fibrosis is a frequent consequence of ARDS, in which polymorphonuclear leukocytes arrest in large numbers in the lung and release large amounts of proteolytic enzymes. Consistent with the hypothesis that basal lamina proteolysis is an important determinant of progression to fibrosis, holes have been shown to appear in the alveolar basal lamina early in IPF (9). This mechanism may be most relevant to ARDS-initiated fibrosis, where initial injury is widespread and severe and leads to significant fibrosis in 50% of survivors. C. Multiple Modes of Tissue Injury

Hatschek and Witschi (10) showed that a combination of inflammation and prevention of epithelial regeneration leads to fibrosis, consistent with the hypothesis that multiple hits are required to tip the balance from repair to fibrosis. D. Slower-Than-Normal Clot Dissolution

The dissolution of the fibronectin-fibrin clot is believed to be important in promoting resolution (11). Alveolar fibrinolytic activity is diminished in human IPF (83), and there is evidence from animal studies that increased susceptibility to fibrosis is associated with a decreased propensity to synthesize plasminogen activator. The implication of this is that the longer a fibrin-rich clot covers the original epithelium, the more likely it is that this will be colonized by fibroblasts and ultimately be co-opted as part of the collagenous matrix of the lung, as opposed to being cleared and architecture returned to normal. E.

The Remodeling Process in the Lung

In a seminal paper comparing wound healing in fetal skin, adult oral mucosa (neither of which leaves a scar), and adult skin (which does scar), Whitby and Ferguson (84) showed that the only clear feature shared by the former two tissues and not by the latter was an ordered sequence of deposition and removal of large amounts of glycosaminoglycan-rich matrix. The authors speculated that the glycosaminoglycan-rich matrix in fetal skin and in the oral cavity allows migration and organization of the tissue by fibroblasts in remodeling, and allows regenerative wound healing. Although the proteoglycans involved have yet to be identified, a critical reading of this manuscript suggests the major proteoglycan in oral wound healing is versican, and our recent studies in oral tissues confirm this


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inference (85). In common with oral and fetal wound healing, glycosaminoglycan deposition in early lung remodeling is dramatic (14,15)—certainly to a greater degree than is seen in skin wound healing. If deposition of large amounts of glycosaminoglycan are characteristic and necessary for regenerative wound healing, as postulated by Whitby and Ferguson (84), our studies in the early remodeling process in the human lung suggest that the ‘‘normal’’ healing process in the lung is regenerative wound healing. Indeed, it would be logical for oral and pulmonary tissues to share pathways of wound healing, as the lungs develop from the presumptive digestive tract during development. As in other systems, including morphogenesis in the nervous system, the precise roles of versican and other proteoglycans in wound healing and fibrosis in the lung remain to be elucidated, and remain a subject of active research in the near future. There is a challenge inherent in this; the lung can be an exceedingly difficult organ in which to determine the function of molecules, yet tissuespecific differences in the wound healing process make it very important to keep the specific cell biology of the lung, and the nature of the serious human disease processes that involve lung remodeling, in focus. Acknowledgments Work in the author’s laboratory is supported by grants from the Medical Research Council of Canada (MT-15171), the British Columbia Lung Association, and the British Columbia Health Research Foundation. Dr. Clive Roberts is a Canadian Institutes of Health Research/British Columbia Lung Association Scientist. References 1. Coultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994; 150:967–972. 2. Tuberculosis. International Union Against TB and Lung Disease Update: http:/ / www.iuatld.org and http:/ /www.cdc.gov/nchstp/tb. 3. Panos RJ. Therapy and management of idiopathic pulmonary fibrosis. Comp Ther 1994; 20:289–293. 4. Crystal RG, Gadek JE, Ferrans VJ, Fulmer JD, Line BR, Hunninghake GW. Interstitial lung disease: current concepts of pathogenesis, staging and therapy. Ann J Med 1981; 70:542–568. 5. Kern DG, Crausman RS, Durand KTH, Nayer A, Kuhn C III. Flock worker’s lung: chronic interstitial lung disease in the nylon flocking industry. Ann Intern Med 1998; 129:261–272. 6. Kuhn C, McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol 1991; 138:1257–1265.

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7. Cantin AM, North SL, Fells GA, Hubbard RC, Crystal RG. Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis. J Clin Invest 1987; 79:1665– 1673. 8. Raghu G, Striker LJ, Hudson LD, Striker GE. Extracellular matrix in normal and fibrotic human lungs. Am Rev Respir Dis 1985; 131:281–289. 9. Haschek WM, Witschi H. Pulmonary fibrosis—a possible mechanism. Toxicol Appl Pharmacol 1979; 51:475–487. 10. McDonald JA. The yin and yang of fibrin in the airways. N Engl J Med 1990; 322: 929–931. 11. Kuhn C, Boldt J, King TE, Crouch E, Vartio T, McDonald JA. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989; 140:1693–1703. 12. Zhang K, Rehkter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis—a combined immunohistochemical and in situ hybridization study. Am J Pathol 1994; 145:114–125. 13. Adler KB, Low RB, Leslie KO, Mitchell J, Evans JN. Contractile cells in normal and fibrotic lung. Lab Invest 1989; 60:473–485. 14. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycan deposition in lung fibrosis. Am J Respir Crit Care Med 1996; 154:1819–1828. 15. Bensadoun ES, Burke AK, Hogg JC, Roberts CR. Proteoglycans in granulomatous lung diseases. Eur Respir J 1997; 10:2731–2737. 16. Roberts CR. Is asthma a fibrotic disease? Chest 1995; 107:111S–117S. 17. Roberts CR, Burke AK. 2001. Localization and synthesis of the proteoglycan versican in the normal and remodeling human lung. (Manuscript submitted.) 18. Roberts CR, Dodge GR. Perlecan in the normal and remodelling human lung. (Unpublished data.) 19. Fraser RG, Pare JA, Pare PD, Fraser RS, Genereux GP. Tuberculosis. In: Diagnosis of Diseases of the Chest. Philadelphia: W.B. Saunders, 3rd ed, 1991. 20. Zimmermann DR, Ruoslahti E. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 1989; 8:2975–2981. 21. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998; 67:609–652. 22. Naso MF, Zimmermann DR, Iozzo RV. Characterization of the complete genomic structure of the human versican gene and functional analysis of its promoter. J Biol Chem 1994; 269:32999–33008. 23. Dours-Zimmermann MT, Zimmermann DR. A novel glycosaminoglycan attachment domain identified in two alternative splice variants of versican. J Biol Chem 1994; 269:32992–32998. 24. LeBaron RG, Zimmermann DR, Ruoslahti E. Hyaluronate binding properties of versican. J Biol Chem 192; 267:10003–10010. 25. Bramley AM, Roberts CR, Schellenberg RR. Collagenase increases shortening of human bronchial smooth muscle in vitro. Am J Respir Crit Care Med 1995; 152: 1513–1517. 26. Roberts CR, Okazawa M, Wiggs BR, Pare PD. Airway wall thickening in asthma. In: PJ Barnes, MM Grunstein, A Leff, AJ Woolcock, eds. Asthma. New York: Raven Press, 1997.


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27. Pare PD, Bai TR, Roberts CR. The structural and functional consequences of chronic allergic inflammation of the airways. In: The Rising Trends in Asthma. Ciba Foundation Symposium 206. New York: John Wiley & Sons, 1997: 71–86. 28. Kuhn C, Mason RJ. Immunolocalization of SPARC, tenascin and thrombospondin in pulmonary fibrosis. Am J Pathol 1995; 147:1759–1769. 29. Yamada KM. Fibronectin. In: Hay ED, ed. Cell Biology of Extracellular Matrix. New York: Plenum, 1991. 30. Yamagata M, Yamada KM, Yoneda M, Suzuki S, Kimata K. Chondroitin sulfate proteoglycan (PG-M-like proteoglycan) is involved in the binding of hyaluronic acid to cellular fibronectin. J Biol Chem 1986; 261:13526–13535. 31. Ujita M, Shinomura T, Ito K, Kitagawa Y, Kimata K. Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chondroitin sulfate proteoglycan. J Biol Chem 1994; 269:27603–27609. 32. Aspberg A, Miura R, Bourdoulous S, Shimonaka M, Heinegard D, Schachner M, Ruoslahti E, Yamaguchi Y. The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety. Proc Natl Acad Sci USA 1997; 94:10116–10121. 33. Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M. Versican interacts with chemokines and modulates cellular responses. J Biol Chem 2001; 276:5228–5234. 34. McQuibban GA, Butler GS, Gong J-H, Bendall L, Power C, Clark-Lewis I, Overall CM. Matrix metalloproteinase activity inactivates the C-X-C chemokine stromal cell-derived factor 1. J Biol Chem 2001; 276:43503–43508. 35. Fukuda Y, Basset F, Ferrans VJ, Yamanaka N. Significance of early intra-alveolar fibrotic lesions and integrin expression in lung biopsy specimens from patients with idiopathic pulmonary fibrosis. Hum Pathol 1995; 26:63–61. 36. Svee K, White J, Vaillant P, Jessurun J, Roongta U, Krumwiede M, Johnson D, Henke C. Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44. J Clin Invest 1996; 98:1713–1727. 37. Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AHK, Miyasaka M. Binding of a large chondroitin sulfate/deramatan sulfate proteoglycan, versican, to L-selectin, P-selectin and CD44. J Biol Chem 2000; 275:35448–35456. 38. Wight TN. Cell biology of arterial proteoglycans. Arteriosclerosis 1989; 9:1–20. 39. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor beta. Am J Pathol 1998; 152:533–546. 40. Evanko SP, Angello JC, Wight TN. Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999; 19:1004–1013. 41. Nara Y, Gao M, Ikeda K, Sato T, Sawamura M, Kawano K, Yamori Y. Genetic analysis of non-insulin-dependent diabetes mellitus in the Otsuka Long-Evans Tokushima fatty rat. Biochem Biophys Res Commun 1997; 241:200–204. 42. Yamagata M, Kimata K. Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican. J Cell Sci 1994; 107:2581–2590.

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43. Zhang Y, Cao L, Yang BL, Yang BB. The G3 domain of versican enhances cell proliferation via epidermal growth factor–like motifs. J Biol Chem 1998; 273: 21342–21351. 44. Zhang Y, Cao L, Kiani CG, Yang BL, Yang BB. The G3 domain of versican inhibits mesenchymal chondrogenesis via the epidermal growth factor–like motifs. J Biol Chem 1998; 273:33054–33063. 45. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol 1998; 202:56–66. 46. Yamagata M, Suzuki S, Akiyama SK, Yamada KM, Kimata K. Regulation of cellsubstrate adhesion by proteoglycans immobilized on extracellular substrates. J Biol Chem 1989; 264:8012–8018. 47. Palecek SP, Loftus JC, Ginsberg MH, Lauffenburger DA, Horwitz AF. Integrinligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 1997; 385:537–540. 48. Iida J, Meijne AML, Oegema TR, Yednock TA, Kovach NL, Furch LT, McCarthy JB. A role of chondroitin sulfate glycosaminoglycan binding site in α4β1 integrinmediated melanoma cell adhesion. J Biol Chem, 1998; 273:5955–5962. 49. Li H, Leung T-C, Hoffman S, Balsamo J, Lilien J. Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan. J Cell Biol 2000; 149:1275–1288. 50. Miura R, Aspberg A, Ethell IM, Hagihara K, Schnarr RL, Ruoslahti E, Yamaguchi Y. The proteoglycan lectin domain binds sulfated cell surface glycolipids and promotes cell adhesion. J Biol Chem 1999; 274:11431–11438. 51. Bandtlow CE, Zimmermann DR. Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol Rev 2000; 1267–1290. 52. Shinomura T, Jensen K, Yamagata M, Kimata K, Solursh M. The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth. Anat Embryol 1990; 181:227–233. 53. Erickson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumours. Annu Rev Cell Biol 1989; 5:71–92. 54. Yeo T-K, Brown L, Dvorak HF. Alterations in proteoglycan synthesis common to healing wounds and tumours. Am J Pathol 1991; 138:1437–1450. 55. Isogai Z, Shinomura T, Yamakawa N, Takeuchi J, Tsuji T, Heinegard D, Kimata K. 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumours is a large chondroitin sulfate proteoglycan, PG-M/versican. Cancer Res 1996; 56:3902–3908. 56. Tremble P, Chiquet-Ehrismann R, Werb Z. The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. Mol Biol Cell 1994; 5:439–453. 57. Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci USA 1996; 93: 9748–9753. 58. Sandy JD, Westling J, Kenagy RD, Iruela-Arispe M, Verscharen C, Rodrique-


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11 Decorin in Asthma

ANTHONY E. REDINGTON Castle Hill Hospital University of Hull Cottingham, East Yorkshire, England

I.

Introduction

Decorin is a prototype member of a group of structurally related molecules referred to as small leucine-rich proteins (SLRPs), most but not all of which are proteoglycans (PGs) (1–3). SLRPs are characterized primarily by a central region comprising a variable number of leucine-rich repeats (LRRs). At least 12 members of the family are known, with biglycan and the recently cloned asporin (4) most closely related to decorin. This chapter will first consider pertinent aspects of the molecular biology of decorin and discuss its ability to bind to multiple components of the extracellular matrix (ECM) and to the fibrogenic cytokine transforming growth factor beta (TGFβ). The presence of airway fibrosis in asthma and its relation to disease pathophysiology will be examined. Studies of the in vivo expression of decorin and TGFβ in normal and asthmatic airways will next be discussed, with emphasis on the colocalization of these two molecules in the mucosa and its significance. Finally, the potential for decorin to provide a novel therapeutic agent in asthma will be briefly considered. A proposal has recently been made to use the term “decoron” to refer specifically to the decorin core protein, with “decorin” reserved for the intact PG 213

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(5). This system has not yet been widely adopted, however, and therefore will not be used in this chapter.

II. Molecular Biology of Decorin Decorin and biglycan were originally identified as two electrophoretically distinct species of small PG isolated from several sources including fetal bovine bone (6), bovine cartilage (7), and human fetal membranes (8). The larger and the smaller of the two species were named dermatan sulfate (DS)-PG I and PG II, respectively (7). Biochemical studies of purified PG II suggested a core protein of molecular weight ⬃ 40 kDa attached to a single chondroitin sulfate (CS)-DS glycosaminoglycan (GAG) chain (6,7). PG II was later renamed decorin on account of its ability to “decorate” the surface of collagen fibrils (see below). Cloning of decorin core protein cDNA from human fibroblasts predicted that the molecule is synthesized as a prepro form of 359 amino acids and molecular weight of 39.7 kDa (9). The primary sequence can be divided into a number of distinct domains: a 16–amino acid signal peptide; a 14–amino acid propeptide that may act as a recognition site for xylosyltransferase (10,11); the GAG attachment site; an N-terminal cysteine-rich globular domain that possesses Zn2⫹-binding activity (12); a central domain composed of 10 tandem LRRs of length 21– 26 amino acids; and a relatively large C-terminal globular domain containing two cysteine residues. Decorin cDNA has been cloned from tissues of several other species, including bovine bone (13), murine 3T3 fibroblasts (14), rat vascular smooth muscle (15), and chicken cornea (16). Comparison of the predicted core protein primary sequences reveals a high degree (⬃80%) of species conservation, particularly in the central LRR region. The posttranslational modifications of decorin are complex and variable. The single CS-DS GAG chain is attached to the serine residue at position 4 of the mature protein (17). The length of this chain varies considerably in decorin extracted from different tissues, as does its degree of epimerization and sulfation (18). CS and DS are the predominant forms in decorin extracted from bone and skin, respectively. Three consensus sites for potential N-linked glycosylation are present at positions 181, 232, and 273 in the central domain of the core protein, but there is differential substitution with either two or three of these sites used (19,20). Oligosaccharides may be of both the complex and the high-mannose types (10,19). Finally, the core protein contains phosphorylated serine (21) and sulfated tyrosine (22) residues. Using somatic cell hybrids and Southern blot analysis, the human decorin gene was first mapped to a relatively broad region of chromosome 12 (23). In situ hybridization studies later refined the chromosomal assignment to 12q21.3 (24–26). The human decorin gene spans over 38 kb of DNA sequence, and is

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therefore relatively large compared to the exonic sequence and to the number of amino acids of the translated protein (25,26). The gene contains two leader exons in its 5′-untranslated region, designated exon Ia and exon Ib, that can be alternatively spliced to exon II (25). Analysis of the 5′-flanking DNA sequence to exon 1a, however, suggests that this region is not functional as a decorin promoter (27). In contrast, the region 5′ to exon Ib exhibits strong basal and inducible promoter activity, and contains several elements known to play a role in transcriptional regulation (27). These include two TATA-like motifs, a CAAT box, and binding sites for the transcription factors AP-1, AP-5, and NF-κB. A 48-bp segment of the proximal promoter, encompassing a consensus AP-1-binding site, has been shown to function as a bimodal transcriptional regulator of decorin gene expression, allowing both induction by interleukin (IL-1) (28) and repression by tumor necrosis factor alpha (TNFα) (29). The proximal promoter also contains a composite element— harboring a vitamin D receptor–retinoic acid receptor motif—that mediates the inhibition of decorin gene expression by TGFβ (see below) (30). Under cell culture conditions, decorin is efficiently internalized by a variety of cells of mesenchymal origin. This process has been studied most intensively in cultured human skin fibroblasts, which recapture a substantial proportion of the secreted PG by receptor-mediated endocytosis (31). Internalization of decorin is followed by transport to the lysosomal compartment where it undergoes degradation to its monomeric constitutents. Endocytosis involves recognition of the core protein (32), and a preferential site for binding to the endocytosis receptor has been localized to the central LRR structures (33). Two proteins of 51 kDa and 26 kDa that are present in endosomes and at the plasma membrane have been considered as endocytosis receptors due to their high-affinity binding of decorin core protein (34). The 26-kDa protein is immunologically related to the 51-kDa protein, and has been proposed therefore to represent a degradation product of the larger molecule (35). The relevance of the secretion recapture pathway in vivo is uncertain. It is conceivable that it provides a mechanism to regulate the amount of extracellular decorin in relation to the decorin-binding capacity of the local ECM constituents.

III. Interactions Between Decorin and the Extracellular Matrix (ECM) An important property of decorin is its ability to bind with relatively high affinity (kd values in the nanomolar range) to multiple components of the ECM. In vitro interactions have been reported with: the fibrillar collagen types I (36,37), II (37), V (38), and VI (39); the nonfibrillar fibril-associated collagen type XIV (40, 41); fibronectin (42,43); tenascin-X (44); thrombospondin-1 (45); and the elastinassociated microfibril protein fibrillin-1 (46).

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Ultrastructural examination of intact tissues has confirmed a close relationship in vivo between decorin and collagen fibrils. Studies using electron microscopy (EM) and the electron-dense dye Cupromeronic Blue first demonstrated an association between DS-PG and collagen fibrils in various nonmineralized type I collagen-rich connective tissues, including tendon, skin, and cornea (47–49). The PG was present on the surface of fibrils of different diameters along their entire length. Studies with antibodies specific for decorin core protein later reported similar appearances in tendon (50) and dermal (51) collagen. In these studies, the axial distance between antigens conformed to the 60–65 nm Dperiodicity of the collagen fibril, with most labeling localized to the d and e bands in the gap region of each D-period (50,51). A more recent report, however, has suggested a region corresponding to the c1 band of the overlap area as the likely site of decorin binding, in very close proximity to one of the two major intermolecular crosslinking sites (52). When stained with Cupromeronic Blue, the GAG side chains of decorin appear as orthogonally arranged filaments, briding between and across collagen fibrils (53,54). These interfibrillar PG ties—so-called “shape modules”—are thought to maintain the orientation of collagen fibrils by holding them at defined distances from one another. Experimental support for this suggestion derives from ultrastructural analysis of the ECM produced in vitro by fibroblasts that are unable to express decorin mRNA (55). The molecular basis of the interaction between decorin and collagen type I has been partially characterized. Despite their significant homology, decorin binds specifically to the α1(I) chain but not the α2(I) chain of collagen type I (52). Early work established that binding is mediated by the core protein, as it was not eliminated by removal of the GAG chain with chondroitinase ABC (56) or by proteolytic cleavage of a 17–amino acid N-terminal fragment of core protein containing the GAG attachment site (57). Several studies using a variety of approaches localized the high-affinity binding sites for collagen type I to LRRs 4–6 (58–60). Molecular modeling of the three-dimensional structure of decorin, based on x-ray crystallographic data for another SLRP, the porcine ribonuclease inhibitor (61,62), predicted that the molecule would be arch shaped (63). This was confirmed by direct visualization using rotary shadowing EM (52,64). The dimensions of the inner concave surface of the arch are adequate to accommodate one triple helix of collagen type I (63). The consequences of interactions between decorin and collagen have been explored in an in vitro fibrillogenesis assay. In this system, decorin exerts a specific inhibitory effect on the rate of collagen type I fibril formation (56). Furthermore, the average diameter of collagen type I fibrils formed in the presence of decorin is reduced (65). Convincing evidence that the binding of decorin to fibrillar collagen has important biological consequences in vivo derives from the phenotype of mice with a targeted disruption at the decorin gene locus (66). These animals are viable, but their skin is thin, lax, and fragile, and has markedly re-

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duced tensile strength. Ultrastructural analysis reveals abnormal collagen morphology: fibrils are coarse, irregular in size and shape, and often haphazardly arranged, apparently owing to uncontrolled lateral fusion. Together, these experiments support an important regulatory role for decorin in the supramolecular organization of collagen during matrix assembly. There is less information regarding the nature and functional significance of interactions between decorin and other ECM components. Binding to collagen type VI in solid-phase assays is mediated by the decorin core protein (39,67), as is the case with collagen type I. An association between DS-containing PGs and collagen type VI filaments (but not collagen fibrils) in vitro has been described in fetal rabbit cornea (68), and a role for decorin in regulating the attachment of type VI collagen networks to banded collagen fibrils has been proposed (39). However, studies in decorin-deficient mice clearly indicate that decorin is not an obligatory component in this interaction (69). The ability of decorin to bind fibronectin (42,43) and thrombospondin-1 (45) in solid-phase assays also appears to be principally a property of its core protein. One consequence of these interactions is inhibition of the ability of fibroblasts to attach to fibronectin-coated (70–72) or thrombospondin-1-coated (45,73) substrates. This effect on cellular adhesion presumably underlies the ability of decorin to impede osteosarcoma cell migration in an in vitro assay (74). There is evidence that the GAG chain of decorin may also contribute to the antiadhesive properties of the PG (74).

IV. Interactions Between Decorin and TGF␤ The TGFβ family consists of three closely related isoforms (TGFβ1, -β2, and -β3) that have broadly similar properties (75). These pleiotropic molecules are believed to play key roles in many processes, including immune regulation and the control of growth and differentiation. TGFβ1, the isoform that has been most studied, is synthesized as a large precursor that then undergoes a complex series of processing steps to yield the mature peptide, a 25-kDa homodimeric molecule. TGFβ1 is secreted from most cells in a latent form that is unable to bind to cell surface receptors or to exert biologic activity (76,77). This state of latency results from the continued noncovalent association, following intracellular processing and secretion, of the mature peptide with the N-terminal propeptide. Activation of latent TGFβ1 by removal of the propeptide is therefore likely a pivotal regulatory step. The mechanisms of activation are not fully understood, although recent interest has focused on possible roles for thrombospondin-1 (78) and for the integrin αvβ6 (79). The property of TGFβ1 that has attracted most attention in relation to the immunopathology of asthma (see below) is its profibrotic effect. Acting at pico-

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molar concentrations, TGFβ1 was first shown to increase the synthesis by various cell types of collagen types I and III and of fibronectin, and the incorporation of these proteins into the ECM (80–83). Many other ECM components are now known to be induced by TGFβ1, including tenascin (84), vitronectin (85), elastin (86,87), and thrombospondin-1 (88). Synthesis of most PGs is similarly enhanced, the response involving both increased synthesis of the core protein and an increase in the molecular mass of the GAG chains (89,90). Decorin, however, is exceptional in that its synthesis is reduced by TGFβ1 (91–93). The integrity of the ECM is dependent upon a balance between the actions of enzymes that degrade ECM components and molecules that specifically inhibit these enzymes. In addition to its direct effects, TGFβ1 is able to promote ECM deposition by inhibiting expression of these proteinases and by inducing expression of their inhibitors. For example, matrix metalloproteinases (MMPs) play an important role in degradation of the ECM, particularly MMP-1/collagenase and MMP-3/stromelysin, which are both inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1). In human lung fibroblasts, TGFβ1 prevents the increases in MMP-1 and MMP-3 mRNA induced by other growth factors, and acts synergistically with these other growth factors to increase TIMP-1 mRNA and protein expression (94). Plasmin, formed from the proenzyme plasminogen by the actions of either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA), also plays a major role in degradation of the ECM. Treatment of various cell types, including lung fibroblasts, with TGFβ1 leads to a decrease in the release of uPa and tPA, and to increased expression of their inhibitor plasminogen activator inhibitor-1 (PAI-1) (95–97). It is now well established that decorin and two other SLRPs—biglycan and fibromodulin—can form complexes with members of the TGFβ family. Yamaguchi et al. (98) first reported the ability of immobilized decorin to bind 125 I-TGFβ1, with a dissociation constant of 1.5 ⫻ 10⫺9 M for this interaction. The interaction involves the core protein of decorin, as pretreatment with chondroitinase ABC does not eliminate, and may in fact increase somewhat, the binding affinity (98,99). Similar interactions have been demonstrated with TGFβ2 and TGFβ3 (99). A study using recombinant decorin core protein fragments has shown that the high-affinity binding site for TGFβ isoforms is localized to the central LRR structures (100). However, the binding sites of decorin for TGFβ and for collagen type I are independent, so that collagen-bound decorin is still able to bind TGFβ (101). There have been conflicting reports regarding the effect of binding to decorin on the biologic activity of TGFβ. Initial studies pointed to an inhibitory effect. Yamaguchi et al. (102) transfected Chinese hamster ovary (CHO) cells (which do not constitutively synthesize decorin) with a human decorin cDNA construct so that they expressed high levels of the PG. This resulted in growth suppression and dramatic associated morphologic changes. In a subsequent re-

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port, the same authors showed that these effects were due, at least in part, to the ability of decorin to bind TGFβ and neutralize its autocrine growth stimulatory activity for CHO cells (98). Other investigators, however, have suggested that complex formation with decorin may only selectively inhibit the properties of TGFβ1 (101). Furthermore, decorin isolated from bovine bone was reported actually to enhance the bioactivity of the growth factor for MC3T3-E1 osteoblastic cells (103). There is compelling evidence that the predominant effect in vivo is indeed inhibitory. This was first demonstrated using a rat model of experimental glomerulonephritis induced by injection of antithymocyte serum (104). In this system, overexpression of TGFβ underlies the pathologic accumulation of ECM in injured glomeruli (104,105). Border et al. (106) showed that administration of four to six daily intravenous injections of either recombinant human decorin or purified bovine decorin was able to suppress the excess deposition of fibronectin, tenascin, and PGs. In later work, direct transfer of a decorin-expressing vector into rat skeletal muscle was similarly shown to attenuate markers of disease, with reductions in TGFβ1 mRNA and protein, glomerular ECM accumulation, and proteinuria (107). In the lung, antifibrotic effects of decorin in vivo have also been demonstrated using a well-established animal model. Intratracheal instillation of the antineoplastic agent bleomycin in rodents induces an initial influx of inflammatory cells followed by the development of severe multifocal interstitial fibrosis (108,109). Increases in lung TGFβ1 mRNA (110,111) and protein (112) content have been demonstrated, and shown to precede the increase in collagen synthesis. Furthermore, the fibrotic response can be partially inhibited by administration of either neutralizing antibodies to TGFβ (113) or a recombinant soluble type II TGFβ receptor (114). Using this model, Giri et al. (115) demonstrated that decorin can exert a similar protective effect against bleomycin-induced lung fibrosis. Repeated intratracheal instillation of recombinant rat decorin significantly attenuated lung collagen accumulation, histologic evidence of fibrosis, and the intensity of immunostaining for collagen type I and fibronectin (115). Several recent reports have confirmed the ability of decorin to neutralize TGFβ-mediated effects in the lung using an adenoviral construct containing human decorin cDNA. This vector induces transient overexpression of the decorin transgene by infected cells over a period of at least 7 days. Pretreatment with a single intranasal injection of the virus has been shown partially to inhibit bleomycininduced lung fibrosis in mice (116), an effect similar to that requiring repeated (twice-weekly) injections in the experiments using recombinant decorin protein (115). Additionally, the construct has been demonstrated to neutralize the inhibitory effect of exogenous TGFβ1 in an ex vivo model of lung branching morphogenesis (117), and to reduce the degree of fibrosis developing when it is administered concomitantly with an adenovirus overexpressing active TGFβ1 (118).

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Airway Fibrosis in Asthma

Recent research in asthma has focused on the presence of certain structural alterations in the airway wall that are referred to collectively as “airway remodeling” (119). A very characteristic feature of the remodeled airway is the deposition of excess connective tissue in the subepithelial region. Airway fibrosis was first recognized in autopsy reports of lung tissue from patients who had died of acute severe asthma (120–122). These reports described broadening and hyalinization of the region below the airway epithelium, appearances referred to at that time as “basement membrane (BM) thickening.” Later studies using bronchoscopy demonstrated similar features in the airways of asthmatics with clinically mild disease (123–126). These changes have been best studied in atopic asthma, but they are also seen in intrinsic asthma (127) and in occupational asthma (128). Examination of the ultrastructure of this region with EM indicated that in asthma the true BM, comprising the lamina rara and lamina densa, is well preserved. In contrast, the lamina reticularis is greatly increased in depth, corresponding to the apparent BM thickening described in light microscopic studies (127,129,130). Initial histochemical analysis suggested excess collagen accumulation in this region, but more detailed information derives from immunohistochemistry based studies. Roche et al. (124) described deposition of abundant collagen type III, collagen type V, and fibronectin in the thickened subepithelial band. Chakir et al. (131) reported collagen type I and collagen type III deposition in this region in asthma, although these investigators could not detect an increase in collagen type V immunostaining. Tenascin-C is weakly and variably expressed in the BM region in biopsy specimens of nonasthmatic subjects (132,133). In asthma, however, Laitinen et al. (133) have described the presence of a broad continuous band of immunoreactive tenascin-C immediately below the laminin layer. Several bronchoscopy-based studies have focused on fibrosis elsewhere in the airway wall. Minshall et al. (134) used Van Gieson’s stain to demonstrate collagen in the lamina propria of biopsy specimens from patients with asthma of varying severity. These authors reported a stepwise increase in the fibrosis score in individuals with mild, moderate, and severe disease. Roche et al. (124) could detect no qualitative differences between asthmatic and nonasthmatic subjects in expression of immunoreactive collagen types I, III, and V in the submucosal region, although Wilson and Li (126) reported increased immunostaining for collagen type III and collagen type V at this site in asthma. Chu et al. (135) could find no difference in either total collagen staining, detected by sirius red staining, or in collagen types I and III evaluated immunohistochemically in asthmatics with disease of varying grades of severity compared to nonasthmatic control subjects. In another study, Godfrey et al. (136) used EM-based morphometric tech-

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niques to determine the proportion of subepithelial area occupied by collagen fibers. No evidence of any alteration was evident in either mild or more severe asthma compared to healthy control subjects. The explanation for the apparent discrepancies among these various studies is unclear. The impact of airway fibrosis on disease expression has been explored principally by mathematical modeling. The fibrotic process is one probable factor contributing to the increased airway wall area in asthma that has been recognized in several morphometric studies (137–139). A small increase in airway wall thickness, having little or no effect on baseline resistance to airflow, is predicted dramatically to accentuate the effect of smooth muscle shortening on airway resistance (140). Similarly, modeling of the pattern of mucosal folding accompanying smooth muscle contraction suggests that thickening and stiffening of the thin inner layer, as likely occur with subepithelial fibrosis, can enhance luminal narrowing (141). Airway fibrosis may therefore be a major contributor to airway hyperresponsiveness, and in suport of this there are now many reports of correlations between subepithelial collagen thickness and airway responsiveness in asthma (142–146). There has been much speculation that airway remodeling/fibrosis may also be an important factor in the accelerated decline in lung function associated with asthma (147–149) and which leads, in a proportion of patients, to the development of irreversible airflow obstruction (150). However, studies of the relation between subepithelial fibrosis and asthma severity, as assessed by physiologic measures such as FEV1 or clinical indices, have produced inconsistent findings (124,142–146). Additionally, most investigators have failed to detect a correlation between subepithelial fibrosis and duration of asthma (124,145,146).

VI. Expression of Decorin and TGF␤ in Normal and Asthmatic Airways A. Expression of Decorin

Surveys of the distribution of decorin in human (151,152) and murine (14) tissues have suggested that the PG is widely expressed in those tissues that are rich in fibrillar collagens, consistent with its proposed role in fibrillogenesis. There is relatively little information, however, relating specifically to pulmonary tissue. The author has performed immunohistochemical analysis of upper- and lower-airway tissue using a rabbit polyclonal antiserum raised against a peptide representing the C-terminal sequence of the human decorin core protein (153). Proximal airway biopsy specimens were obtained by fiberoptic bronchoscopy from subjects with atopic asthma and from healthy nonsmoking volunteers with no history of lung disease. Immunostaining for decorin was evident throughout

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the thickness of the submucosal region, from the subepithelial BM to the airway smooth muscle, and was sometimes seen in a pericellular distribution (Fig. 1). The airway epithelium also demonstrated immunoreactivity. Appearances were indistinguishable in tissue from asthmatic and nonasthmatic subjects. No significant immunostaining was evident in negative control sections where the primary antiserum was omitted or replaced by nonimmune rabbit serum. The asthmatic subjects in that study had mild disease, with few symptoms, near-normal lung function, and bronchodilators as their only treatment. However, consistent findings have been described in postmortem lung tissue from six individuals who had died with severe asthma, with prominent decorin immunostaining in the submucosal layer and around smooth muscle cells (154). A similar pattern of immunostaining is also seen in the mucosa of resected nasal polyps,

Figure 1 Immunostaining for decorin in bronchial biopsy specimen from a subject with mild asthma. Immunoreactivity is seen throughout the thickness of the submucosal layer. Staining of the bronchial epithelium is also evident, but the thickened subepithelial basement membrane (*) and the airway smooth muscle layer (sm) are not immunostained.

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another tissue characterized by severe allergic inflammation (A.E. Redington, unpublished information). These findings are consistent with several other studies of decorin expression in the airways, and peripheral lung tissue, of humans and rodents. Pulkkinen et al. (24) found strong expression of decorin mRNA in human pulmonary tissue by Northern blot analysis. Bensadoun et al. (155) localized immunoreactive decorin to the airway subepithelial connective tissue and the adventitia of blood vessels in tissue from patients with normal lung function undergoing lung resection for peripheral tumors. Using in situ hybridization and immunohistochemistry, Bianco et al. (152) reported decorin mRNA and core protein expression in the “small” and “large” interstitium of human fetal pulmonary tissue. Furthermore, expression of decorin mRNA and/or core protein has been described in human nasal mucosa (156) and in pulmonary tissue of normal rats (157–159) and mice (14). There have been two conflicting reports. Dolhnikoff et al. (160) performed PG extraction on tissue obtained from patients undergoing therapeutic lung resections for tumors. When samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, no expression of decorin core protein was detectable. The authors speculated that this negative result may have reflected the scarcity of airways in the peripheral lung tissue examined. Using an immunohistochemical approach, Huang et al. (161) could detect only minimal decorin immunostaining in a minority of bronchial biopsies from either healthy volunteers or subjects with atopic asthma. Immunostaining of cartilage was not reported either, however, perhaps raising questions about the sensitivity of the antiserum. B. Expression of TGF␤

Many studies have examined the presence and cellular localization of TGFβ in pulmonary tissue. This section will first describe reports of TGFβ mRNA and protein expression in nonasthmatic human and rodent tissue. Comparative studies in asthma will then be discussed. Early work indicated that TGFβ1 mRNA was detectable by Northern blot analysis in pulmonary tissue of humans (162) and mice (163). Coker et al. (164) used digoxigenin-labeled riboprobes to perform a detailed survey of TGFβ1 gene expression in histologically normal peripheral lung tissue from patients undergoing surgical resection of tumors. Hybridization signal was predominantly detected in bronchiolar epithelial cells and alveolar macrophages, with some localization to mesenchymal cells, presumed to be fibroblasts, and to endothelial cells. In the same study, a very similar pattern of TGFβ1 mRNA distribution was found in pulmonary tissue from normal adult mice (164). Other investigators have been unable to detect TGFβ1 mRNA in the airway epithelium of adult mice (163) and

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rats (165), despite the localization of signal to connective tissue cells and airway smooth muscle. In both those reports, however, TGFβ1 immunoreactivity was localized to airway epithelial cells (see below). Immunohistochemical localization of TGFβ1 protein has been facilitated by the use of two polyclonal antisera anti-LC(1-30) and anti-CC(1-30) raised against different synthetic preparations of a peptide sequence corresponding to the N-terminal 30 amino acids of mature TGFβ1 (166). Both these antisera recognize TGFβ1 in a number of immunoassays (166,167). However, immunohistochemical studies in various murine tissues reveal that anti-LC(1-30) produces an intracellular staining pattern whereas immunostaining with anti-CC(1-30) is principally extracellular (166,168), suggesting that they react with different epitopes of the molecule. In human pulmonary tissue, immunostaining with antiLC(1-30) has been localized to the airway epithelium, the submucosa, and airway smooth muscle (146,162). With anti-CC(1-30), on the other hand, strong immunoreactivity was associated with the subepithelial connective tissue and airway smooth muscle, but the bronchial epithelium was not stained (162). The author has reported localization of immunoreactive TGFβ in endobronchial biopsy specimens obtained from healthy nonsmoking volunteers using a commercially available mouse monoclonal antibody that recognizes all three isoforms of TGFβ (153). A prominent feature was extracellular staining, which was evident throughout the depth of the subepithelial layer apart from the zone immediately below the epithelial BM, where it was either absent or less marked (Fig. 2). Staining of bronchial epithelial cells and submucosal cells was also apparent, but airway smooth muscle was not immunostained. The existence of a pool of extracellular TGFβ protein in these studies is consistent with findings in rodent airways, where immunoreactive TGFβ has been localized both to the bronchial epithelium (163,165) and to the underlying connective tissue (165) in large conducting airways, and with a study in normal human skin describing variable staining of the ECM but no cellular staining (169). Several other studies, however, have apparently failed to detect extracellular immunoreactivity in human airways. Magnan et al. (170), for example, reported preferential localization of TGFβ immunostaining to the bronchial epithelium and to a lesser extent airway smooth muscle in resected lung tissue, but did not describe extracellular immunoreactivity. Other investigators have described immunostaining of epithelial and subepithelial cells for TGFβ1 in a proportion of bronchoscopic biopsies from healthy control subjects, but not reported extracellular staining (134,135,171). These apparent discrepancies likely relate at least in part to differences in the epitopes recognized by the various antibodies employed. In the first study of TGFβ expression in asthma, Aubert et al. (162) found no difference in total TGFβ1 mRNA, detected by Northern blot analysis, in re-

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Figure 2 Immunostaining for TGFβ in a bronchial biopsy specimen of a subject with mild asthma. Prominent extracellular immunoreactivity is apparent in the subepithelial region. There is also some staining of the bronchial epithelium and of inflammatory cells in the submucosa. The thickened basement membrane (*) is not immunostained.

sected and postmortem lung specimens from patients with asthma compared to control tissue from smokers without airflow obstruction. Later studies have examined TGFβ1 expression in asthma at the cellular level using in situ hybridization. Ohno et al. (172) reported an increased number of subepithelial TGFβ1 mRNA⫹ cells in bronchoscopic biopsies from patients with severe asthma, but not mild disease, compared to healthy control subjects. An increase in subepithelial TGFβ1 mRNA⫹ cells in severe and moderate asthma was also described by Minshall et al. (134), who additionally reported a correlation between the number of TGFβ1 mRNA⫹ cells and depth of subepithelial fibrosis. Eosinophils were identified as a prominent source of TGFβ1 gene expression in these studies, particularly in patients with severe asthma, with other cells exhibiting a phenotype consistent

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with macrophages and fibroblasts (134,171,172). Localization of hybridization signal to the airway epithelium has also been described in asthmatic tissue in one report (171). Immunohistochemical studies of TGFβ in asthma have produced some inconsistent findings. In the study of Aubert et al. (162), no difference was detected in either intracellular [i.e., anti-LC(1-30)⫹ ] or extracellular [anti-CC(1-30)⫹ ] immunostaining pattern between asthmatic and nonasthmatic subjects. That conclusion was questioned because of the variability in specimen processing, smoking history, and antiasthma treatments that subjects had been receiving. However, the author has also reported a comparable pattern of TGFβ immunostaining in bronchial biopsies from mild nonsmoking subjects with atopic asthma and healthy control subjects (153). Epithelial immunoreactivity in that study, assessed by a semiquantitative scoring system, was similar in the two groups, although other reports have confusingly described both increased (171) and reduced (173) epithelial TGFβ staining in asthma. Extracellular staining was very prominent (153), and this feature has been reported in one other study of bronchial tissue from asthmatics (171). Several investigators have quantitated subepithelial TGFβ1 immunoreactive cells in asthma. Some have found no significant change compared to control subjects (135,146), whereas others have reported an increased number of TGFβ1⫹ cells in asthmatic tissue (134,171,174). Some of these discrepancies are difficult to reconcile. In particular, they do not appear easily explicable by factors such as epitope or isoform specificity, disease severity, or antiasthma treatment. In any case, interpretation of immunohistochemistry studies is limited by the fact that available reagents cannot distinguish between active and latent forms of TGFβ. Measurement of the free growth factor in bronchoalveolar lavage fluid may provide complementary information, and the author has reported elevated levels of TGFβ1 in asthma (175). An alternative approach that has not yet been widely exploited would be to study molecules such as the Smad proteins that are involved in TGFβ-dependent signaling pathways (176). C. Colocalization of Decorin and TGF␤

To examine the spatial relationship between TGFβ and decorin in the airways, the author has compared their respective patterns of immunostaining in sequential 2-µm sections of bronchial biopsies (153). In tissue from both asthmatic and nonasthmatic subjects, colocalization of decorin and TGFβ was evident in the subepithelial region (Fig. 3). This observation points to the likelihood that extracellular TGFβ is bound to decorin in vivo, but its functional significance is uncertain. One possibility is that binding to decorin provides a means to limit the bioactivity of free TGFβ1 by sequestration in the ECM, so preventing it from interacting with its cellular

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Figure 3 Two pairs of serial 2-µm sections showing colocalization of immunoreactive TGFβ (upper panels) and decorin (lower panels) in the bronchial submucosa. In both cases, there is a similar pattern of interstitial staining. Airway smooth muscle (sm) is not immunostained (left), but the adventitia of small blood vessels (arrows) does show reactivity (right). (From Ref. 153.)

receptors. The consequences of unregulated activity of the growth factor are illustrated by animal models of TGFβ1 overexpression. Targeted expression of TGFβ1 to the developing respiratory epithelium of transgenic mice leads to arrested lung morphogenesis and perinatal lethality (177). Localized overexpression of active TGFβ1 in the lungs of adult rats, achieved by intratracheal instillation of an adenoviral construct expressing a mutated TGFβ1 cDNA, results in a widespread, severe, and apparently irreversible fibrotic response (178). A physiologic mechanism to remove TGFβ1 from the extraceullar milieu after release may therefore be of critical importance. The colocalization of immunoreactive TGFβ and decorin also suggests the existence of an extracellular “reservoir” of matrix-bound growth factor that can be released in a bioactive form in response to certain stimuli. One possible mechanism would involve the actions of MMPs. Decorin is susceptible to proteolytic degradation by MMP-2, MMP-3, and MMP-7, all of which produce multiple cleavages in the central LRR region (179). Furthermore, these three MMPs can release immunoreactive TGFβ1 bound to immobilized decorin (179). In a similar manner, collagenase/MMP-1 treatment can result in release of TGFβ1 from decorin immobilized on reconstituted type I collagen fibrils, and the biologic activity of the released growth factor has been confirmed (100). Increased expression

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of MMPs, as has been described in asthmatic airways (180,181), could therefore alter the equilibrium between matrix-bound and soluble TGFβ1 to favor fibrosis. Another factor that may be of relevance in this context is mast cell heparin, which has been reported specifically to solubilize decorin from preparations of rat skeletal muscle ECM (182). By increasing the availability of decorin-TGFβ complexes, heparin may, therefore, provide a further influence to promote a fibrotic response in asthma. VII. Therapeutic Implications If airway fibrosis does indeed contribute to the pathophysiology of asthma, and more information will be required before this postulate can be accepted, an important question will be the extent to which the fibrotic process can be modulated by treatment. Current antiasthma therapies appear to have rather limited activity in this regard. Inhaled corticosteroids, for example, are highly effective as airway anti-inflammatory agents in asthma, but their effects on airway fibrosis are inconsistent (183–185) and may require prolonged treatment (186). There may therefore be a need to develop alternative therapeutic strategies to target the remodeling process. The evidence from rodent models suggests that decorin could form the basis of such a novel treatment. Decorin may be particularly suitable for treating pulmonary disease because of its propensity to accumulate in the lungs after intravenous administration. Furthermore, no obvious toxicity has been described in animal studies, and the fact that it is a naturally occurring human molecule may mean there is little risk of initiating adverse immunologic reactions. Initial studies will likely involve patients with pulmonary fibrosis rather than asthma, with decorin administered either as a recombinant protein, by intravenous infusion or perhaps by inhalation, or using some form of gene therapy approach. Ultimately, decorin may prove a useful treatment for asthma and perhaps for many other conditions associated with TGFβ-dependent fibrosis for which there are currently few or no effective therapies. References 1. Kresse H, Hausser H, Scho¨nherr E. Small proteoglycans. Experientia 1993; 49: 403–416. 2. Hocking AM, Shinomura T, McQuillan DJ. Leucine-rich repeat glycoproteins of the extracellular matrix. Matrix Biol 1998; 17:1–9. 3. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998; 67:609–652. ¨ nnerfjord P, Bayliss MT, Neame PJ, Heinga˚rd D. Identi4. Lorenzo P, Aspberg A, O

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12 Role of Proteoglycans in the Development and Pathogenesis of Emphysema

YMKE M. VAN DER GELD, JEANETTE F. M. VAN STRAATEN, DIRKJE S. POSTMA, and WIM TIMENS University Hospital Groningen Groningen, The Netherlands

I.

Introduction

A. Emphysema as a Component of COPD

Chronic obstructive pulmonary diseases (COPD) is defined according to criteria of the American Thoracic Society (ATS) as a disease state characterized by the presence of airway obstruction due to chronic bronchitis or emphysema. The airflow limitation is generally progressive, is often accompanied by airway hyperresponsiveness, and is usually but not always persistent (1). There is some inconsistency in the definition of COPD in that COPD cannot be considered as one disease entity but rather as a complex of conditions that contribute to airflow limitation. COPD is defined functionally, whereas chronic bronchitis is defined by symptoms, and emphysema by gross pathology. Chronic bronchitis is, according to ATS criteria, defined as a disease with a chronic productive cough for 3 months in each of two successive years in a patient in whom other causes of chronic cough have been excluded (1). Emphysema is, according to these criteria, defined as a condition of the lung characterized by abnormal permanent enlargement of the air spaces distal to the terminal bronchioles accompanied by destruction of their walls and without obvious fibrosis (1,2). Given these defini241

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tions, it is clear that chronic bronchitis can be present in a patient with predominantly emphysema and vice versa. Cigarette smoking is the major risk factor for COPD (1). Smoking results in a reduction of lung function, generally measured as forced expiratory volume in 1 s (FEV1) (3), and the more severe this obstruction in an individual, the higher the number of packs and years of smoking. Only a small percentage of heavy smokers, however, actually develop pathologic airway obstruction (FEV1 percentage predicted ⬍80%) (4). Other risk factors for the development of COPD include passive smoking (5,6), exposure to ambient air pollution and occupational factors (7–11), male sex (12), white race (1), low socioeconomic status (12), adenoviral infections (4), hyperresponsiveness and atopy (13). Finally, individuals with homozygous α1-antitrypsin (α1AT) deficiency develop emphysema (14) and comprise ⬃ 1–5% of all patients with emphysema. B. Types of Emphysematous Lung Lesions

Emphysema is classified in four types according to its anatomic presentation in relation to the acinus of the lung (15–18). The first type of emphysema is centrilobular (centriacinar, proximal acinar) emphysema (CLE). Central or proximal parts of the acini are affected, while distal alveoli are spared. As a consequence, both normal alveolar architecture and enlarged spaces with alveolar wall destruction are situated immediately adjacent to each other. CLE is a gradually progressive disease, and progression occurs both by extension of individual sites and by development of new sites of destruction. CLE is usually more common and more severe in the upper lung lobes and is associated with cigarette smoking. The second type of emphysema is panlobular (panacinar, generalized) emphysema (PLE). Acini are uniformly enlarged from the level of the respiratory bronchiole to the terminal blind alveoli. The whole acinus is involved in PLE from the onset of the disease and throughout its progression to the point of lethal respiratory failure. PLE is rather evenly distributed over the entire lung, although often somewhat more severe in the lower lobes. PLE is associated with α1AT deficiency. Patients with CLE or PLE can have similar severity of airway obstruction and parenchymal destruction. CLE or PLE can be present in its pure form in an individual lung. However, in ⬃ 50% of patients with emphysema, features of the two types of emphysema overlap, with generally one type being clearly predominant (19,20). Part of this overlap is likely explained by α1AT deficiency patients who smoke. The third type of emphysema is localized emphysema including bullous, distal acinar, and paraseptal emphysema. The proximal portion of the acinus is normal, and predominantly the distal part is affected. There is only one, or there are at most a few site(s) of severe destruction of alveoli; the remaining pulmonary architecture is normal. Commonly, this lesion is found in the extreme apex of

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both upper lobes, but it can occur in any other location, even at the base of a lower lobe. Although this type of emphysema initially is localized, it tends to be progressive and can lead to larger areas of destruction, often called bullae. The fourth type of emphysema is perifocal (paracicatricial, irregular) emphysema. The acinus is irregularly affected, and the air space enlargement usually occurs in the vicinity of focal lesions in the lung such as scarring granulomas. In most cases, these foci of perifocal emphysema are merely anatomical and do not present themselves by airway obstruction or symptoms. C. Diagnosis of Pulmonary Emphysema

The clinical diagnosis of emphysema is based on history taking, chest radiographs, and pulmonary function tests—i.e., increased total lung capacity and residual volume, reduced diffusion capacity, and reduced flow rates during forced expiration (21). However, the definite diagnosis, classification and assessment of emphysema according to ATS criteria are made only in those situations when tissue is available for microscopical and macroscopical analysis by a pathologist. Emphysema is then identified according to the presence of enlarged air spaces, evidence for a destructive process, and the absence of obvious fibrosis. The characteristic histologic evidence for emphysema is the finding of isolated or “freelying” segments of viable alveolar septal tissue or isolated cross sections of pulmonary vessels (15). The architecture of normal lung tissue requires all alveolar septal tissue to connect with other septal tissue at least at one end if not both (15). Lung tissue from a normal subject and from a patient with severe emphysema is shown in Figure 1.

II. Pulmonary Interstitium in Emphysema A. Fibroblasts

Fibroblasts are mesenchymal cells that are ubiquitously present in the pulmonary interstitium (35–40% of the cells). They consist of subpopulations with unique phenotypes and functions (22). Fibroblasts are the main source of the extracellular matrix (ECM) proteins present in the lung tissue. For instance, lung fibroblasts have been reported to synthesize types I and III collagen (23), elastin (24), fibronectin (25), heparan sulfate proteoglycans (26), chondroitin/dermatan sulfate proteoglycans (27), and hyaluronic acid (28). Furthermore, fibroblasts produce factors that influence tissue remodeling processes, such as ECM-degrading enzymes and their inhibitors (29,30). Additionally, these fibroblasts may have a role in inflammatory cell influx via production of a variety of cytokines [plateletderived growth factor (PDGF); transforming growth factor-beta (TGFβ); interleukin (IL)-8, IL-6, IL-1; monocyte chemotactic protein-1 (MCP-1); and tumor ne-

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(A)

(B) Figure 1 (A) Lung tissue from a normal subject; (B) lung tissue from a patient with severe emphysema. Hematoxylin-eosin stain; original magnification 100⫻.


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crosis factor-alpha (TNFα) (31–33)]. Also, fibroblasts interact with cells of the immune system, such as T-lymphocytes and mast cells, indirectly mediated by cytokines, and directly via cell-cell contacts (34–36). In pulmonary emphysema, the fibroblast may be intrinsically dysfunctional, resulting in a hampered production of ECM in a quantitative and/or qualitative way. This hampered production of ECM may also affect the binding, release, or regulation of the activity of matrix-bound cytokines. In these intrinsically dysfunctional fibroblasts, modulation of tissue repair by production of ECM-degrading enzymes and inhibitors of ECM-degrading enzymes might be affected. Additionally, the role of these fibroblasts in inflammation might be changed. In our own laboratory, we found that the proliferative capacity of fibroblasts isolated from lung tissue of patients with pulmonary emphysema is different from that of control subjects. Fibroblasts from control subjects incorporated more BrdU after incubation with IL-1β than cultures from patients with emphysema, and TGFβ decreased incorporation of BrdU more in fibroblast cultures from control subjects than from patients with emphysema (Noordhoek et al., preliminary results). Changes in fibroblast function have been reported in other lung diseases as well. In asthma (37), fibrotic lung disorders (38–41), and sarcoidosis (42) they show increased proliferation and/or ECM production in vitro. B. Extracellular Matrix

The structural alterations in pulmonary emphysema, such as loss and/or rearrangement of lung tissue architecture are the consequence of quantitative and/or qualitative changes in the composition of the ECM. These changes in ECM can affect the structural integrity of tissue as well as binding, release, or regulation of matrix-bound cytokines. Changes in synthesis or turnover of interstitial ECM components have been reported to be associated with lung injury, as seen in pulmonary fibrosis (43,44) and pulmonary hypertension (43). Pulmonary emphysema has been reported to be related to a decreased elastin (45), hyaluronic acid (46), chondroitin sulfate content (47), increased collagen content (45,48), disrupted elastin sheets (49), and disorganized collagen fibrils (49). However, studies have not been conclusive in determining the exact ECM components affected in pulmonary emphysema, and only a small subset of the lung ECM components have been studied so far. One of the problems in investigating emphysema lies in the fact that one cannot simply follow up individuals during development and progression of disease, given the restraints on obtaining lung tissue. Thus, one can investigate lung tissue only at one time point during an individual’s life. Hence, it is generally not clear whether the observed changes in ECM are the consequence of the disease or they have a causative role in development of disease.

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The most prominent structural components in interstitial lung ECM are collagen and elastin. Collagens are known to stabilize pulmonary tissue: collagen I and III types as part of the interstitium, and collagen IV type as a constituent of basement membranes (44,50). Elastin plays an important role in the elasticity of pulmonary tissue. Less abundantly present, but functionally very important, are fibronectin and a variety of proteoglycans in the interstitium, and laminin, entactin, and proteoglycans in basement membrane (44,50). The proteoglycans play a pivotal role in maintaining the structural integrity of the ECM. C. Proteolytic Enzymes

Proteolytic enzymes are categorized as metalloproteinases, serine proteases, and cysteine proteases (51,52). Matrix metalloproteinases (MMPs) cleave the triple helix of collagens I, II, and III types (collagenases: MMP-1, MMP-8, MMP-13), and degrade cleaved collagens I, II, and III types and collagens IV, V, VII, IX, XI types, elastin, decorin, entactin and fibronectin (gelatinases: MMP-2, MMP-9). Furthermore, they degrade collagens IV, V, IX, and X types, elastin, laminin, decorin, proteoglycan core protein, and fibronectin (stromelysins: MMP-3, MMP-10, MMP-11, metalloelastase: MMP-12, and matrilysin: MMP-7), and degrade progelatinase A (membrane-type MMPs: MT-MMPs) (52,53). MMP expression can be regulated via the activity of tissue inhibitors of metalloproteinase (TIMPs) (52). TIMPs form a complex with all activated MMPs as well as with the proenzyme form of MMP-2 (TIMP-2) and MMP-9 (TIMP-1). Furthermore, several MMPs, among others MMP-1, -2, -3, -7, directly modulate the activity of several growth factors and chemokines, such as TGFβ, TNFα, insulinlike growth factor-1, epithelial growth factor (EGF), and MCP-3 (54–60). The serine proteases, including elastase, proteinase 3, cathepsin G, plasminogen activator, tryptase, and chymase, have elastin, fibronectin, laminin, entactin, vitronectin, and collagen IV type substrate specificity and they can activate MMPs (51). Inhibition of the activity of these serine proteases is performed by α1AT and secretory leucocyte protease inhibitor. The cysteine proteases comprise cathepsin B, L, and S. Cathepsin B can degrade collagen, proteoglycans, and laminin, whereas cathepsin L and S have elastinolytic activity (51). In physiological circumstances, the balance between proteolytic enzymes and their inhibitors is a crucial factor in maintaining homeostasis of ECM proteins. In pathological tissue-destructive conditions, there is an imbalance between proteolytic enzymes and their inhibitors (61–64). Pathologic changes in metalloproteinases and their inhibitors have been reported in diffuse alveolar damage and idiopathic pulmonary fibrosis (65). The net lung tissue loss and/or rearrangement of the architecture of lung tissue in pulmonary emphysema is likely also (partly) the consequence of an imbalance between proteolytic enzymes and


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their inhibitors. This may be caused by a relative increase of protease activity or a relative decrease (or noncompensating increase!) of protease inhibitors. From murine models of cigarette smoke–induced emphysema it is known that macrophage metalloelastase is important in emphysema, because macrophages have the capacity to cause emphysema upon recruitment and activation by cigarette smoke. In a mouse macrophage elastase knockout model, no development of emphysema was observed after smoking (66). Human studies showed increased quantities of matrix-degrading enzymes, in particular collagenases and gelatinases, in bronchoalveolar lavage fluid (67) and lung tissue (68–70) and increased production of elastolytic and collagenolytic enzymes by alveolar macrophages (71) in patients with emphysema. Immunohistochemical studies in our own laboratory revealed increased immunoreactivity for MMP-2 in macrophages in lung tissue from patients with severe emphysema (unpublished observations).

III. Proteoglycans in Emphysema A. Tissue Proteoglycans in Normal Lung

Proteoglycans constitute only a small, but essential, part of the total protein content of the ECM. They are produced mainly by fibroblasts, type II alveolar epithelial cells, and endothelial and smooth muscle cells. Proteoglycans are a large family of proteins, consisting of a protein core and side chains with highly variable sugar moieties. This unique composition enables proteoglycans to play a central structural role in the architecture of the interstitial and basement ECM, as well as an important functional role in dynamic interactions between cells and tissues. The structural role of proteoglycans is formed mainly by bridging and connecting other ECM proteins. The chondroitin/dermatan sulfate proteoglycans (C/DSPG), such as decorin and biglycan, interact with fibrillar collagens, contribute to collagen fibril formation and orientation (72–74), interact with fibronectin (75), and, by virtue of their large hydration capacity and anionic properties, can exert swelling pressure (76,77). It has been shown that decorin-deficient mice have a more loosely packed collagen fiber network in the skin with abnormal collagen morphology and relatively coarse, irregular fiber outlines. Furthermore, the animals have reduced tensile strength of the skin, indicating an important role for decorin in stabilization of the fibrillar matrix in vivo (78). Studies on lung tissue of decorin-deficient mice have not been published in this respect, but may likely show effects as described above. Heparan sulfate proteoglycans (HSPGs) connect collagen IV type network, entactin, and laminin in the basement membrane (79), and are also important in the pericellular matrix. The keratan sulfate proteoglycan lumican interacts with collagen fibrils and may influence the interaction of collagen fibrils with other components of the ECM (74,80).

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The functional role of proteoglycans can be divided into two main parts. First, proteoglycans can function as an integral cell surface receptor molecule for outside-in signals from ECM proteins, other cells, or soluble mediators. The HSPG molecule syndecan, for example, is essential as a coenhancer for integrinmediated adhesion of fibroblasts to fibronectin (81). The HSPG betaglycan is an integral transmembrane proteoglycan, which functions as the type III receptor for TGFβ (see below). The second functional role of proteoglycans is a regulatory one in inflammation and repair of destroyed pulmonary tissue. This functional characteristic mainly results from various cytokine-binding capacities (40,82– 84) (Table 1; discussed in Sec. III.C). B. Tissue Proteoglycans in Emphysema

In human lung tissue, there is evidence for an important role of proteoglycans in the development of emphysema. In particular, in lung tissue from patients with severe emphysema, and to a lesser extent in patients with mild emphysema, a diminished immunohistochemical staining of the interstitial proteoglycans decorin and biglycan was found in the peribronchiolar area, as compared to lung tissue from control subjects or from patients with pulmonary fibrosis (85). Immunoreactivity for decorin in human lung tissue from a normal subject and from a patient with severe emphysema is shown in Figure 2. In addition, diminished and discontinuous staining of heparan sulfate proteoglycans was found in the alveolar basement membranes in lung tissue from patients with mild emphysema, severe emphysema, and fibrosis, as compared to lung tissue from control subjects. 1.

Chondroitin/Dermatan Sulfate Proteoglycans

The functional role of the diminished peribronchiolar staining of the interstitial proteoglycans decorin and biglycan in lung tissue from patients with severe emphysema is unclear. The diminished staining of decorin and biglycan may be due to either a decreased presence of the proteoglycans or a qualitative alteration of the core protein of the proteoglycan. For decorin it is most likely that a decreased presence exists as two antibodies directed against different parts of the core protein of decorin both showed the diminished binding (85). Furthermore, using Western blot analysis, no (qualitative) differences in the size of decorin and biglycan were found between lung tissue from control subjects and patients with severe emphysema (preliminary results). Interstitial proteoglycans are known for their interactions with fibrillar collagens and fibronectin in vitro (86–88), for their colocalization with collagens in situ (88), and for their stabilization of the fibrillar collagen matrix in vivo (78). We found strong collagen staining in the peribronchiolar area in lung tissue from patients with severe emphysema, but diminished decorin and biglycan staining. These alterations in interstitial proteoglycans may represent the parenchymal ab-

Proteoglycans and Emphysema Table 1

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Growth Factor–Binding ECM Molecules

Core protein PROTEOGLYCANS HSPG Syndecan Betaglycan-Cell Betaglycan-Sol HSPG

DSPG KSPG CSPG

Biglycan, Decorin Fibromodulin

OTHER ECM MOLECULES Fibronectin

Heparin

Growth factor–binding potential

Functional role of proteoglycan/ cytokine complex

bFGF, TGFβ, VEGF TGFβ

Enhance activity Presentation to type I and II receptor Blocking activity Chemotaxis Blocking activity Depot function Depot function Blocking activity Depot function Enhance activity

TGFβ CTAP-III, NAP-2 IFNγ, RANTES, IL-8 IP10 GM-CSF, IL-3, IL-4 TGFβ IFNγ IL-8 TGFβ GM-CSF IL-3 TNFα bFGF HGF

Hyaluronan Collagen IV Fibrin(ogen) Thrombospondin

IFNγ IL-1β TGFβ TGFβ TGFβ

SPARC

PDGF

Chemotaxis Depot function Depot function Depot function Depot function, adhesion Enhance activity Release matrixbound cytokine Blocking activity Depot function Depot function Depot function(?) Depot function(?), protection Depot function, blocking activity

Abbreviations: bFGF ⫽ basic fibroblast growth factor; Cell ⫽ cell surface–associated molecule; CSPG ⫽ chondroitin sulfate proteoglycan; CTAP-III ⫽ connective tissue–activating peptide III; DSPG ⫽ dermatan sulfate proteoglycan; GM-CSF ⫽ granulocyte macrophage colony-stimulating factor; HGF ⫽ hepatocyte growth factor; HSPG ⫽ heparan sulfate proteoglycan; IFNγ ⫽ interferon gamma; IL ⫽ interleukin; IP-10 ⫽ (IPN) inducible protein-10; KSPG ⫽ keratan sulfate proteoglycan; NAP-2 ⫽ neutrophil-activating peptide 2; PDGF ⫽ platelet-derived growth factor; RANTES ⫽ regulated upon activation, normal T-cell expressed and secreted; Sol ⫽ soluble protein; SPARC ⫽ secreted protein acidic and rich in cysteine; TGFβ ⫽ transforming growth factor beta; TNFα ⫽ tumor necrosis factor alpha; VEGF ⫽ vascular endothelial growth factor. Source: Refs. 83, 99, 100, 125, 126.

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(A)

(B) Figure 2 Immunoreactivity for decorin in human lung tissue. Lung tissue was fixed and processed for immunohistochemical staining using a monoclonal antibody against decorin. (A) Lung tissue from a normal subject; (B) lung tissue from a patient with severe emphysema. Note the diminished immunoreactivity in the peribronchial area in lung tissue (arrowheads) from patients with severe emphysema compared to normal lung tissue. B, bronchiolus; V, vessel; arrow indicates carbon pigment. Hematoxylin-eosin stain; original magnification 400⫻. (From Ref. 85.)


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normality causing damage to peribronchial attachments, inadequate lung tissue recoil, and, in this way, increased uncoupling of airways, which may lead to airway collapse on expiration (89). Alternatively, it cannot be excluded that the parenchymal changes are merely the effect of continuous damage. Independent of the origin, this parenchymal abnormality can be considered to be responsible for part of the functional obstructive problems in the airways of patients with emphysema. Furthermore, the diminished presence or qualitative alterations in interstitial proteoglycans may be responsible for disruption of the collagen network, which is supposed to be essential for the marked increase in total lung capacity that occurs in emphysema (90). The diminished peribronchiolar presence of decorin and biglycan was more prominent in patients with severe emphysema than in patients with mild emphysema. This selective difference may reflect the cumulative result of quantitatively and/or qualitatively impaired decorin and biglycan production in patients with severe emphysema over a long period of time, ultimately contributing to inferior lung tissue. This explanation is consistent with the slowly progressive character of the disease. The observation that decorin and biglycan alterations are detected only in the peribronchiolar area and not in the alveolar septa does not imply that there are no changes in interstitial proteoglycans in alveolar septa of lung tissue from patients with emphysema. Loss of alveolar tissue may also be caused by disruption of alveolar attachments, owing to alterations in proteoglycans. Since immunohistochemical studies only assess alterations in residual lung tissue, and not in the apparently vulnerable tissue that is lost during the development of emphysema, this may explain the lack of difference in the presence of interstitial proteoglycans in the remaining alveolar septa. The quantitative or qualitative alterations in interstitial proteoglycan expression might indirectly affect binding and regulation of cytokines, in particular TGFβ (91–93). TGFβ is a main regulator of tissue repair and fibroblast activity (94). The quantitative alterations may result in less core protein available for TGFβ binding, neutralization, and/or storage, or qualitative alterations might involve the TGFβ-binding site. In this way, an altered presence of interstitial proteoglycans may influence structural integrity as well as the ongoing inflammatory process. 2. Heparan Sulfate Proteoglycans and Heparan Sulfate

Next to alterations in interstitial proteoglycans, diminished staining for HSPG and HS in the basement membranes of the alveolar compartment of lung tissue was found for both patients with emphysema and patients with fibrosis, as compared with lung tissue from control subjects (85). This diminished staining might represent an early indication of alveolar damage in pulmonary tissue. It appears

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that there is a selective absence of HSPG and HS from the alveolar basement membranes, since diminished staining was not observed for other basement membrane components like collagen IV type and laminin. HSPGs interact with other basement membrane components (44), which is necessary for barrier integrity of the basement membrane in pulmonary tissue. Diminished presence of HSPG and HS in lung tissue from patients with emphysema and fibrosis may affect basement membrane integrity. In addition, reduced levels of HS may result in altered regulatory effects on cytokine activities, e.g., basic fibroblast growth factor (bFGF), IL-3, and granulocyte macrophage colonystimulating factor (GM-CSF) (25,91,95), in patients with emphysema or fibrosis. Thus, similar to interstitial proteoglycans, a qualitatively or quantitatively altered presence of basement membrane-related HSPG and HS can influence structural integrity as well as the ongoing inflammatory process. This concept is supported by the findings in rats that specific inhibition of proteoglycan synthesis induces emphysematous lesions (96) and that HSPGs are very vulnerable toward elastase degradation (97). In the former two animal models for emphysema (96,97), an increase in the urinary GAG content was observed, mainly owing to elevated levels of chondroitin sulfate and dermatan sulfate. The increase in urinary GAG content was positively correlated with the extent of emphysema. In addition, glycosaminoglycans have been analyzed in urine of patients with emphysema. Decreased contents of a specific epitope of HS and an unchanged content of total HS were reported (98). Differences between emphysema and fibrosis, especially in the role of interstitial proteoglycans decorin and biglycan, might imply an alternative regulation of the repair reaction after pulmonary injury in emphysema, as compared to the development of fibrosis. HSPG alterations in alveolar tissue seem to be common to the development of both emphysema and fibrosis and might best be considered as a general consequence of local damage. 3.

Cytokine-Binding Capacities

ECM proteins can also perform regulatory functions in inflammation and in repair of destroyed pulmonary tissue by various cytokine-binding capacities, which are mainly performed by proteoglycans (83,99). Interactions of matrix proteins with cytokines lead to local storage of cytokines (depot function), neutralization of the cytokine activity (blocking activity), chemotaxis, and/or presentation of the cytokine to high-affinity cytokine receptors (presentation/enhancement of activity; see Table 1) (83,99,100). For instance, decorin and biglycan bind TGFβ, resulting in local storage in the ECM and neutralization of TGFβ action (92,93). Fibronectin binds IL-8, functionally inactivating its neutrophil chemotactic activity (101), and TGFβ, GM-CSF, IL-3, and TNFα, resulting in local storage (100, 102). Alterations in individual components of the ECM can affect the binding,


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release, and regulation of matrix-bound cytokines, and in this way they can influence the regulation of a tissue repair reaction. Intrinsic dysfunctioning of pulmonary fibroblasts may further increase alterations in tissue repair responses, since in particular these cells produce the ECM components. Cytokines in the lung are mainly produced by alveolar macrophages, polymorphonuclear leukocytes, lymphocytes, epithelial cells, and fibroblasts. These cytokines can act both in an autocrine and in a paracrine manner. TGFβ, PDGF, GM-CSF, and bFGF are the main fibrogenic cytokines involved in regulation/ interaction processes of fibroblasts (76,84,103,104). TGFβ is specifically involved in the repair phase after injury (105), since it binds to locally produced proteoglycans (23,76,82,84), such as cell surface betaglycan. Betaglycan is the type III receptor (among others on fibroblasts) for TGFβ, which binds TGFβ without intracellular signaling. Type I and II TGFβ receptors normally show only low-affinity binding; primary binding of TGFβ to betaglycan is essential for crosslinking and high-affinity reaction with the type I and II receptors, which are then capable of inducing intracellular signal transduction and subsequent activation of fibroblasts, leading to proliferation and ECM production (104). In contrast to these activation pathways, binding of TGFβ to proteoglycans, such as decorin and soluble betaglycan, neutralizes TGFβ action (82,84,106). Another important cytokine-proteoglycan interaction is between bFGF and HSPG. This binding is necessary for bFGF binding to its high-affinity receptor on the fibroblast cell surface. With respect to bFGF, and also GM-CSF, inadequate binding causes insufficient facilitation/activation of various fibroblast functions (84, 104,105). Two factors are crucial in the exposure to cytokines: timing and concentration. TGFβ can, in conjunction with other cytokines, either stimulate or inhibit proliferation of growth-arrested fibroblasts, depending on the timing and sequence of exposure to the respective cytokines (84). Similar timing-related synergistic and antagonistic actions of cytokines and extracellular matrix proteins on fibroblasts can be found, mediated by cell surface receptors (83). Furthermore, TGFβ is known to stimulate fibroblast proliferation in low concentrations, while higher concentrations inhibit proliferation (84). Insufficient binding of TGFβ to proteoglycans can, in this way, lead to untoward inhibition of tissue repair by fibroblasts. Proteoglycan-bound cytokines constitute a reservoir of mediators, readily available in case of tissue destruction, resulting in a variety of inhibitory, activating, or coenhancing effects on cells (82,83,100). However, at the same time, the described binding capacities may also play a role in preventing fibrosis by neutralizing excess fibrogenic cytokines, produced in an inflammatory response. The complex regulation of tissue repair described above in the ideal situation results in tissue homeostasis (adequate repair). When dysregulation occurs in this delicate balance among different cells, cytokines, and ECM proteins, it may result

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in excessive matrix deposition, as seen in fibrosis, or in inferior ECM construction, as seen in emphysema. Apart from binding “matrix-regulating” cytokines, fibroblast-deposited ECM can also bind and influence chemotactic proteins, responsible for the attraction of specific leukocytes. Thus, ECM can also play a regulating role in lung inflammation itself. Fibronectin fragments can specifically bind the chemotactic cytokine IL-8, and in this way inactivate its neutrophil chemotactic activity (101). Subtle changes in the molecular composition of fibronectin can affect these binding properties and thus the ability to inactivate the cytokine. The interrelationship between proteoglycans and chemotactic proteins is illustrated by characterization of two platelet-derived heparan sulfate–degrading enzymes, connective tissue– activating peptide III (CTAP-III), and neutrophil-activating peptide 2 (NAP-2) (107). These two proteins are members from the CXC chemokine family. Their intrinsic heparanase activity may enhance the release of cyto- or chemokines from heparan sulfate and so exert complex effects on local inflammatory cells. This dual function of CTAP-III/NAP-2, as both a heparanase and a neutrophil chemoattractant, suggests that a complex relationship exists, where each activity may change the activity or bioavailability of the other function.

IV. Pathogenesis of Pulmonary Emphysema A. History

In the early 1960s, it was hypothesized that the protease-antiprotease imbalance, in favor of the proteases, caused emphysema. This hypothesis was based on two studies: subjects with a deficiency of the major serum serine protease inhibitor α1AT were susceptible to the development of pulmonary emphysema (108); and instillation of papain, an elastolytic plant enzyme, induced pulmonary emphysema in experimental animals (109). In addition, from the 1980s, a disturbance of the balance between oxidants and antioxidants, in favor of the oxidants, was also proposed to be involved in the pathogenesis of emphysema (110,111). The involvement of proteases and oxidants in tissue destruction in the pathogenesis of pulmonary emphysema is beyond all doubt (112–114). But, as the activity of proteases as well as oxidants can be expected to be quite similar in all smokers, these factors do not explain why only 10–20% of all smokers develop (clinical) pulmonary emphysema. B. Current Concept and New Developments

Smoking is not the only factor determining the development of severe emphysematous lung lesions. In smoking individuals emphysematous lung lesions of variable severity are found, in addition to variations in the clinical manifestations of


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the disease. The mild lesions in patients with mild emphysema may imply that the lung tissue in these patients is less susceptible to damaging agents or is more adequately repaired. More insight into the responsible process may have therapeutic consequences. Although smoking cessation will always be the most fruitful “therapy” to prevent fast deterioration in the function of the lungs, a protection against damaging agents in cigarette smoke might be applied by supporting protective mechanisms against oxidative stress such as glutathione S-transferase (115) and microsomal epoxide hydrolase (116). But, when the intrinsic risk factors of a subject to develop emphysema upon smoking can be characterized, a more adequate therapy can be offered, and, even more important, subjects can be pointed out the risks and consequences of starting or prolonging smoking and the benefits of smoking cessation. Intrinsic risk factors for development of emphysema might imply a disturbance of an adequate tissue repair. The damage caused by smoking, even in heavy smokers is, in a given time frame, expected to be limited. This is supported by the fact that ⬎80% of smokers do not develop emphysema, indicating sufficient tissue repair. It is plausible that inadequate tissue repair is involved in the development of severe emphysema. This hypothesis is sustained by our finding that the residual lung tissue from patients with severe emphysema had alterations in the individual components of the ECM: the interstitial proteoglycans decorin and biglycan (85). Furthermore, additional factors implicated in tissue repair such as neutrophils and CD8 T-lymphocytes in the alveolar tissue and nitric oxide synthase expression in macrophages were specifically associated with the extent of emphysematous lesions (117). No single specific component in the tissue remodeling mechanism is yet found to be primarily responsible for the development of severe pulmonary emphysema. Therefore, it can be concluded that there is a complex interaction among the individual components, and that the balance and coordination in these interactions finally determine the net tissue repair and thus the resulting destruction. Altogether, the alterations in lung tissue from patients with severe emphysema imply the presence of a dysfunctional tissue repair system, although the exact nature of the dysfunction has to be further characterized. The following hypothesis is put forward (Fig. 3). Upon passage through the lung compartments, smoke is supposed to cause injury to epithelial cells and to the basement membrane. This damage is followed by an inflammatory phase, accompanied by attraction of inflammatory cells (macrophages and neutrophils). These inflammatory cells attempt to clear the small inhaled particulate materials (smoke) and to scavenge tissue debris. When the damaging agent is eliminated, damaged tissue will eventually be replaced by new tissue. Fibroblasts are activated and/or attracted, in order to produce new tissue components (i.e., tissue repair), and renewed epithelialization occurs in order to reestablish tissue integrity, with gradual re-formation of the basement membrane. The net result is adequate integrity of the lung tissue. In case of chronic exposure to smoke in healthy

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Figure 3 Schematic representation of the main tissue components participating in pulmonary destruction and tissue repair processes with emphasis on the development of emphysema. Injury to epithelial cells and basement membrane was caused by cigarette smoke in the lung containing structural cells (fibroblasts) and extracellular matrix components. Inflammatory cells (neutrophils and macrophages) are attracted to the site of injury. Inflammatory mediators (modulating cytokines), proteolytic enzymes [matrix metalloproteinases (MMP)], and tissue inhibitors of MMP (TIMP) are produced by inflammatory cells as well as fibroblasts. Upon smoke-induced injury of the pulmonary tissue, the interplay between these separate components will determine the net tissue repair and the development of pulmonary emphysema. (From Ref. 126.)

individuals, the tissue repair response is repeatedly and more intensively activated but will, in normal individuals, finally result in complete recovery of the tissue. It can be hypothesized that in those smoking individuals who are prone to develop emphysema, the tissue repair may restore integrity after limited damage but may not be sufficient to compensate for the repeated damage by cigarette smoke, finally resulting in the development of emphysematous lung lesions (Fig. 4). Thus, inadequate tissue repair in certain prone individuals may constitute a further explanation for the pathogenesis of pulmonary emphysema, including its characteristic of the slowly progressive course. Many different components participate in pulmonary destruction, inflammatory and tissue repair processes. Some of these components are considered to be essential in the development of emphysema, and therefore deserve further consideration.


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Figure 4 Schematic representation of hypothesized dysbalance between tissue damage and adequate tissue repair in smokers. Insufficient tissue repair in a subset of susceptible smokers will lead to the development of pulmonary emphysema. Net balance between damage and repair in favor of ongoing damage. (From Ref. 126.)

C. Future Research

Further qualitative analysis of differences in ECM proteins, decorin, and biglycan, found in the immunohistochemical studies, has to be performed. Knowledge of the nature of the diminished immunoreactivity for the interstitial proteoglycans decorin and biglycan in lung tissue from patients with severe emphysema may have important implications for understanding the development and progression

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of emphysema. Therefore, further characterization of the nature of this reduction has to be performed. This characterization may include protein and carbohydrate analyses of the peribronchiolar proteoglycan matrix to assess whether the observed alterations are caused by qualitative or quantitative alterations of these proteoglycans. Second, the role of the peribronchiolar fibroblast in the production of these interstitial proteoglycans has to be analyzed by characterization of the proteoglycans produced in vitro. In this respect, modulation of this production by cytokines associated with early repair (i.e., TGFβ) and inflammatory processes (i.e., IL-1), and by cytokines that are present in increased levels in COPD (i.e., IL-8), as well as by T-lymphocyte-derived cytokines (i.e., IFNγ)-derived cytokines, has to be evaluated. It has been reported that TGFβ upregulates biglycan expression while it downregulates that of decorin in several models (27,118). Third, animal models may be helpful in confirming the relevance of our immunohistochemical findings in human lung tissue for the development of pulmonary emphysema. However, most of the animal models do not resemble the slow progression of emphysema observed in humans. Usually, acute models are applied in which a single high, nonphysiological dose of an external destructive agent (e.g., intratracheally instilled elastase) is administered. Emphysematous lesions in these animal models can be seen within a few weeks, which is a sharp contrast to the human situation, where development of emphysema follows several decades of exposure to cigarette smoke. Recently, cigarette smoke–induced emphysematous lesions have been described in guinea pigs (119) and mice (66). Furthermore, mice that overexpress MMP-1 spontaneously develop pulmonary emphysema (120), and mice with a targeted expression of IL-13 in the airway epithelium develop airway and lung parenchymal inflammation and emphysema after induction of the IL-13 gene (121). These animal models more closely resemble the slow progression of emphysema observed in humans and can be used to further study the role of proteoglycans in emphysema. Knockout models for relevant proteins, using decorindeficient mice (78) and biglycan-deficient mice (122), may be helpful in studying the role of these proteins. Recently, decorin and biglycan double-knockout mice were described (M.F. Young, et al., preliminary data). These knockout mice may also help in pathogenetic studies since decorin and biglycan have been shown to be affected in lung tissue from patients with emphysema. Analysis of the influence of decorin and biglycan deficiency on morphology, strength, and function of lung tissue, in particular after cigarette smoke exposure, might confirm the importance of an adequate production of these glycoproteins in the lung. Another way to better understand the development of different types of emphysema may be the measurement of peptide fragments of elastin, collagen, and proteoglycans and their actions. For instance, elastin peptide fragments are


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capable of attracting inflammatory cells—e.g., neutrophils—thereby maintaining the inflammatory process. Detection and characterization of these fragments may also contribute to further insight. Finally, attention should be paid to better characterization of the central cells in the production of matrix proteins, i.e., the fibroblasts, since this provides more insight in the adequacy of the tissue repair processes in vivo. Characteristics of special interest in determining the potential for fibroblasts to perform an adequate tissue repair reaction are the proliferation capacity and the potency to produce matrix proteins (both quantitative and qualitative). Additionally, the influence of modulating factors, e.g., cytokines and inflammatory cells, on these responses must be determined, as well as the influence of cigarette smoke. This approach seems to be feasible since after exposure to cigarette smoke, an inhibition of cell proliferation and migration (123) and of proteoglycan and procollagen production (124) by lung fibroblasts has been reported. This inhibition might impair lung repair following lung injury, and may thus contribute to the development of pulmonary emphysema. Susceptibility of fibroblasts for this smokeinduced inhibition may vary between nonsusceptible smokers and susceptible smokers who develop emphysema.

V.

Conclusions

The pathogenesis of pulmonary emphysema is still unclear. There is support in the literature that both a protease-antiprotease imbalance, in favor of the proteases, and an oxidant-antioxidant imbalance, in favor of the oxidant, contribute to the development of emphysema. But, as the activity of proteases as well as oxidants can be expected to be quite similar in all smokers, these factors do not explain why only 10–20% of all smokers develop (clinical) pulmonary emphysema. In lung tissue from patients with severe emphysema, and to a smaller degree in patients with mild emphysema, a diminished presence of the interstitial proteoglycans decorin and biglycan was found in the peribronchiolar area and diminished and discontinuous presence of HSPGs in the alveolar basement membranes, as compared to lung tissue from control subjects. The differences between emphysema and fibrosis, especially in the staining of interstitial proteoglycans decorin and biglycan, might imply an alternative regulation of the repair reaction after pulmonary injury in emphysema as compared to the development of fibrosis. Proteoglycans play a central structural role in the interstitial and basement ECM, as well as an important functional role in cell and tissue dynamics in the lung. The structural role of proteoglycans is mainly formed by bridging and connecting other ECM proteins. Diminished presence of interstitial proteoglycans or qualita-

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tive alterations in interstitial proteoglycans may be responsible for disruption of the collagen network, which is supposed to be essential for the marked increase in total lung capacity that occurs in emphysema. In pulmonary emphysema, the fibroblast may be intrinsically dysfunctional, resulting in a hampered production of ECM in either a quantitative or qualitative way. This hampered production of ECM also affects the binding, release, or regulation of the activity of matrix-bound cytokines. Furthermore, modulation of tissue repair by production of ECM-degrading enzymes and inhibitors of ECMdegrading enzymes might be affected. The net result of increased lung tissue degradation and insufficient repair is parenchymal destruction and emphysematous lesions (Fig. 4).

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13 Proteoglycans in Normal and Pathological Bronchial Mucus

K. RAMAKRISHNAN BHASKAR Beth Israel Deaconess Medical Center Boston, Massachusetts, U.S.A.

I.

Introduction

Mucus, the viscous secretion of epithelial glands, is a ubiquitous substance found in even the most primitive life forms. In mammals, mucus is secreted as a protective barrier between the epithelium and the environment by several organs: in the stomach it inhibits diffusion of secreted HCl (1); in the intestine it prevents damage from bacteria, viruses, and parasites (2); in the cervix it restricts passage of sperm to fertile period (3); and in the eye it helps to keep the corneal surface from drying (4). One of the main functions of bronchial mucus is to protect the lung from airborne particles by trapping them and facilitating their clearance by the ciliary movement (5). The viscous properties crucial for the functional role of mucus are generally attributed to mucin, a polymeric glycoprotein secreted by epithelial cells. Mucin has indeed proved to be a major macromolecular component responsible for the viscoelastic properties of gastric (6,7) and cervical mucus (8) and in sputum from chronic bronchitic (9) and asthma patients (10). Also, histochemical techniques have long identified acidic glycoproteins in most of the mucous cells of the human tracheobronchial tree (11). 269

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Airway secretion is a product of several cell types besides mucous cells (12,13): serous cells of submucosal glands as well as ciliated cells of the surface epithelium. Pure cultures of bovine tracheal serous gland cells have been shown to secrete mainly chondroitin sulfate, hyaluronic acid, and N-linked glycoproteins of complex type (14). Primary cultures of hamster tracheal surface epithelial cells (15) and immortalized human biliary epithelial cells (16) have also been shown to secrete chondroitin sulfate. That these are not artifacts of the cell culture system is suggested by the recent studies from several laboratories that have indicated that proteoglycans are present in both normal (17,18) and pathological mucus (19,20). In cystic fibrosis in particular, chondroitin sulfate appears to be a significant contributor to sputum insolubility (21). This chapter will review the current knowledge about proteoglycans present in bronchial mucus in health and disease. II. Cellular Sources of Bronchial Mucus Multiple cell phenotypes, several of which are secretory, constitute the airway epithelium that includes the surface epithelium and the submucosal gland (13). The two major types of mucus-secreting cells are the mucous and serous cells. Another cell type that could be a potential contributor of proteoglycans to bronchial mucus is the ciliated cell. A. Mucous Cells

Mucous cells are predominant in the surface epithelium and when distended with mucus, these cells are referred to as goblet cells. Histochemical studies have identified acidic glycoproteins in the mucous cells (11). The number of mucussecreting cells increases in chronic bronchitis in humans as well as in animal models of SO2-induced bronchitis (12). Although both mucous and serous cells can be identified in the human fetus, the latter are sparse in adult surface epithelium presumably because of their transformation into mucous cells under the influence of air pollutants (22). B. Serous Cells

Serous cells are far more numerous in the submucosal glands that are a major source of tracheobronchial mucus. The term “serous” was originally applied to these cells as they were thought to secrete primarily proteins with antibacterial and antiproteinase activity (23). Histochemical studies of serous secretion from the submucosal gland suggested that like the mucous cells, serous cells also contained complex carbohydrate (24). Consistent with autoradiographic evidence showing high radioactive sulfate uptake by serous cells (23), serous acini react

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with stains for sulfated glycoconjugates. Serous cells, however, failed to show reactivity to antibodies raised to airway mucin ruling out sulfated mucin as their secretory product (24). In vitro culture of bovine tracheal serous cells has since demonstrated that chondroitin sulfate proteoglycans are the major secretory product of these cells (see below). C. Ciliated Cells

Ciliated cells are the dominant cells of the surface epithelium and are crucial for the protective effect of bronchial mucus (22). It is the sweeping motion of the cilia that provides the force to move the mucous blanket from the peripheral airways into the pharynx. This “ciliary escalator” is an important defense mechanism since it helps clear inhaled particles trapped by mucus. It has been observed that with a combination of Alcian Blue (AB) and periodic acid Schiff (PAS), the surface layer on the tips of the cilia stain a turquoise blue that is typical of cartilage, suggesting proteoglycan, unlike the heliotrope purple-red typical of the mucous granule in a goblet cell (25). It is not known whether the cell surface proteoglycan is released or to what extent it contributes to bronchial mucus. III. Proteoglycans in Normal Bronchial Mucus Normal bronchial mucus is difficult to obtain because in the healthy airway mucus is constantly transported by the mucociliary system to the larynx and swallowed. Several approaches have been used to obtain and characterize normal bronchial mucus, chief among which are (1) bronchoalveolar lavage from healthy volunteers, (2) in vitro culture of airway mucosal explants, and (3) characterization of glycoconjugates secreted by airway epithelial cells in culture. IV. Bronchoalveolar Lavage (BAL) from Healthy Volunteers Saline aspiration using fiberoptic bronchoscopy of mucus from healthy volunteers with no smoking history provides material closest to secreted normal bronchial mucus although its scanty amount limits detailed characterization, especially on individual aspirates. Mucus aspirated from normal subjects when examined individually by analytical density gradient ultracentrifugation in cesium bromide (CsBr) did not appear to contain any material of buoyant density ⬃1.5 g/mL, typical of mucin but rather a trace component of higher density ⬃1.6 g/mL (17). Gas chromatographic analysis revealed the presence Xyl, a component of proteoglycans in addition to sugars found in mucin, i.e., Fuc, Gal, GlcNAc, GalNAc, and Ac Neu, but because of insufficient material more detailed characterization was not possible. The larger amounts recovered from healthy volunteers with a

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smoking history allowed further characterization of the high-density component. In gel filtration, it eluted as a single peak near the void volume of a Biogel A5M column, suggesting a single macromolecular species, yet fractions from this peak still contained appreciable amounts of Xyl in addition to sugars typical of mucin. The major amino acids in the high-density component—Asp, Thr, Ser, Glu, Pro, Gly, and Ala—were the same that make up ⬃80% of the total amino acids in bronchial mucins (9,10) but their relative distributions were different. In bronchial mucins, Thr and Ser to which oligosaccharide chains are attached, are the preponderant amino acids with Thr always in excess of Ser (9,10). In the highdensity glycoconjugate Ser and Gly were equimolar and considerably higher than Thr thus resembling proteoglycans in which the sequence Ser-Gly is consistently found at the site of attachment of chondroitin sulfate (CS) chains (26). Twodimensional cellulose acetate electrophoresis of the component gave rise to an Alcian Blue–positive spot comigrating with CS that was lost upon chondroitinase digestion, confirming the presence of CS in the high-density component. The high buoyant density, elution as a single peak in gel filtration, and presence of sugars typical of both mucins and proteoglycans suggest that the high-density component is a “hybrid” molecule similar to that reported in porcine ovarian follicular fluid (27).

Table 1 Carbohydrate Composition of Glycoconjugates Isolated from Canine Bronchial Aspirate (Pre-SO2 )

Fuc Xyl IdoA Man Gal Glc GlcNAc GalNAc NeuAc

Dog 11953

Dog 12602

Dog 34033

Dog 8999

1.0 0.1 0.3 1.6 3.4 0.6 4.2

1.0 0.1 0.3 1.0 2.7 1.1 4.9 0.6 1.0

1.0 0.2 0.3 1.2 3.5 2.4 3.5 1.0 1.4

1.0 0.3 n.d. 0.8 2.3 1.3 2.8 2.5 0.8

1.1

Values are given as molar ratios with respect to Fuc as 1.0. Glycoconjugates separated by densitygradient ultracentrifugation were digested with papain prior to analysis by GC, except in the case of dog 8999. The identification of Xyl and IdoA confirms the presence of glycosaminoglycans in every case, in addition to sugars typical of epithelial glycoprotein. The limited amount of the glycoconjugate did not permit sugar analysis as a percentage of sample weight. Further, the presence of glycosaminoglycans interfered with the analysis, especially on hexosamines. Source: Ref. 30.


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Figure 1 Two-dimensional electrophoresis of glycoconjugate from normal canine bronchial mucus. High-density glycoconjugate from normal canine (pre-SO2 ) bronchial aspirate with standard heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate AC (CSAC), chondroitin sulfate B (CSB, dermatan sulfate) (right panel), standards alone (left panel). Glycoconjugate in canine bronchial aspirate gave rise to an Alcian blue–positive spot (indicated by arrow in right panel) between HA and HS partially overlapping the latter. (From Ref. 18.)

Similar findings have been reported for normal aspirates from a canine model of SO2-induced bronchitis (18). Examination of multiple bronchial aspirates from airways of a number of dogs obtained prior to SO2 exposure did not reveal material with buoyant density typical of mucin, only a high-density component containing appreciable amounts of Xyl and IdoA, in addition to sugars typical of mucin (Table 1). Examination by two-dimensional electrophoresis following the procedures of Schmidt et al. (28) further supported the notion that the glycoconjugate is a hybrid. As shown in the right panel of Figure 1, the glycoconjugate from pre-SO2 aspirate gave rise to an Alcian blue–positive spot between standard HA and HS, partially overlapping the latter. V.

In Vitro Culture of Airway Mucosal Explants

Explant culture studies of human and animal airway mucosa have been used for many years both to characterize mucus and study factors regulating its secretion. An early study by Kent and coworkers (29) found that the major protein-polysaccharide complex secreted by calf tracheal epithelium contained sugars typical of both mucin and CS. Upon fractionation of the spent medium on Sephadex G-200, radiolabeled sulfate and glucosamine, used as metabolic precursors, were found exclusively in the void volume, and in cellulose acetate electrophoresis a single polyanionic component was observed. Although these features were suggestive of homogeneity, gas chromatography revealed the presence of GlcA and

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Xyl as well as mucin sugars. This component could not be detected in spent culture medium using explants from which the epithelium had been removed, indicating its epithelial origin. A more recent study of in vitro secretion by human bronchial and canine tracheal (30) explants also found glycosaminoglycans to be the major secreted glycoconjugates accounting for up to 80% of bound 14 C-glucosamine. Cervical trachea from mongrel dogs and human tissue obtained from lobectomy specimens resected due to carcinoma but judged to have a normal tracheal architecture with no neoplastic tissue, were used to prepare explants of airway mucosa free of cartilage and parenchymal tissue. Explants were incubated with culture medium containing antibiotics for an initial period of 3 h to wash off any adherent mucus and blood after which the explants were transferred to new flasks and cultured for 24 h in medium containing 1 µCi/mL D-(1-14 C)-glucosamine hydrochloride. Harvested media were dialyzed in the cold against distilled water and analyzed. Examination of nondialyzable material from several individual specimens of canine and human tissues indicated that the secreted glycoconjugate had a higher buoyant density than is typical of mucin (⬃1.5 g/mL in CsBr) and contained Xyl in addition to sugars typical of mucin. A pool of secretions from several specimens was subjected to density gradient ultracentrifugation in a CsBr gradient to remove lipid, and protein constituents and five glycoconjugate subfractions of varying density were recovered. Sugars

Table 2 Amino Acid Composition of Glycoconjugates Secreted by Human Airway Mucosal Explants In Vitroa Amino acid Fraction 1 2 3 4 5 Proteoglycan of pig laryngeal cartilageb Bronchial epithelial glycoprotein (9) a

Asp

Thr

Ser

Glu

Pro

Gly

Ala

11 9 12 14 14 12

6 10 11 11 8 9

15 15 15 15 11 15

22 19 18 16 22 19

8 11 13 13 15 14

28 26 20 18 17 18

9 10 10 13 9 12

8

26

17

11

15

12

12

Values are given as moles amino acid/100 moles of amino acids listed. These 7 acids account for 60– 70% of the protein and include ones that show marked differences in distribution between epithelial glycoprotein and proteoglycan. Fractions 1–5 are glycoconjugate subfractions obtained by density gradient ultracentrifugation, in decreasing order of density. b Hardingham TE, Muir H. Hyaluronic acid in cartilage and proteoglycan aggregation. Biochem J. 1974. 139: 569–581. Source: Ref. 30.


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Figure 2 Identification of glycosaminoglycans secreted in vitro by human bronchial explants using two-dimensional electrophoresis. Glycoconjugate subfraction 1 (left panel), which accounted for 25% of incorporated 14 C-glucosamine radiolabel, gave rise to an intense alcian blue–positive spot corresponding to HA and weaker spots for CS A, B, and C. Right panel shows the same fraction at twice the concentration in the presence of standard CS C. At the higher concentration the CS B band is visible. CS AC was the predominant component in fraction 2 that incorporated 44% of radiolabel (not shown here). (From Ref. 30.)

typical of epithelial glycoprotein (mucin) were identified only in the glycoconjugate subfraction 5 of lowest density with the least radiolabel incorporation (5% in the case of human and 12% in canine), but glycosaminoglycans were also present in this fraction. In both human and canine explant secretion, the highest incorporation of radiolabel was in the two fractions of highest density (⬎1.55 g/mL). Amino acid composition of the fractions did not show preponderance of Ser and Thr typical of mucin, but showed similarities to that of proteoglycan (Table 2). Two-dimensional electrophoresis of the fractions confirmed the presence of HA and CS A, B, and C in these fractions (Fig. 2). Radioscanning of the electrophoresis strips detected radioactivity in the region of the alcian blue spots, confirming incorporation of glucosamine into glycosaminoglycans (Fig. 3). VI. Glycoconjugates Secreted by Airway Epithelial Cells in Culture Media collected from explants include secretions from several cell types. Advances in cell culture techniques in recent years have allowed studies of monolayer cultures of surface epithelial and submucosal gland cells. Serous cells of

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Figure 3 Radioscanning of two-dimensional electrophoretograms of glycoconjugate fraction 1 (shown in Fig. 2). Arrows in the electrophoresis strips (lower panel) and scanner charts (upper panel) indicate location and direction of scanning, respectively. Two channels differing in sensitivity by a factor of 3 were used simultaneously for each scan. Redand blue-colored pens used by the two channels result in differing intensities of scans. Radioactivity was detected only over the alcian blue–positive spots as seen in the two peaks in the upper panel. A larger peak, indicating higher radioactivity, was associated with the spot corresponding to Ha than with CS, presumably because the disaccharide of HA contains GlcNAc, the radiolabel ( 14 C-glucosamine) used. (From Ref. 30.)


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the submucosal glands are thought to be a major contributor to bronchial mucus and have therefore been the subject of several studies. Bovine tracheal serous cells were prepared by stripping away the surface epithelium followed by dissection of the soft tissue from cartilage and then incubating the tissue with collagenase and elastase (31). Cells dissociating from the tissue clumps were recovered by centrifugation and plated on collagen-coated plastic plates. The cells displayed features characteristic of serous cells and were reactive to specific monoclonal antibodies. Cells were cultured in the presence of 14 C-glucosamine, and radiolabeled glycoconjugates secreted into the medium were subjected to gel filtration on a Sepharose Cl-4B column in the presence of 4 M guanidine HCl. About half the incorporated radiolabel eluted in the void volume, indicating high molecular glycoconjugates (⬎106 ). Anion exchange chromatography on ECTEOLA cellulose of the void volume fraction gave a major peak eluting with 0.5 M NaCl that contained 89% of the radiolabel. Almost all the radiolabeled material in this peak was susceptible to digestion by chondroitinase ABC or AC, and the products of digestion were identified as unsaturated disaccharides (0-sulfate), the absence of sulfation suggesting hyaluronic acid. Further verification that this is hyaluronic acid came from the identification of glycosamine in the disaccharides. A small amount of radiolabel associated with O-linked glycoprotein was also present in the same fraction. The minor peak (11% of radiolabel) that eluted with 2M NaCl was also digested by chondroitinase ABC or AC, and radioactivity was found to be predominantly in sulfated disaccharides (⬃40% di-6S, ⬃30% di-4S, ⬃30% di0S), which had galactosamine as the hexosamine, indicating this fraction contained chondroitin sulfate proteoglycans. A small percentage of the incorporated label was not digestible by chondroitinase ABC, and this fraction contained Fuc and Gal, suggesting that mucin-type oligosaccharides are present. The results from this study of bovine tracheal serous cells in culture parallel those from human bronchial explant culture reported above (30). In both systems the secreted glycoconjugates are primarily proteoglycans (hyaluronic acid and chondroitin sulfate), and mucin appears to be a minor component inseparable from the glycosaminoglycans under the fractionation conditions used. Decorin, the small dermatan sulfate–containing proteoglycan, also known as PG II or PG40, has since been identified as the major proteoglycan secreted by bovine tracheal gland cells (32). Serous cell phenotype of the cells was established by immunocytochemical identification of lysozyme and lactoferrin, known secretory products of these cells. In addition, a rabbit antihuman decorin antibody was found to stain all the cells. When medium from cells cultured in the presence of [ 35S]sulfate was subjected to gel filtration on Sepharose CL-6B, a single peak was observed, which was completely degraded by chondroitinase ABC, confirming the radiolabeled product was chondroitin and/or dermatan sulfate. To identify the protein core, cells were also cultured in the presence of [ 35S]methionine, and

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the medium was subjected to gel filtration before and after chondroitinase ABC treatment. The majority of radiolabeled proteoglycan in the medium eluted at a Kav ⬃0.35, corresponding to a molecular weight of ⬃100,000 daltons, which shifted to a Kav of ⬃0.5 (molecular weight ⬃40,000) after chondroitinase ABC digestion. This approximates the reported values for the decorin core protein from bone and tendon (33). Examination of radiolabeled medium by SDS-PAGE showed a band ⬎200 kDa that shifted to a predominant band at 45 kDa after chondroitinase ABC treatment, confirming the presence of decorin. Partial amino acid sequencing of the 45-kDa band gave a sequence (Ser-Ser-Gly-Ile-Glu-Asn-Gly-Ala-Phe-GlnGly-Met-Lys) identical to amino acids 159–171 of the deduced amino acid sequence of the cDNA for bone decorin (34). To verify the expression of decorin message in the bovine tracheal serous cells, RNA extracted from confluent cultures was hybridized to a human decorin cDNA probe. A band at ⬃2.0 kb was observed, in conformity with the transcripts of 1.6 and 1.9 kb previously described for decorin (35). In situ hybridization of a decorin riboprobe to bovine tracheal tissue confirmed that decorin is expressed, albeit at low levels, in the epithelium of the submucosal glands. Higher levels of expression were observed in the mesenchymal cells on the periphery of the submucosal glands which is consistent with the known presence of decorin in connective tissue. While the reasons for the low levels of expression in the epithelium remain to be determined, it is clear from this study that tracheal gland cells in culture can and do synthesize and secrete decorin. Proteoglycans were also identified in the secretions from in vitro culture of human tracheal gland cells (36) isolated by enzymatic digestion of tracheal mucosa from healthy young adults who died of head trauma. Unlike in the case of the bovine tracheal gland cells (31), hyaluronic acid was not among the radiolabeled glycoconjugates secreted by the human cells, but up to 50% of the incorporated sulfate label was susceptible to digestion by chondroitinase ABC. It has been long held that mucins are the major glycoconjugate component of normal as well as pathological mucus, and the suggestion that proteoglycans are the dominant glycoconjugate in normal mucus has generated considerable controversy (37,38). Admittedly, there have been reports describing mucin as the major glycoconjugate in normal mucus as in the case of secretions collected in the canine tracheal pouch (39) or from patients undergoing minor dental surgery (40) among others. In our studies of normal mucus from a variety of sources described above, the major glycoconjugate component had unmistakable features of a proteoglycan. Studies from other groups indicate that proteoglycans constitute the major secreted glycoconjugate during in vitro culture of tracheal gland cells, which are a major contributor to airway secretion. It is clear that proteoglycans are an important glycoconjugate constituent of normal mucus and need to be studied in greater


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detail to have a better understanding of the pathological changes in mucus in airway diseases. VII. Proteoglycans in Pathological Bronchial Mucus Bronchial mucus makes its presence felt especially, if not only, in its pathological state because of the considerable physical distress it causes to patients with obstructive airway diseases. It thus drew the attention of clinicians long before attracting basic researchers to this field. Early attempts by clinicians to solubilize sputum led to empirical use of thiol-reducing agents as mucolytics even before the role of disulfide bond aggregation in mucin aggregation and polymerization was established. A. Chronic Bronchitis

Chronic bronchitis is characterized by the production of copious amounts of viscous mucus that leads to obstruction of airways in many patients. Even before they were isolated and characterized, epithelial glycoproteins were recognized as major contributors to the viscosity of such mucus (41). Later studies have confirmed that epithelial glycoproteins or mucins isolated from chronic bronchitic sputum are, indeed, of large molecular weight and can give rise to solutions of high viscosity (9). Since viscosity of mucus has a major role in pulmonary obstruction, the identification of large polymeric epithelial glycoproteins in chronic bronchitic sputum restricted the attention of most researchers to these mucin molecules. The canine model of bronchitis (18) offered an opportunity to analyze secretion from the same airway before and after the onset of bronchitis, and here, too, hypersecretion of mucin has been observed. Whereas the glycoconjugate of pre-SO2 (control) aspirates appeared to be a hybrid molecule with features of a glycoprotein as well as proteoglycans, appreciable amounts of mucin of typical buoyant density and chemical composition were present in post-SO2 aspirates from the same dog. The hybrid glycoconjugate was still present in the post-SO2 aspirate, and in a few cases additional discrete bands were seen in the electrophoresis strips corresponding to HA and CSB. Theocharis (42), in a study devoted to investigating proteoglycans in bronchial mucus, reported hyaluronic acid as the major glycosaminoglycan in sputum expectorated by chronic bronchitic (CB) patients. Sputum samples collected from seven chronic bronchitic patients were immediately mixed with 10 volumes of acetone and after overnight standing in the cold, were centrifuged, washed once with acetone, and air-dried. Weighed amounts of the dry mucus samples were digested with papain, and the released glycosaminoglycans were recovered by precipitation with ethanol. Chemical analysis revealed the presence of uronic acid

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and hexosamines; uronic acid assay of fractions from gel filtration chromatography on Sephadex G-100 showed two peaks, both of which were completely digested by hyaluronidase and chondroitinase ABC. Cellulose acetate electrophoresis identified HA as the major glycosaminoglycan, with a minor component that appeared to be undersulfated CS. B. Pulmonary Alveolar Proteinosis

Pulmonary alveolar proteinosis (PAP) is a rare but potentially deadly disease of unknown etiology characterized by the accumulation of surfactant in alveolar spaces eventually leading to impairment of gas exchange (43). Clinical symptoms of PAP include severe nonproductive cough and shortness of breath on exertion, relief from which may be obtained by bronchoalveolar lavage (BAL) to remove accumulated phospholipid material. In some patients the condition is cured after lavage; in others it recurs, requiring repeated lavage. Biochemical studies of BAL fluid have mostly focused on lipid and protein constituents, but there have been a few reports of the presence of glycosaminoglycans (44,45). Sahu and Lynn (44) reported finding hyaluronic acid as the only glycosaminoglycan constituent of the pulmonary secretions of patients with alveolar proteinosis. The GAG component had a hexuronate/hexosamine ratio of ⬃1:1 with GlcNAc accounting for 98% of hexosamine, migrated as a single band on cellulose acetate electrophoresis with the same mobility as that of standard HA, and was susceptible to digestion by hyaluronidase. This HA was associated with small amounts of protein in which aspartic and glutamic acids, glycine, alanine, and leucine were predominant. Satoh et al. (45) found differences in the GAG content of BAL from two PAP patients who had very different clinical courses. In one patient three successive lavages brought about a complete recovery, and the BAL from this patient did not contain any GAG—only glycoprotein. In contrast, HA; CS A, B, and C; and HS were all identified, in addition to glycoprotein, in the BAL from the other patient in whom symptoms persisted even after repeated therapeutic lavages. Whether the presence of GAGs in BAL fluid can be used as a prognostic marker for PAP remains to be established by more studies. C. Asthma

Sahu and Lynn (20) analyzed the glycosaminoglycan content of bronchoalveolar lavage collected from four asthmatic patients. Insoluble material in the lavage fluid was separated by centrifugation, and the supernatant was dialyzed, lyophilized, and subjected to proteolysis with pronase. Proteins were precipitated with 5% TCA, and the glycosaminoglycans in the supernatant were precipitated with an ethanol/acetic acid mixture (46) or as a cetylpyridinium complex (47). Cellulose acetate electrophoresis of the isolated glycosaminoglycan showed a single


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Alcian Blue–positive spot that comigrated with standard hyaluronic acid and was no longer seen after digestion with testicular hyaluronidase. Chemical analysis indicated that the hyaluronic acid contained 48% by weight of hexuronic acid and 42% of hexosamine, 98% of which was glucosamine, the rest galactosamine. A small amount of protein (3%) was also present which remained even after extensive proteolysis. The fact that a single glycosaminoglycan component was present in the lavage fluid from asthmatic patients led the authors to speculate that this may be a specific marker for this disease. A recent study (48) has found that when healthy nonsmoking volunteers with no history of lung disease were challenged with inhaled histamine, there was a marked (twofold) increase of hyaluronan in bronchoalveolar lavage fluid. The authors explained the increase as due to histaminemediated leakage of interstitial fluid, rich in HA, to the alveolar space. Since allergic asthma is associated with mast cell degranulation, this could explain the presence of increased amounts of HA in BAL fluid from asthmatic patients. D. Bronchial Mucus in Acute Quadriplegic Patients

Accidents causing severe spinal cord injury result in quadriplegia with associated paralysis of muscles of respiration, making it difficult for patients to clear mucus from their lungs. In about one in five such patients, there is a sudden onset of bronchial mucus hypersecretion, the cause of which remains unknown. Accumulation of mucus persists in many patients, often proving fatal owing to complications from airway obstruction, atelectasis, and respiratory failure, whereas in some there is spontaneous recovery from mucus hypersecretion within months, sometimes even as early as weeks. The sudden onset of mucus hypersecretion in acute quadriplegia is in striking contrast to hypersecretion leading to sputum production seen in chronic bronchitis among chronic smokers. This and the spontaneous recovery rule out glandular hypertrophy as the cause of hypersecretion and suggest a role for disturbed neuronal control. Mucus from acute quadriplegic patients was found to contain appreciable amounts of mucin with typical biochemical and biophysical features (49). A highdensity glycoconjugate component was also present in the mucus, gas chromatographic (GC) analysis of which revealed sugars typical of mucin but they only accounted for ⬍25% by weight. Digestion of this component with papain prior to methanolysis followed by GC revealed, in addition, xylose and uronic acid, suggesting that it contained glycosaminoglycans. The amino acid profile was different from that of the mucin fraction. Threonine constituted 195 and 249 moles/ 1000 moles of AA in the mucin fraction from two different samples, but only 116 and 112 moles/1000 moles in the respective high-density components and thus was no longer the most predominant amino acid as in the mucin fraction. Gly was higher in the two high-density components (250 and 203 moles/1000

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moles) than in the corresponding mucin fractions (106 and 96 moles/1000 moles). This high-density component, when examined by two-dimensional electrophoresis, gave rise to an Alcian Blue–positive spot lying between hyaluronic acid and heparan sulfate, but no discrete glycosaminoglycan was identified. This is similar to the spot seen in pre-SO2 canine tracheal aspirates described above and might represent the “hybrid” glycoconjugate of normal bronchial mucus. The high Gal and GlcNAc content was suggestive of the presence of polylactosamine chains as in keratan sulfate, and the high-density component did indeed exhibit reactivity to a monoclonal antibody to keratan sulfate whereas the mucin fraction did not. It is worth noting here that in the proteoglycan aggregate O-linked oligosaccharides are present in the region where keratan sulfate chains occur. These findings are supported by the report that rabbit surface epithelium secretes, in addition to glycosaminoglycans, a novel high-molecular-weight glycoprotein resembling keratan sulfate but no mucins (50). In mucus from one of the patients, discrete bands were seen in two-dimensional electrophoresis comigrating with CS A, B, and C; HS; and trace amounts of HA. Unlike the other patients in the study, who recovered, this patient died, which again raises the question about the role of proteoglycans in the pathophysiology of mucus hypersecretion. E.

In Vitro Culture of Respiratory Carcinoma Cell Lines

The ease of passage and rapid growth of cell lines derived from carcinomas of epithelial origin have led to their use as model systems to study mucin secretion and gene regulation. One such recent study examined glycoconjugates secreted by six carcinoma cell lines of respiratory tract origin (51). Cells were metabolically labeled with [ 35S]SO4 for 20–24 h, and the radiolabeled secretions were dialyzed for two successive 24-h periods. One-half of the nondialyzable material was directly subjected to gel filtration chromatography on Sepharose CL-4B; the other half was digested with bovine testes hyaluronidase prior to chromatography. In secretions from all the six cell lines, a small amount of radiolabel was found in the excluded fractions, indicating that the cells were secreting large-molecularweight sulfated glycoconjugates. In hyaluronidase-digested secretion from five of the six cell lines there was no radiolabel in the excluded fractions, while in the case of the sixth cell line, Calu-3, only 50% of the radiolabel remained, suggesting that the large-molecular-weight sulfated glycoconjugates were predominantly glycosaminoglycans. It is important to emphasize that the secretion from the majority of the cell lines consists of proteoglycans although the study was concerned mainly with the small percentage of mucin from one of the cell lines. F. Cystic Fibrosis

Cystic fibrosis (CF) is the most common autosomal-recessive disorder in Caucasians worldwide. The major pathological manifestations in CF are obstruction of


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pulmonary, gastrointestinal, and pancreatobiliary ducts by accumulation of mucous secretion (52). The basic cellular defect in CF is abnormal chloride transport resulting from mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (53), which encodes a protein required for the normal function of a cAMP-regulated chloride channel (54) present in secretory and other cells throughout the body. The connecting link between the defect in the CFTR gene and the cellular/physiological basis of the mucous abnormalities in CF remains to be elucidated. Approximately 95% of CF patients develop lung disease related to airway obstruction, inflammation, and recurrent infections that are eventually lethal. A number of studies carried out over the years have, however, failed to identify any qualitative or quantitative abnormality of mucin secretion specific to CF. Although mucin has been considered to be the major secreted glycoconjugate constituent of mucus, accumulating evidence indicates that proteoglycans are also secreted by the airway mucosa. Proteoglycans could also account for the increased sulfation of glycoconjugates in CF reported in some studies (55). Our recent study of immortalized intrahepatic biliary epithelial cells not only showed that chondroitin sulfate is the major secretory product but also that cells derived from CF patients secreted increased amounts of CS (16). In the bile duct epithelium, CFTR is localized to these cells, raising the possibility that a similar situation might arise in the airway mucosa where CFTR has been localized to serous cells, cells that have been shown to secrete chondroitin sulfate proteoglycans. Studies have indeed identified CS in sputum from CF patients. In one study by Rahmoune et al. (19), sputum samples from 13 CF patients were examined by agarose electrophoresis for the presence of glycosaminoglycans. Sputa were dialyzed against distilled water and centrifuged at 3000g for 30 min, and the supernatants after lyophilization were digested with pronase. The mixtures of glycopeptides, glycosaminoglycans, and nucleic acids were treated separately or with a mixture of nucleases and glycosaminoglycan-degrading enzymes—i.e., chondroitinase and/or heparinase. Agarose (1%) electrophoresis in veronal buffer pH 8.2, performed according to the procedures of Scheidegger (56), gave rise to toluidine blue–positive bands in 11 of the 13 samples; susceptibility to chondroitinase ABC and AC but not heparinase identified the band as due to CS. There was very little nucleasesensitive material in sputa from two of the CF patients, suggesting they contained only trace amounts of DNA; interestingly, CS was not detected in these two samples. Since the presence of nucleic acids usually indicates infection, the presence of CS appears to be related to infection status of sputum. Proteoglycans are known to give rise to viscous solutions and could very well contribute to sputum insolubility, as has been recently found with DNA. We have recently carried out a collaborative study of the effect of protease-free chondroitinase ABC on solubility of CF sputum (21). Aliquots of sputa collected from a large number of CF patients were incubated in duplicate with buffer alone

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Figure 4 Identification of CS in CF sputum using an anti-CS monoclonal antibody. Aliquots of CF sputum were incubated with buffer alone or with chondroitinase (Ch) ABC, and the resulting supernatants (SN) were subjected to 7% SDS-PAGE followed by Western blotting to anti–chondroitin-4-sulfate monoclonal antibody (MAB 2030, Chemicon, CA). Several intense immunoreactive bands are seen in the enzyme-treated SN but not in the control SN, confirming the presence of CSPG in the sputum.

or buffer containing chondroitinase ABC (EC 4.2.2.4 Proteus vulgaris, Seikagaku) for 18 h at 37°C. Samples were then centrifuged at 12000 rpm for 20 min, and the supernatant was recovered by aspiration for analysis. Compared to buffer controls, the size of the pellet in aliquots incubated with the enzyme was markedly lower, suggesting that enzyme digestion had resulted in solubilization of sputa. Examination of the supernatants from the enzyme-digested aliquots by SDSPAGE followed by Western blotting to anti–chondroitin-4-sulfate monoclonal antibody (MAB 2030, Chemicon, CA) showed several intense immunoreactive bands that were not seen in the supernatant from the control aliquot (Fig. 4). Digestion with chondroitinase ABC leaves a modified disaccharide, delta unsaturated GlcA-GalNAc, attached to the core protein through the linkage tetrasaccharide, hexuronic acid β1,3 Gal β1,3 Gal β1,4 Xyl (57). The antibody used in the Western blots has been shown to recognize the unsaturated disaccharide resulting from the chondroitinase ABC digestion (58). Examination of sputa from a large number of CF patients has shown a similar set of anti-CS immunoreactive bands in all cases. It was clear from these blots that appreciable amount of CSPGs were present in the sputa and, more importantly, that enzymatic digestion of CSPG may offer a novel and potentially significant adjunct to mucolytic therapy to relieve airway obstruction in CF patients.


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The studies described above indicate that proteoglycans constitute a significant glycoconjugate component of airway mucus requiring further study. Their role in the pathophysiology of airway diseases remains to be elucidated (59). Studies of normal mucus appear to suggest that in the normal, the glycoconjugate component is not typical mucin but a mixed molecule with features of glycocoprotein and proteoglycan. The fact that mucin genes are not detected except in the irritated airway supports this notion. In the canine model of bronchitis where secretion from the same animal before and after irritation could be examined, typical glycoprotein was detected after the onset of bronchitis, whereas the control pre-SO2 aspirates contained what appeared to be a hybrid molecule. This was supported by the finding that antibody to purified bronchial mucin stained tissue from SO2-exposed dogs but not from control animals (60). Histochemical studies have shown the transformation of serous cells to mucous cells upon irritation. Since serous cells, in vitro, secrete mainly proteoglycans, and mucous cells stain predominantly for acidic glycoprotein, i.e., mucin, such transformation can explain the change from a proteoglycan in the normal to a typical mucin in the irritated airway of a chronic bronchitic patient with hypertrophy of the bronchial epithelium. Although no clear pattern can be seen for the type of glycosaminoglycan associated with a specific lung disease, in sputum from CF patients chondroitin sulfate appears to be the dominant glycosaminoglycan. Since sputum from CF patients is often infected and purulent, there are a number of possible sources for proteoglycans, such as protease digestion of the epithelium or connective tissue proteoglycans, secretion or release of human peripheral leukocytes which have been shown to contain CSPG (61). On the other hand, studies with tracheal serous cells indicate that CSPG is the major glycoconjugate secreted by these cells in vitro in the absence of infection. Interestingly, in the lung CFTR is localized to the serous cells pointing to a potential link between the two.

References 1. Bhaskar KR, Garik P, Turner BS, Bradley JD, Bansil R, Stanley HE, LaMont JT. Viscous fingering of HCl through gastric mucin. Nature 1992; 360:458–461. 2. Forstner JF. Intestinal mucins in health and disease. Digestion 1978; 17:234–263. 3. Chantler E. Structure and function of cervical mucus. In Chantler EN, Elder JB, Elstein M, eds. Mucus in Health and Disease. New York: Plenum Press, 1981:251– 263. 4. Kurpakus WM, Kernacki KA, Hazlett LD. Corneal cell proteins and ocular surface pathology. Biotech Histochem 1999; 74(3):146–159. 5. Lopez-Vidriero MT, Reid L. Bronchial mucus in health and disease. Br Med Bull 1978; 34:63–74.

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6. Allen A. Structure and function of gastrointestinal mucus. In: Johnson LR, ed. Physiology of the Gastrointestinal Tract. New York: Raven, 1981;617–639. 7. Bhaskar KR, Gong D, Bansil R, Pajevic S, Hamilton JA, Turner BS, LaMont JT. Profound increase in viscosity and aggregation of gastric mucin at low pH. Am J Physiol 1991; 261 (Gastrointest Liver Physiol 24):G827–G832. 8. Sheehan JK, Carlstedt I. Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride. Biochem J 1984; 217(1):93–101. 9. Creeth JM, Bhaskar KR, Horton JR, Das I, Lopez-Vidriero MY, Reid L. The separation and characterization of bronchial glycoproteins by density-gradient methods. Biochem J 1977; 167:557–569. 10. Bhaskar KR, Reid L. Application of density gradient methods for the study of mucus glycoprotein and other macromolecular components of the sol and gel phases of asthmatic sputa. J Biol Chem 1981; 256:7583–7589. 11. Lamb D, Reid L. Histochemical types of acidic glycoproteins produced by mucous cells of the tracheobronchial glands in man. J Pathol 1969; 98:213–229. 12. Jones R, Reid L. Secretory cells and their glycoproteins in health and disease. Br Med Bull 1978; 34:9–16. 13. Jeffrey PK. Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J 1997; 10:1655–1662. 14. Basbaum CB, Forsberg LS, Paul A, Sommerhoff C, Finkbeiner WE. Studies of tracheal secretion using serous cell cultures and monoclonal antibodies. Biorheology 1987; 24:585–588. 15. Kim KC, Opaskar-Hincman H, Bhaskar KR. Secretions from primary hamster tracheal surface epithelial cells in culture: mucin-like glycoproteins, proteoglycans, and lipids. Exp. Lung Res 1989; 15:299–314. 16. Bhaskar KR, Turner BS, Grubman SA, Jefferson DM, LaMont JT. Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-F508) cystic fibrosis transmembrane conductance regulator. Hepatology 1998; 27:54–61. 17. Bhaskar KR, O’Sullivan DD, Seltzer J, Rossing TH, Drazen JM, Reid LM. Density gradient study of bronchial mucus aspirates from healthy volunteers (smokers and non-smokers) and from patients with tracheostomy. Exp Lung Res 1985; 9:289– 308. 18. Bhaskar KR, Drazen JM, O’Sullivan DD, Scanlon PM, Reid LM. Transition from normal to hypersecretory bronchial mucus in a canine model of bronchitis: changes in yield and composition. Exp Lung Res 1988; 4:101–120. 19. Rahmoune H, Lamblin G, Lafitte J-J, Galabert C, Filliat M, Roussel P. Chondroitin sulfate in sputum from patients with cystic fibrosis and chronic bronchitis. Am J Resp Cell Mol Biol 1991; 5:315–320. 20. Sahu S, Lynn WS. Hyaluronic acid in pulmonary secretions of patients with asthma. Biochem J 1978; 173:565–568. 21. Khatri IA, Bhaskar KR, LaMont JT, Sajjan SU, Ho CKY, Forstner JF. Solubilizing effect of chondroitinase ABC on purulent sputum from cystic fibrosis and other patients. (Manuscript in preparation.) 22. Murray JF. The Normal Lung. The Basis for Diagnosis and Treatment of Pulmonary Disease, 2nd ed. Philadelphia: W.B. Saunders, 1986.


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14 Vascular Proteoglycans

¨ RVELA ¨ INEN HANNU JA

THOMAS N. WIGHT

University of Turku Turku, Finland

The Hope Heart Institute Seattle, Washington, U.S.A.

I.

Introduction

Proteoglycans form a versatile group of complex macromolecules consisting of a core glycoprotein covalently linked to linear chains of carbohydrates termed glycosaminoglycans (GAGs). Proteoglycans can be found in different amounts in the extracellular space, on the cell surface, and inside the cells in all tissues and organs. In blood vessels, proteoglycans constitute only a minor component but are critically involved in a variety of physiological and pathophysiological events that occur within the vascular wall. Proteoglycans are essential for proper matrix assembly, and may play an important role in maintaining the structural integrity of the vascular wall. In addition, proteoglycans participate in more dynamic processes. They contribute to hemostasis and thrombosis by interacting with clotting and fibrinolytic factors. Proteoglycans are also centrally involved in cell adhesion, migration, and proliferation through their ability to bind to and interact with a number of other extracellular matrix (ECM) molecules, growth factors, and cytokines. Furthermore, interactions of proteoglycans with lipoproteins and enzymes regulating lipoprotein metabolism can promote extra- and intracellular lipid accumulation in the vascular wall during the progression of vascular disease. An increasing 291

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amount of evidence is available suggesting that proteoglycans also play an important regulatory or modulatory role in inflammation and neovascularization. In this review we will discuss in more detail the structural characteristics and classification of vascular proteoglycans as well as their distribution and potential functions within the vascular wall. Examples of the importance of proteoglycans to vascular diseases such as atherosclerosis and restenosis will also be given. While this chapter focuses on vascular proteoglycans, many of the observations discussed may relate to other soft tissues and diseases that involve ECM remodeling such as those observed in the lung.

II. Proteoglycans: Structure and Classification The term proteoglycan designates complex macromolecules that are composed of a core glycoprotein to which one or more GAG side chains are covalently attached through a specific linkage region (Fig. 1). Proteoglycans normally exist bearing GAGs, but there are a number of glycoproteins such as collagen types IX, XII, XIV, XV, and XVIII, thrombomodulin, the lymphocyte homing receptor CD44, and the macrophage colony-stimulating factor M-CSF-1 that can exist in both GAG-bearing and nonbearing forms. These molecules have been termed “part-time” proteoglycans (1). The cloning of the genes for proteoglycan core proteins during the past decade has dramatically increased the number of the known proteoglycans species present in the vascular wall. Each individual proteoglycan molecule differs in size and amino acid sequence of the core protein as well as the type, number, length, and fine structure of the GAG(s) attached to the core protein (2–5). Wide structural variation in the GAG side chains further leads to structural diversity within a given species of proteoglycan. The GAGs, previously called acid mucopolysaccharides, are linear, negatively charged polysaccharides of various length that consist of repeating disaccharide units where one of the two sugar residues is an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and the other a hexuronic acid (glucuronic acid or its C5-epimerized homolog, iduronic acid) or galactose (2,3). Of the known GAG chains, two are galactosaminoglycans and four glucosaminoglycans (Fig. 2). All the GAGs except one, hyaluronan (HA), are sulfated. Hyaluronan is also the only GAG that does not exist in a proteoglycan form, but is instead a free chain. Hyaluronan can bind to the N-terminal globular domain of large extracellular proteoglycans called hyalectans/lecticans (see below) to form large aggregates. The interaction between HA and hyalectans/lecticans is further stabilized by link protein (6). Heparin and HS GAGs are structurally very similar but not identical. Both of them are glucosaminoglycans (Fig. 2), but in heparin the disaccharides are much more heavily substituted with sulfate groups. Heparin is essentially a highly sulfated subclass of HS (3,7).

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Figure 1 Schematic structure and overall composition of a typical proteoglycan molecule. Typical size range of proteoglycan core proteins is 15–400 kD. The chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin GAG side chains are shown to be attached to the serine (Ser) residue of the core protein via galactosylgalactosyl-xylosyl (Gal-Gal-Xyl) linkage, whereas keratan sulfate (KS) GAG chains are shown to be attached to the asparagine (Asn) or serine (Ser)/threonine (Thr) residues of the core protein via N- or O-linkages, respectively. The site of the potential phosphate (P) substitution on the Gal-Gal-Xyl linkage is also indicated. Gal, galactose; GalNAc, Nacetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; Man, mannose; SA, sialic acid; Xyl, xylose; NH 2 and COOH designate the amino-terminal and carboxyl-terminal ends of the core protein, respectively. (From Ref. 3.)

Proteoglycans were originally classified into four categories based on the dominant type of the GAG chains attached to the core protein. These four categories are chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) proteoglycans. Proteoglycans have also been categorized into three main groups according to their cellular location, i.e., extracellular proteoglycans, cell surface proteoglycans, and intracellular proteoglycans. The current classification of proteoglycans is a combination of both the cellular location and the core protein structure of individual proteoglycans. Such a classification

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Figure 2 Composition of GAGs belonging to either galactosaminoglycans or glucosaminoglycans. Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcNSO 3 , N-sulfoglucosamine; GlcA, glucuronic acid; IdoA, iduronic acid; 2SO 4 , 3-SO 4 , 4-SO 4 and 6-SO 4 denote the position of ester-O-linked sulfate substituents; chain linkages are also indicated. (From Ref. 3.)

gives rise to distinct families and subfamilies such as the CS-containing hyalectans (8)/lecticans (9), the CS/DS or KS-containing small leucine-rich proteoglycans (SLRPs) (10), and the HS-containing proteoglycans such as syndecans (11) and glypicans (12). Other proteoglycans are found in basement membranes, i.e., agrin, perlecan, bamacan, and the “part-time” proteoglycan, type XVIII collagen, and are usually classified separately (13). Serglycin, whose core protein was the


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first proteoglycan gene to be cloned (14), is the only proteoglycan that belongs to the class of intracellular proteoglycans.

III. Proteoglycans Identified in Blood Vessels Of the different proteoglycans and their splice variants characterized so far, ⬎20 different proteoglycans have variously been identified in blood vessels in vivo or synthesized by vascular wall cells in vitro. These proteoglycans including the “part-time” proteoglycans are listed in Table 1. Versican, a large CS proteoglycan of the hyalectan family (8,15), interacts with HA and link protein-forming large aggregates, and is a predominant proteoglycan in the vascular wall (16). Versican exists in at least four different splice variant forms—V0, V1, V2, and V3 (17)—two of which, V0 and V1, are known to reside in the ECM of blood vessels (4). In addition, cultured vascular smooth muscle cells (SMCs) have been shown to be able to express the V3 isoform of versican (18), but whether this isoform is present in the vascular wall in vivo remains to be confirmed. Cultured vascular endothelial cells (ECs) also synthesize a large aggregating CS proteoglycan that most likely represents versican (19). The location of versican in blood vessels varies depending on the type of the vessel. In coronary arteries and in veins, versican is present in relatively high levels in the subendothelial space (20), while in muscular arteries versican is more abundant in the outermost layer of the vessel wall, the adventitia (17,21). Biglycan and decorin, the two highly homologous members of the SLRP gene family (10,22,23), are perhaps the second most quantitatively significant group of proteoglycans present in the ECM of blood vessels (21,24,25). Biglycan, which is produced by both vascular ECs and SMCs (24,26,27), is normally found throughout the vascular wall (21), while decorin, which is primarily synthesized by vascular SMCs (24,27,28), is usually concentrated more in the outer layers of the vascular wall (21). Recently, it was shown that decorin is also an integral component of newly formed microvessels (29). During vascular diseases such as atherosclerosis, marked changes in the distribution of all the three aforementioned proteoglycans within the arterial wall can be observed (21,25,30,31). Other members of the SLRP gene family identified in blood vessels include keratocan (32), a KS proteoglycan originally isolated from the cornea (33,34), lumican (35,36), another KS proteoglycan also originally found in the cornea (37), and osteoglycin (38), the smallest member of the SLRP gene family which was first isolated and characterized as an osteoinductive factor from bovine bone (39,40). The human form of osteoglycin is termed mimecan (41). Little is known regarding the distribution of the above proteoglycans within vascular wall. Osteoglycin is expressed in significant amounts in both the media and intima (38),

1.

Agrin

Keratocan Lumican Osteoglycin/mimecan Basement membrane proteoglycans Perlecan

Decorin (PG-40, PG-II)

Small leucine-rich proteoglycans (SLRPs) Biglycan (PG-I)

Extracellular proteoglycans Hyalectan/Lectican family Versican (multiple isoforms)

HS (2-3)

HS (3)

⬃400

225

KS KS (2-3) KS

CS/DS (1)

CS/DS (2-1)

CS (0-23)

GAG composition

37 37/48 ⬃34/25

36

38

72-370

M r (kDa) of protein core

Capillary EC

EC, SMC

SMC SMC SMC

ECs of neovessels, SMC

EC, SMC

EC, SMC

Cellular origin in blood vessels

Structural component of BMs Binds growth factors and cytokines Regulates cell adhesion and proliferation Interacts with lipoproteins and influences their catabolism Is involved in hemostasis and thrombosis See perlecan

Influences cell adhesion and migration Binds lipoproteins Binds type I collagen and TFG-β Regulates collagen and fibronectin fibril formation Binds TGFβ and blocks its activity Is involved in the regulation of cell proliferation Binds lipoproteins Is involved in angiogenesis Unknown Regulates collagen fibrillogenesis Structural component of vascular wall

Binds HA and link protein, and forms large aggregates Provides the vasculature viscoelasticity and turgor Influences cell adhesion, migration and proliferation Binds lipoproteins Influences elastin synthesis and fibril formation

Potential function(s) in vascular wall

Proteoglycans Identified in Vascular Wall In Vivo or Shown to Be Synthesized by Vascular Wall Cells In Vitro

Proteoglycan

Table 1

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2.

64

Glypicans Glypican-1 (glypican) HS

CS (0-1)

CS

⬃300

57

CS/HS (0-4) CS/HS (0-4)

CS/DS, HS (1) CS/HS

HS (2)

⬃100 37

⬃100 18

⬃180

Thrombomodulin a

NG2/HMP

Cell surface proteoglycans Betaglycan a CD44 a

Other extracellular proteoglycans PG-100 a Testican

Type XVIII collagen a

EC, SMC

EC, SMC

EC, pericyte, SMC

EC, SMC EC, SMC

EC, SMC EC

Capillary EC, SMC

Interacts with growth factors such as FGFs VEGF and modulates their activity Controls cell growth Is involved in cell adhesion and migration Is involved in endo- and transcytosis Possesses anticoagulant activity

Presents TGF-β to signalling receptors Is the principal receptor for HA Interacts with a variety of ECM molecules Is involved in cell adhesion, migration and proliferation Is involved in inflammation Participates in SMC response to growth factors Is involved in angiogenesis Interacts with other ECM molecules Interacts with cellular ligands such as CD44 and α 4 β 1 integrin Modulates the functional properties of angiostatin and plasminogen Functions as a high-affinity receptor for thrombin on ECs Exhibits mitogenic activity for vascular SMCs

Controls cell proliferation Is involved in cell-cell and cell-matrix interactions Contributes to cell proliferation

Structural component of BMs Its C-terminal fragment (endostatin) inhibits EC proliferation and blocks angiogenesis

Vascular Proteoglycans 297

Intracellular proteoglycans Serglycin ⬃15

23 43 22

33

57 ⬃60

Heparin/HS/CS (8)

HS HS HS/CS (3)

HS (1-4)/CS CS (1-2)

HS HS

GAG composition

EC

SMC SMC EC, SMC

EC, SMC

SMC SMC

Cellular origin in blood vessels

Is involved in platelet-EC interactions Interacts with ECM molecules such as collagen and fibronectin Participates in inflammation

Is involved in cell-cell and cell-ECM interactions Binds growth factors such as FGFs and modulates their activity Regulates lipoprotein metabolism through interactions with LPL and apolipoproteins B and E Plays a role in hemostasis and thrombosis, i.e., inhibits coagulation Is involved in angiogenesis Plays a role in cell signal transduction See syndecan-1 See syndecan-1 See syndecan-1 Plays a role in the formation of focal contacts

Unknown, see glypican-1 Unknown, see glypican-1

Potential function(s) in vascular wall

Abbreviations: BMs, basement membranes; CS, chondroitin sulfate; DS, dermatan sulfate; EC(s), endothelial cell(s); ECM, extracellular matrix; FGF(s), fibroblast growth factor(s); GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; kD, kilodaltons; KS, keratan sulfate; LPL, lipoprotein lipase; M r , relative molecular weight; SMC(s), smooth muscle cell(s); TGFβ, transforming growth factor-β; VEGF, vascular endothelial growth factor. a “Part-time” proteoglycan.

3.

Syndecan-2 (fibroglycan) Syndecan-3 (N-syndecan) Syndecan-4 (amphiglycan, ryudocan)

Glypican-4 (K-glypican) Glypican-6 Syndecans Syndecan-1 (syndecan)

M r (kDa) of protein core

Proteoglycans Identified in Vascular Wall In Vivo or Shown to Be Synthesized by Vascular Wall Cells In Vitro

Proteoglycan

Table 1

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but studies on the distribution of keratocan and lumican in normal blood vessels are lacking. PG-100, which is the proteoglycan form of the macrophage colonystimulating factor M-CSF-1 (42), and testican, originally published as a testisderived proteoglycan (43), are also vascular extracellular proteoglycans, since vascular ECs are capable of synthesizing both of them (44,45), at least in culture. Furthermore, vascular SMCs have been shown to synthesize and secrete biologically active M-CSF-1 (46). Basement membranes, the highly organized molecular networks present in all tissues and organs, including the vasculature, contain several different ECM components, four of which are proteoglycans, including the HS proteoglycans, perlecan, agrin, and type XVIII collagen (13), and the CS proteoglycan bamacan (47). Perlecan, the best-characterized member of the basement membrane– associated proteoglycans (13,48,49), is synthesized by both ECs and SMCs and is present in basement membranes throughout both the intimal and medial layers of blood vessels (16). There is growing evidence suggesting that agrin, originally isolated from extracts of the electric organ of electric ray Torpedo californica as a 150-kDa polypeptide that caused acetylcholine receptors and acetylcholinesterase to assemble in patches (50), is also an important vascular basement membrane–associated proteoglycan (51). This is also true for type XVIII collagen, which is readily detected in all vascular basement membranes (13,52). Studies to identify the presence of bamacan in basement membranes of blood vessels are lacking. Proteoglycans identified on the surface of vascular cells include betaglycan, CD44, NG2/human melanoma proteoglycan (HMP), thrombomodulin, and the two HS proteoglycan families, glypicans and syndecans. Betaglycan, also known as the TGF-β type III receptor, is an integral membrane protein to which HS and/or CS GAG chains are attached (53,54). This proteoglycan has been shown to be synthesized by both vascular ECs (55,56) and SMCs (57), and its distribution is identical to that of the TGFβ type I and type II receptors (55). CD44, originally known as the lymphocyte homing receptor, belongs to a larger group of HA-binding proteins, called hyaladherins (58). It can exist in several different splice variant forms and each of them is highly glycosylated, containing both Nand O-linked carbohydrate side chains as well as CS or HS GAG chains (59,60). CD44 synthesis in ECs and SMCs seems to be closely related to inflammation; i.e., inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin1α (IL-1α), and -1β (IL-1β) are the main regulators of CD44 expression by these cells (61–63). The rat proteoglycan NG2 (64) and its human homolog HMP (65) are membrane-spanning CS proteoglycans. The expression of this proteoglycan is developmentally regulated; i.e., NG2/HMP has not been detected in the quiescent adult vasculature, but is expressed in the vasculature of normally developing tissues (66) as well as in the neovasculature of tumor stroma (67) and the granulation tissue of healing wounds (68). In the neovasculature, NG2/HMP expression

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is mainly limited to the pericytes (69), although in the developing brain, capillary ECs are also capable of expressing NG2/HMP (66,70). Furthermore, SMCs in arteries have been found to express this proteoglycan (66). Thrombomodulin, another integral membrane-bound glycoprotein that can carry a CS chain, is constitutively expressed by ECs and thus is distributed throughout the vasculature (71,72). Thrombomodulin has also been localized on vascular SMCs (73). As mentioned above, there are two other cell membrane-integrated HS proteoglycan families known to date, glypicans (12) and syndecans (11,74). The glypican family consists of six distinct but highly homologous proteoglycans— glypican-1 (glypican) (75), glypican-2 (cerebroglycan) (76), glypican-3 (OCI-5) (77), glypican-4 (K-glypican) (78), glypican-5 (79), and glypican-6 (80). They all are linked to the cell surface via a glycosylphosphatidylinositol anchor. So far, only little is known about the glypicans in the vasculature. However, there are studies demonstrating that vascular ECs and/or SMCs synthesize glypican-1, -4, and -6 (78,80–83). The syndecan family consists of four transmembrane HS proteoglycans— syndecan-1 (syndecan), the prototypic member of the syndecan family that was originally isolated from mouse mammary epithelial cells (84); syndecan-2 (fibroglycan) (85); syndecan-3 (N-syndecan) (86); and syndecan-4 (amphiglycan, ryudocan) (87,88). All of them are variably expressed by vascular ECs and/or SMCs and can be found in all layers of the vascular wall, the intima, the media, and the adventitia (88–93). The intracellular proteoglycan family consists of only one member, serglycin, which is a heparin/HS or CS-containing proteoglycan (94). Serglycin was initially discovered as a secretory and membrane-associated product of rat L2 yolk sac tumor cells (95), and was later found to be expressed primarily by hematopoietic cells (96). Serglycin has therefore been referred to as a hematopoietic cell-specific proteoglycan (94). In the vasculature, most attention has focused on serglycin derived from mast cells (97,98). However, recent studies have demonstrated that ECs (61,99) are also capable of expressing serglycin in high levels. Thus, serglycin can be regarded as a vascular proteoglycan.

IV. Functions of Proteoglycans Found in the Vascular Wall Proteoglycans form a versatile group of ECM molecules, not only structurally, but also functionally. Proteoglycans fill the extracellular space and are critically involved in matrix assembly by regulating collagen (100–102), fibronectin (103), and elastin (104,105) fibrillogenesis. This regulation of tissue assembly by proteoglycans imparts compressibility, viscoelasticity, and mechanical strength to the vascular wall. In addition, proteoglycans are involved in more dynamic pro-


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cesses within the vascular wall: they contribute to hemostasis and thrombosis; influence cell adhesion, migration, and proliferation; participate in lipoprotein metabolism; and regulate inflammation. Moreover, proteoglycans are involved in the formation of new capillary blood vessels through several mechanisms that include novel functions for proteoglycans such as the activation of matrix metalloproteinases (106,107) and the prevention of EC apoptosis (108) (Fig. 3). A. Hemostasis and Thrombosis

The principal vascular proteoglycans that are involved in hemostasis and thrombosis are the heparin/HS-containing proteoglycans—particularly syndecans, perlecan, and serglycin (92,98,109–111) and the CS proteoglycan thrombomodulin (112). Heparin/HS GAGs interact with the serine proteases, e.g., thrombin and Factor Xa, that are responsible for triggering the coagulation cascade. The same GAGs also interact with the serine protease inhibitors (serpins) such as antithrombin III (ATIII). Through these interactions, heparin/HS accelerates the rate at which ATIII induces the inactivation of thrombin and Factor Xa, thereby suppressing coagulation reactions (5,92). Other mechanisms whereby heparin/HS prevents thrombosis include their ability to potentiate the anticoagulant activity of activated protein C, a serine protease that can inactivate coagulation Factors V and Va, and VIII and VIIIa (113,114). Moreover, the heparin proteoglycan serglycin has been shown to clearly interfere with platelet-collagen interactions (98). Thrombomodulin also functions as an anticoagulant through binding to thrombin, which results in the activation of protein C and leads to the enhancement of proteolytic inactivation of Factors Va and VIIIa in a manner similar to that described above for heparin/HS (72). Thrombomodulin–thrombin complex can also activate a plasma protein called thrombin-activatable fibrinolysis inhibitor, which causes an inhibitory action on fibrinolysis (72). Thus, thrombomodulin is involved not only in the control of blood coagulation but in fibrinolysis as well. It is also evident that DS chains play a role in thrombosis and hemostasis, one potential mechanism being the activated protein C enhancing activity (115). Furthermore, DS chains (116) and DS proteoglycans (117) have been shown to increase the rate of thrombin inhibition by heparin cofactor II, a plasma proteinase inhibitor that has been detected within the artery wall. The importance of specific species of proteoglycans, e.g., HS proteoglycans such as perlecan and the CS proteoglycan thrombomodulin, in the prevention of thromboembolic complications is well established (72,111,118), and unfractionated heparin, as well as lowmolecular-weight heparins, is widely used in the prophylaxis and treatment of arterial thromboses. However, it has also to be remembered that the use of heparin may cause, although rarely, immune-mediated thrombocytopenia, called heparininduced thrombocytopenia, that can be associated with an enhanced thrombosis

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Figure 3 Potential functions of proteoglycans in the vascular wall. (1) Bind enzymes [e.g., lipoprotein lipase (LPL)] and influence their enzymatic activities; (2) bind clotting and fibrinolytic factors and modulate their functions; (3) influence platelet-collagen interactions during vascular injury; (4) bind growth factors and cytokines and modulate their activity; (5) influence cell-cell associations; (6) influence cell-matrix interactions; (7) participate in the organization of ECM structures such as BMs; (8) influence EC migration and proliferation; (9) modulate SMC migration and proliferation; (10) participate in inflammatory processes; (11) influence intra- and extracellular lipid deposition and turnover


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(119). Thus, there is still much to learn about the action of proteoglycans and GAGs in thrombotic processes. B. Regulation of Cell Phenotype

Proteoglycans also regulate cell adhesion, migration, and proliferation through their ability to interact with a variety of ECM proteins, growth factors, and cytokines that are central to these cellular events (120). In addition, certain proteoglycans have been shown to be directly involved in the above cellular functions, either as adhesion receptors or matrix ligands for such receptors. For example, the syndecan family of cell surface HS proteoglycans can regulate cell adhesion by binding via their HS chains to the heparin-binding domains of other ECM molecules, such as fibronectin and collagens (74). Syndecans, particularly syndecan-4, can also interact via their core protein with molecules at the cell surface (121). Moreover, the core proteins of the syndecan class are able to participate in intracellular signalling mechanisms that control the adhesive phenotype of cells (122). Glypicans, unlike syndecans, have been shown to be anti-adhesive rather than adhesive molecules (123). The role of perlecan, the basement membrane–associated HS proteoglycan, in vascular cell adhesion processes seems to be dual, i.e., adhesive or antiadhesive, depending on the structural characteristics of the HS chains attached to its protein core and the composition of the ECM surrounding the cell (124,125). Besides HS proteoglycans, CS and DS-containing proteoglycans such as versican and biglycan also participate in cell adhesion by interfering with other ECM molecules (126–128). The effect of versican on cell adhesion is mainly mediated by the GAG chains, since chondroitinase ABC treatment has been shown to abolish the effect, while in the case of biglycan the intact proteoglycan is necessary for the activity. Cell migration is closely associated with and dependent on cell adhesion, requiring both adhesion at the leading edge of the cell and deadhesion at the trailing edge of the cell. Thus, it is not surprising that the same proteoglycans that are involved in the regulation of cell adhesion also participate in the regulation of cell migration (120,129–131). However, additional proteoglycans have also been shown to possess migration regulatory functions for vascular cells (103).

(e.g., LDL); (12) regulate matrix assembly, e.g., collagen, fibronectin, and elastin fibrillogenesis; (13) maintain viscoelastic properties and turgor; (14) are involved in angiogenesis, e.g., by preventing EC apoptosis and activating specific MMPs. ATIII, antithrombin III; BM, basement membrane; EC, endothelial cell; LPL, lipoprotein lipase; PDGF, plateletderived growth factor; SMC, smooth muscle cell; MMPs, matrix metalloproteinases. (From Ref. 19.)


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Two discoveries that can be regarded as cornerstones in establishing explicitly the importance of proteoglycans in cell proliferation are (1) the finding that fibroblast growth factors (FGFs) require HS for high-affinity binding to their receptors (132,133), and (2) the notion that binding of transforming growth factor-β (TGFβ) to decorin abolishes TGFβ action (134). Proteoglycans are known to be able to bind and modulate the activity of the signaling pathway of a variety of additional growth factors and cytokines central in vascular biology including platelet-derived growth factor (PDGF) (66,135–138), vascular endothelial growth factor (VEGF) (139), and interferon-γ (INFγ) (140,141), to name just a few. The role of proteoglycans in the regulation of cell proliferation can also be more direct. For example, the member of the SLRP gene family, decorin, has been shown to induce p21, a protein that complexes with cyclins and reduces cyclin-dependent kinase activity, thus blocking cell division (142). Furthermore, evidence exists suggesting that decorin can activate the epidermal growth factor receptor, at least in tumor cells, which results in the suppression of tumor cell growth (143). It remains to be established whether decorin has a similar effect on vascular cells. C. Lipid Metabolism and Lipid Accumulation

The pioneering work on the role of proteoglycans in lipoprotein metabolism was performed in the 1960s and 1970s by investigators such as Iverius, Hollander, Camejo, and Berenson, who demonstrated that lipoproteins are able to form soluble and insoluble complexes with proteoglycans (144–147). These findings led to the “Response to Retention Hypothesis” that states that lipoproteins entering the subendothelial space are bound and retained through ionic interactions between the positively charged residues on the lipoproteins and negatively charged residues on the GAGs of proteoglycans present in the ECM or on the surface of vascular cells (148). While the above interactions between the GAGs and apolipoproteins are considered to be direct, the interaction between proteoglycans and lipoproteins can also be indirect through bridging molecules such as lipoprotein lipase (149–151). Furthermore, protein-protein interactions between lipoproteins and proteoglycans have been described (152). Regarding other characteristics of proteoglycan-lipoprotein interactions, in vitro binding assays have indicated that versican isolated from cultured arterial SMCs binds low-density lipoprotein (LDL) with saturable kinetics and the binding affinity of 2.3 ⫻ 10⫺8 M (153). Similar findings have been made in vivo (154,155). The calculated stoichiometry of the interaction suggests that at least five LDL particles can be bound to a single CS chain on versican. One of the versicanbinding sites in the apoprotein B of LDL is located at amino acid residue 3363,


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since replacing this charged residue with a neutral amino acid by site-directed mutagenesis eliminates versican binding to LDL in vitro (156). Vascular DS proteoglycans, such as biglycan and decorin, are also able to bind lipoproteins and promote the retention of lipoproteins, especially in the extracellular space (150,151,153). Heparin/HS proteoglycans such as serglycin, syndecans, and perlecan are mainly involved in the uptake of lipoproteins by cells within the vascular wall (97,157,158) leading to intracellular lipid accumulation. Other roles that proteoglycans play in lipoprotein metabolism include their influence on lipoprotein oxidation. In vitro experiments have demonstrated that lipoproteins bound to C-6-S-rich proteoglycans are more readily oxidized than nonbound lipoproteins and taken up more rapidly by macrophages (159). On the other hand, C-4-S GAGs inhibit copper-mediated LDL oxidation, and this GAG may thereby act as an antioxidant (160). The reason for these opposing activities is not known, but likely involves the ability of the CS chains to chelate copper. However, once the lipoproteins are oxidized, their ability to bind to proteoglycans is reduced (153,161). Furthermore, proteoglycans such as decorin and biglycan are able to bind and enhance the activity of phospholipase A2, an enzyme that can lipolyze LDL particles (153,162,163), resulting in the induction of particle fusion and the enhancement of the retention of LDL molecules to arterial proteoglycans (164). Macrophage plasma membrane CS proteoglycan is also believed to play an important role in the cellular uptake of lipoproteins and the formation of cholesterol-loaded foam cells (165). In conclusion, proteoglycans are able to regulate lipoprotein metabolism through several mechanisms, each of which can promote either extra- or intracellular accumulation of lipoproteins within the vascular wall. D. Proteoglycans as Mediators of Inflammation

Besides the functions described above, proteoglycans found in the vascular walls are involved in the regulation of tissue processes such as inflammation and angiogenesis/neovascularization. Regarding inflammation, HS and/or CS proteoglycans on ECs, particularly syndecan-1, have been shown to be potential ligands for E-selectin (166), endothelial-leucocyte adhesion molecule-1, which participates in leucocyte rolling and adhesion to the endothelium during inflammation (167). The GAGs of cell surface and ECM proteoglycans have also been shown to be able to differentially bind various chemokines, a major class of leucocyteactivating factors. This raises the possibility that the GAGs mediate the presentation of chemokines to leukocytes by vascular ECs and thus determine the specificity of leukocyte recruitment (168). Interactions of chemokines and GAGs of cell surface proteoglycans can also modulate vascular wall cell behavior (141). The importance of proteoglycans in inflammatory processes within the vascular


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wall is further emphasized by a number of studies demonstrating that inflammatory cytokines such as IL-1β and TNFα markedly modulate proteoglycan expression by vascular wall cells (44,61,62,169). E.

Angiogenesis and Neovascularization

It has become evident that not only growth factors and cytokines, but also molecules of the ECM including proteoglycans, are of great importance in the angiogenic process (170). For example, the induction of decorin expression by ECs has been shown to be associated with angiogenesis in vitro (171). More recently, decorin has been shown to be required during angiogenesis in vitro (108). Decorin is known to be involved also in angiogenesis in vivo, especially inflammationassociated angiogenesis (29). However, the functional role that decorin plays in angiogenesis/neovascularization is not known at the moment, but several potential mechanisms can be proposed. It is possible that by interacting with other angiogenesis-associated ECM molecules such as type I collagen (100,101,172) and fibronectin (173,174), and by influencing the organization of these molecules (102,103) decorin stabilizes the ECM in a way that provides a template for ECs to form capillary tubes (175,176). On the other hand, a recent study demonstrated that decorin alone or in combination with thrombospondin-1 actually inhibits endothelial tubelike structure formation (177). Thus, the role of decorin in angiogenesis seems to be highly dependent on the composition of the ECM surrounding the ECs undergoing angiogenesis. Decorin may also be involved in angiogenesis by interacting with and regulating the activity of angiogenic growth factors (134,178). Furthermore, the apoptosis-preventing effect of decorin on ECs (108) and the effect of decorin on matrix metalloproteinase activity in ECs (107) have been suggested to be potential mechanisms whereby decorin could play a role in angiogenesis. Other proteoglycans that have variously been connected to angiogenesis, include syndecan-1 (179), syndecan-4 (180), glypican-1 (139), perlecan (181), the “part-time” proteoglycan CD44 (182), and NG2/HMP (68). However, similar to decorin, the mechanisms of these proteoglycans in angiogenesis are not completely understood, although several specific functional roles can be attributed to each of the aforementioned proteoglycans in angiogenesis. Furthermore, breakdown products of HA stimulate angiogenesis (183–185). In contrast, the C-terminal fragment of the “part-time” proteoglycan type XVIII collagen, called endostatin, inhibits EC proliferation and blocks angiogenesis (186). Although the list of functions described above for vascular proteoglycans is not complete, it clearly indicates the versatility that proteoglycans play within the vasculature. Therefore, changes in the structure and composition of vascular proteoglycans can lead to severe hemostatic and hemodynamic disturbances lead-


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ing to a number of different vascular diseases, some of which are briefly discussed below.

V.

Proteoglycans in Vascular Diseases

Atherosclerosis, a progressive disease affecting large and medium-sized arteries, is the primary cause of heart disease and stroke. In the Western world, it is the leading cause of death (187). The pathogenetic mechanisms of atherosclerosis are still not completely understood. However, events such as repeated injuries to the endothelium, followed by platelet adhesion and aggregation, release of growth factors and cytokines from platelets and other cells gathered to the site of injury, i.e., inflammatory cells, and induction of SMC migration from the media to the intima and proliferation therein are thought, together with changes in the lipoprotein metabolism leading to intra- and extracellular lipid accumulation, to be central steps during the diease process (148,188,189). Proteoglycans, as described above, can have a great impact on all the mentioned events, and thus the role of proteoglycans in atherogenesis is likely to be crucial. Indeed, enhanced accumulation and changes in the composition of proteoglycans in the areas of developing vascular lesions are clearly the early hallmarks of atherosclerosis (16,21,31,190). This is true not only for atherosclerosis, but also for restenosis, the renarrowing of blood vessels after vascular lesions have been treated by reconstructive techniques such as endarterectomy, bypass grafting, percutaneous transluminal coronary angioplasty (PTCA), or intravascular stenting (191–193). The role of proteoglycans in the development of atherosclerosis and restenosis is not solely harmful, as might be expected based on their pathogenicity in the lipid retention within the vascular wall. For example, it has recently been demonstrated that gene transfer of the ECM proteoglycan decorin into an injured arterial wall significantly reduces intimal ECM volume by altering the composition of the ECM (194). Abdominal aortic aneurysm (AAA) is a frequent complication of severe atherosclerosis. Therefore, it is not surprising that alterations of the ECM, including proteoglycans, occur in AAA, thereby contributing to the altered viscoelastic and compressive properties and the deformity and dilatation of the aorta (195). In addition, a familial form of thoracic aortic aneurysms and dissections has recently been mapped to a region on chromosome 5q13-14 (196), which includes the versican encoding gene (197). It remains to be determined whether versican is specifically affected in this familial form of thoracic aortic aneurysms and dissections. Proteoglycans not only play an important role in macrovascular diseases, but are also likely involved in microvascular pathology occurring in diseases


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15 Degradation of Lung Matrix Proteoglycans in Bronchiectasis

DAISY KWOK-YAN SHUM and MARY SAU-MAN IP The University of Hong Kong Hong Kong S.A.R., China

I.

Introduction

Bronchiectasis refers to the pathologic lung condition in which the walls of medium-size bronchi are damaged and subsequently dilated. In the white population, the major cause of bronchiectasis is cystic fibrosis, whereas in other ethnic groups bronchiectasis is often of postinfectious or unknown etiology. Pathogenetic mechanisms have been postulated to be a combination of congenital and acquired processes. In the majority of cases, the pathogenetic components include chronic bronchial infection and persistent airway inflammation attributable to a vicious cycle of persistent host-defense mechanisms (1,2). II. Central Role of the Neutrophil in Bronchiectasis In patients with bronchiectasis, pulmonary inflammation is dominated by neutrophils (3). Although neutrophils are primarily recruited to the airways as part of the host defense against infecting bacteria, the airway epithelium and neutrophils can become sources of proinflammatory cytokines that sustain the chemotactic recruitment of neutrophils (4,5). Recent investigations show that neutrophils in 323

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the processes of chemotaxis and diapedesis do not cause indiscriminant proteolysis of the matrix architecture (6,7). Rather, secretion of granule proteases has been shown to be polarized, and these products are retained on the surface of not only neutrophils, but also endothelial cells along the migration path of the neutrophils (8). This defies the abundant elastolytic activity detectable in the airways and the gradual yet persistent damage to bronchial tissues of patients with bronchiectasis. We reasoned that neutrophils recruited in bronchiectasis were locally activated to damage bronchial tissues in their vicinity. Indeed, the expectorated bronchial secretions of these patients were found to contain not only neutrophil elastase activity, but also proinflammatory mediators that can promote the pericellular proteolytic action of neutrophils (9,10). Given that the inflamed bronchial environment is replete with antiproteases, it is not entirely clear how neutrophilderived protease activity can remain dominant (11,12). This remains a key issue in the understanding of how dynamic homeostatic mechanisms to maintain tissue integrity become inadequate, leading to decrements in pulmonary function and eventual respiratory failure in the protracted course of the disease.

III. Neutrophil Elastase and Antielastases Among the neutrophil-derived proteases, human neutrophil elastase is potent in the stimulation of airway secretion, acceleration of airway inflammation, and destruction of the airway mucosal tissue in both acute and chronic pulmonary diseases (13). The mature serine protease is produced in granulocyte precursor cells, stored in neutrophil azurophilic granules, and released from neutrophils upon surface activation, phagocytosis, or cell death (14,15). The extracellular proteolytic activity of human neutrophil elastase in inflamed pulmonary tissue is counterbalanced essentially by secretory leukoprotease inhibitor (SLPI), a nonglycosylated polypeptide produced locally by the bronchial epithelium and submucosal glands (16–18). When the local production of SLPI is overwhelmed, as with chronic airway inflammation, the role of the plasma-derived antiprotease, α 1-antitrypsin (α 1-AT), becomes important (19). α 1-AT is a plasma glycoprotein synthesized mainly by hepatocytes (20). Local production by lung-derived epithelial cells (21) and neutrophils (22) has also been reported, but the antiprotease effect of these latter is readily overwhelmed in the event of a major neutrophil degranulation. Kinetic studies with purified products show rapid binding of both α 1-AT and SLPI to neutrophil elastase and consequent inhibition of elastase activity. The rates of inhibition could, however, be differentially modulated by polyanions introduced into the reaction mixture such that the antielastase effect of SLPI was accelerated but that of α 1-AT was depressed (23–25). This observation found in vivo relevance in that heparin administered as a polyanionic drug in

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combination with SLPI was effective in protecting the airway against elastasebased diseases (25,26). Neutrophil elastase activity has been detected in the airway secretions of subjects with various inflammatory airway diseases, including asthma, emphysema, bronchiectasis, and cystic fibrosis (13,27,28). Excessive elastolytic activity due to an imbalance of elastase and antielastases has been incriminated in the degradation of elastin in the lung tissue matrix, thus leading to lung function impairment (13,29). Most work on lung tissue degradation has focused on the fibrillar elastin and collagen components of the lung extracellular matrix (30). It is interesting that elastin can compete with the antielastases in binding neutrophil elastase, which then cannot be inactivated by α 1-AT, but that the elastin-bound enzyme can be inhibited by SLPI (31). Similarly, neutrophils adherent on a fibronectin substratum exhibit pericellular elastase activity that cannot be inhibited by α 1-AT (32). While it can be argued that α 1-AT is restricted from access to neutrophil elastase deposited in the tissue path (5) or retained in the pericellular environment of activated neutrophils during extravasation (33), the mechanism by which neutrophil elastase activity is preserved in lung fluids that are replete with α 1-AT remains intriguing and worthy of further study.

IV. Degradation of Lung Matrix Proteoglycans A. Proteoglycans as a First Line of Defense

The lung tissue matrix is a composite of collagen fibers and elastin fibers with variously associated proteoglycans, some specifically bound through their core proteins to the protein fibrils and others filling the spaces between the fibers (34). The small, leucine-rich repeat proteoglycans that bridge the fibrillar meshwork of collagens and elastins with their interacting glycosaminoglycan chains offer one level of defense of the matrix components against invading proteases (35– 37). On another level, defense against cell damage caused by enzymatically produced oxygen radicals can be provided by hyaluronans which associate with large aggregating proteoglycans in the pericellular environment and the spaces between the fibrous proteins (38). This “protective” barrier to tissue destruction, together with dynamic repair processes, may well explain the relatively slow decline of lung function despite the high levels of free elastolytic or collagenolytic activity detected in airway secretions. Understanding the degradation of proteoglycans is therefore an important step in the understanding of extracellular matrix homeostasis in chronic inflammatory airway disease. Changes of lung matrix proteoglycans in situations of chronic lung inflammation provide clues to the balance between proteoglycan degradation and resynthesis in long-term tissue remodeling. The large aggregating proteoglycan versican was found to be abundant in the interstitial matrix of the lung paren-

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chyma of both granulomatous and nongranulomatous fibrotic lung conditions (39). In atopic asthmatics, versican and the small leucine-rich proteoglycans lumican and biglycan were found to be enriched in the subepithelial matrix of the airway wall (40). In rats with bleomycin-induced lung injury, while increases in extractable hyaluronan, versican, and the small leucine-rich proteoglycans fibromodulin and biglycan were found to be in phase with the inflammatory response, intense immunostaining of the small proteoglycans in areas of fibrosis remained after the inflammatory response had resolved (41–43). Contrasting these observations in the fibrotic lung, lungs of patients with severe emphysema show diminished immunostaining for the small proteoglycans decorin and biglycan in the peribronchiolar area, whereas the staining for the matrix proteins— collagens, laminin, and fibronectin—remains unaffected (44). Taken together, the results indicate that the inflammatory and fibrotic phases of lung tissue repair involve net increase of interstitial proteoglycans, but when high protease load overwhelms the availability of the physiological antiproteases, as in severe emphysema, catabolism and loss of these proteoglycans appear, preceding overt degradation of the matrix proteins. Evidence is thus provided for a role of the proteoglycans as a first line of defense against proteolytic attack on the lung matrix. B. Cytokine Activation of Neutrophil Degranulation

In bronchiectasis, we provided evidence that tumor necrosis factor-α (TNFα) is a key mediator in the bronchial cytokine network that enhances neutrophil degranulation and indiscriminant release of proteases which act on lung tissue proteoglycans (9). Priming of neutrophils with recombinant TNFα has indeed been shown to enhance both neutrophil-activating peptide (NAP-2)- and recombinant IL-8-induced neutrophil degranulation (45). With recombinant human TNFα, we also demonstrated that accompanying sputum sol or mediators therein are required to stimulate neutrophil-mediated proteoglycan degradation (9). The expectorated bronchial secretions of patients with bronchiectasis and cystic fibrosis have indeed been found invariably to contain TNFα, interleukin (IL)-1β and IL-8 (4,46,47), and these have been implicated in the influx of neutrophils into and the sustenance of an intense local inflammatory response in the affected bronchial tree. Although bacteria infecting the airways initially generate the factors that trigger the host defense system, the airway epithelium and recruited neutrophils may become activated to be sources of the proinflammatory cytokines. TNFα, IL-6, IL-8, and intercellular adhesion molecule-1 have been shown to be constitutively synthesized and released by human bronchial epithelial cells in culture (48). Expression and release of these mediators were upregulated when the cells


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were exposed to purified bacterial endotoxins (49). Such cells can also synthesize and modulate release of IL-1β in response to antigen challenge (50). Neutrophils recruited to bronchial sites may also be involved in cytokine-mediated autocrine loops that promote recruitment of additional neutrophils (5). The combination of chronic airway infection and inflammation thus leads to the persistent recruitment and accumulation of neutrophils in the inflamed airways. Proteolytic damage to the bronchial tissues in the vicinity follows when the bronchial cytokine network activates neutrophil degranulation and release of proteases. C. Proteolytic Attack on the Core Protein

Studies of proteoglycan degradation in inflammatory processes related to rheumatoid arthritis revealed that serine proteinases, collagenase (matrix metalloproteinase-8; MMP-8), and gelatinase-B (MMP-9) derived from neutrophils act on a variety of sites in the interglobular domain (IGD) between the G 1 and G 2 regions of the core protein of aggrecan and thus release glycosaminoglycan-containing fragments into the tissue fluids (51). Mice genetically deficient in either stromelysin (MMP-3) or MMP-9 have been used to affirm the roles of these MMPs in an acute lung injury model (52) which involves macrophage-dependent neutrophil recruitment (53). Little, however, is known regarding contributions of MMPs-3 and -9 to versican turnover in bronchioalveolar tissue. More recently, the ADAMTS (a disintegrinlike and metalloproteinase thrombospondin type 1 motif ) family is realized to contribute significantly to postinjury loss of aggrecan into tissue fluids (54). By alignment of consensus cleavage sequences, susceptible sites were identified in the expanded glycosaminoglycan attachment region of versican and relevant proteolytic products were recovered from extracts of human aorta (55). The relevance of ADAMTS to bronchial tissue turnover remains to be investigated. In contrast, turnover of the small leucine-rich proteoglycans appears to depend more on intralysosomal than extracellular digestive mechanisms (37,56,57). The sputum-stimulated neutrophil-mediated proteoglycan degradation observed in our study of patients with bronchiectasis is, in the majority of cases, attributable to neutrophil elastase; metalloprotease activity, when significant, was found to contribute to ⬃20% of the proteoglycan-degrading activity (9). Neutrophil elastase has also been shown to be the major source of neutrophil-degrading activity on proteoglycans secreted by rat aortic smooth muscle cell or lung fibroblasts (58). The glycosaminoglycan chains of the proteoglycans are largely preserved in both these incubation systems in which pH is maintained physiological. Although neutrophil granules were found to contain exoglycosidase and sulfatase that act on the nonreducing terminals of chondroitin sulfate chains of the proteoglycans, the low pH optimum of these activities (59) suggests intracellular rather

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than extracellular degradation in subcellular vesicles. The glycosaminoglycans reported in the sputum sol may therefore in part be products of proteolytic actions of neutrophils on matrix proteoglycans of the airways bathed in the stimulatory cytokine environment. Among the glycosaminoglycans, chondroitin sulfates were observed in sputum samples from patients with chronic bronchitis and cystic fibrosis, (60) and hyaluronan from patients with asthma (61). It remains of interest to determine if sputum glycosaminoglycans can be monitored as an indicator of net proteolytic activity due to persistently activated neutrophils in the bronchial environment of patients with bronchiectasis.

Figure 1 Neutrophil-mediated degradation of lung proteoglycans in bronchiectasis. Bacteria infecting the airways or unknown factors acting via activation of the bronchial epithelium trigger the release of chemotaxins and cytokines. These in turn mediate recruitment of neutrophils to the infected bronchial tree. Under the local cytokine environment, neutrophils are activated to release pro-inflammatory cytokines as well as elastase. When the indiscriminant release of neutrophil elastase overwhelms the prevailing antielastases, tissue repair processes will give way to proteolytic damage to the bronchial epithelium and the matrix proteoglycans.

Degradation of Lung Matrix Proteoglycans V.

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Summary

In bronchiectasis, a condition with persistent airway inflammation, there is evidence that cytokines present in bronchial secretions interact to activate release of neutrophil elastase which degrades lung matrix proteoglycans, and may thus contribute to tissue damage (Fig. 1). Therapeutic strategy has traditionally targeted treatment of bacterial infection and, more recently, airway inflammation. It is conceivable that modulation of elastase activity may also be a target for investigation, in the context of achieving optimal proteolytic activity for bacterial defense and tissue repair but avoiding excessive tissue destruction.

Acknowledgments This work was supported by grants from the CRCG of the University of Hong Kong (10203342) and the Hong Kong Research Grants Council (HKU 7242/ 99M) to D.K.Y. Shum and M.S.M. Ip.

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16 Effect of Hypoxia on Glycosaminoglycan Synthesis by Lung Cells

ELENI PAPAKONSTANTINOU and GEORGE KARAKIULAKIS

MICHAEL ROTH

Aristotle University Thessaloniki, Greece

University Hospital Basel Basel, Switzerland and Royal Prince Alfred Hospital Camperdown, New South Wales, Australia

I.

Introduction

A. Glycosaminoglycan Chemistry

Glycosaminoglycans are linear acidic polysaccharides of variable length and composition that are abundant in the extracellular matrix and the pericellular space. They consist of repeating disaccharide units with one monosaccharide usually being either glucuronic or iduronic acid, and the second either N-acetyl-Dglucosamine or N-acetyl-D-galactosamine. On the basis of their composition, glycosaminoglycans are grouped into four major categories: hyaluronic acid; heparin and heparan sulfate; chrondroitin and dermatan sulfates; and keratan sulfate. With the exception of hyaluronic acid, all glycosaminoglycans can be sulfated, and in addition they can carry neutral sugars, such as D-fucose, D-galactose, Dglucose, D-mannose, or D-xylose (1–6). Glycosaminoglycans, except hyaluronic acid, are attached to a protein core to form proteoglycans (7–10). Some proteoglycans can be linked together with hyaluronic acid molecules forming large networks in the extracellular matrix. For example, it has been demonstrated that keratan sulfate or chondroitin sulfate proteoglycans assemble in the extracellular space into large noncovalently bound 335

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complexes, where the protein core is bound to one large chain of hyaluronic acid (11–13). Glycosaminoglycans deposited in the extracellular matrix undergo daily considerable turnover. The synthesis of glycosaminoglycans is controlled by specific enzymes which are mainly located in cell membranes and which act mainly as glycosyltransferases. Syntheses for sulfated glycosaminoglycan are localized in the Golgi apparatus, while hyaluronic acid is synthesized by the respective enzymes located in the plasma membrane (14–16). Like the synthesis, the degradation of glycosaminoglycans is controlled by a large number of enzymes, and as a result the daily turnover of glycosaminoglycans is assumed to range between 10% and 20% (10,14,15,17–22). A large variability in the structure and length of the glycosaminoglycan chains has been described for different cell types and species. The control mechanism determining the length and structure of glycosaminoglycan chains is not well understood. It has been suggested that this in part depends on the nature of the proteoglycan to which the polysaccharide chains are linked. This finding led to the hypothesis that the regulation of sulphated glycosaminoglycan synthesis is not regulated solely in the Golgi apparatus (20–23). Glycosaminoglycans play a role in organizing tissue structure by regulating collagen fibrillogenesis and tensile strength, and controlling the action of growth factors. Animal studies suggest that the relative proportion of collagens to proteoglycans determines the mechanical properties of the extracellular matrix (24– 28). Extracellular matrix designed to resist high tensive forces is rich in collagen and low in total proteoglycan content. The proteoglycan in such extracellular matrix consists mainly of dermatan sulfate–linked proteins. In contrast, extracellular matrix subjected to compressive forces has a greater content of chondroitin sulfate–linked proteoglycans (25). Furthermore, glycosaminoglycans have the ability to trap large amount of water molecules forming hydrated gels, thereby regulating the consistence of tissues (8,26,29–31). Glycosaminoglycans also provide structural links between fibrous and cellular elements, contribute to viscoelastic properties, regulate permeability and retention of plasma components within the matrix (8,26,29–33), inhibit vascular cell growth (34), affect hemostasis and platelet aggregation (22,35,36), and interact with lipoproteins (37) and various growth factors (9,27,38–40). B. Glycosaminoglycan Types 1.

Hyaluronic Acid

Hyaluronic acid or hyaluronan is a nonsulfated glycosaminoglycan which consists of repetitive alternating N-acetyl-D-glucosamine and glucuronic acid molecules. The molecular mass of this polysaccharide varies from 2 ⫻ 10 5 to 10 ⫻ 10 7 (14,41). It is the length of hyaluronic acid molecules that determines its biological

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function. Therefore, it is crucial to understand the mechanisms controlling the synthesis of hyaluronic acid, which is regulated by three independent hyaluronan synthases (42). Based on animal models it is postulated that hyaluronan synthase1 and -2 are required for the synthesis of high-molecular-weight hyaluronic acid, while hyaluronan synthase-3 produces shorter forms (42,43). Hyaluronic acid is the only glycosaminoglycan not covalently bound to any protein core and is secreted into the extracellular space where it is involved in the regulation of the water content of the extracellular matrix. Therefore, hyaluronic acid is abundantly found in all body fluids. In humans the daily turnover of hyaluronic acid is estimated to be about one-third (⬃5 g) of the total hyaluronic acid mass. Based on its physical properties one disaccharide unit of hyaluronic acid can bind up to 10 molecules of water (14,41). Hyaluronic acid has multiple roles: in addition to providing tissue hydration and facilitation of gliding and sliding of tissue, it also forms an integral component of large proteoglycan aggregates in pressure-resisting tissues (25). 2. Heparin

Heparin is a small, highly sulfated glycosaminoglycan with specific anticoagulant properties. Its molecular weight ranges from 6000 to 25,000. It consists of either D-glucuronic acid or l-iduronic acid molecules alternating with N-acetyl-Dglucosamine. Each disaccharide unit can bind two to three sulfate groups. The polysaccharide-protein linkage of glycosaminoglycan chains has the structure: D-GlcA-D-Gal-D-Gal-D-Xyl-L-Ser/L-Thr. Heparin is mainly produced by a subset of mast cells where it is located in granules bound to histamine. Large amounts of heparin are found in the lung, liver, and skin (3,29,44). Heparin has been shown to inhibit inositol-triphosphate-controlled calcium channels and therefore, to affect intracellular calcium concentration, and, it is assumed, to bind and activate ryanodine receptors (45,46). Heparin is frequently used in the prevention or therapy of blood clotting diseases (45,47,48). 3. Heparan Sulfate

Heparan sulfate is a glycosaminoglycan with an average molecular mass of 15,000, and is formed by alternating molecules of either D-glucoronic acid or l-iduronic acid and N-acetyl-D-glucosamine. One disaccharide unit binds up to two sulfate groups. The carbohydrate-protein linkage of heparan sulfate glycosaminoglycan chains has the same linkage tetrasaccharide as heparin chains. High concentrations of heparan sulfate have been described in the basement membrane of the lung and arteries, and on cell surfaces in general. The highest concentration of heparan sulfate is found on the cell surface of endothelial cells (29,44). The expression of heparan sulfate is not only controlled by de novo synthesis but also by specific proteases, and heparanases (47,49).

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Heparan sulfate has been reported to interact with L-selectin, at least in the kidney (50–52). It is associated with heparin-binding molecules affecting their biological action (47). Heparan sulfate binds bFGF, thereby protecting bFGF against physical denaturation and protease degradation (8,50). The high concentrations of heparan sulfate in basement membranes have implicated these matrices as storage sites for bFGF in vivo (47). Heparan sulfate also achieves its action via syndecans and glypicans, which are cell surface proteoglycans. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, heparan sulfate mediates the interactions with a variety of extracellular ligands, such as growth factors and adhesion molecules (53). In regard to tumorigenesis, heparan sulfate proteoglycans can inhibit or promote cell invasion. Their function, in this case, is determined by their location on either the cell surface or in the extracellular matrix, and by the structural characteristic of the proteoglycan to which they are bound (47). 4.

Chondroitin Sulfates A and C

Chondroitin sulfates consist of alternating D-glucuronic acid and N-acetyl-Dgalactosamine. These two sugars can form polysaccharide chains of a molecular weight ranging between 5000 and 50,000. One disaccharide unit can bind up to two sulfate groups. The polysaccharide-protein linkage of these glycosaminoglycan chains is the same as that of heparin glycosaminoglycan chains. Chondroitin sulfates have been isolated from cartilage, cornea, bone, skin, and arteries. Chondroitin sulfates seem to play an essential role in the regulation of prenatal and early postnatal stages, while they diminish progressively with advancing development of mammalian organisms (54–57). In this context it is interesting that chondroitin sulfates seem to play a central role in neuron development, which is critical during embryogenesis (58–60). 5.

Dermatan Sulfate/Chondroitin Sulfate B

Dermatan sulfate, also called chondroitin sulfate B, is a polysaccharide chain of molecular weight ranging between 15,000 and 40,000. It consists of repeating disaccharide units of D-glucuronic acid or l-iduronic acid alternating with Nacetyl-D-galactosamine. One disaccharide can bind one or two sulfate groups. The linkage region of these glycosaminoglycans chains to protein contains the same carbohydrate residues as heparin glycosaminoglycan chains. Dermatan sulfate has been identified in skin, vessels, and the heart (57). Like the other chondroitin sulfates, high expression of dermatan sulfate is associated with neuron development, while it diminishes in neurodegenerative diseases (57). Therefore it is not surprising that high amounts of dermatan sulfate are expressed during the embryonal phase, while it disappears with aging of the organism (58–60). Its role in wound repair, tissue remodeling, and tumorigenesis


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is not clear (61). A growth-stimulatory role of dermatan sulfate has been reported in mesothelioma cell cultures (62). 6. Keratan Sulfate

Keratan sulfate is formed by alternating molecules of D-galactose and N-acetylD-glucosamine, so it is the only glycosaminoglycan that does not contain any uronic acid. Its molecular weight ranges between 4000 and 19,000. The sulfatebinding capacity of keratan sulfate varies between one to two sulfate groups per disaccharide (63). Keratan sulfate is characteristic for tissues that lack vessels and therefore get their oxygen supplies by diffusion. These tissues include cartilages, intervertebral disks, and corneal stromas (63,64). It is hypothesized that keratan sulfate is a functional substitute for chondroitin sulfates when the oxygen supply is low. Keratan and chondroitin/dermatan sulfates have similar functions in corneal stroma and probably in the other connective tissues. Both glycosaminoglycans swell the collagenous matrix, keeping the fibrils apart, and orient the fibrils visa`-vis each other by specific interactions of their proteoglycan protein cores with the fibrils (64). II. Role of Glycosaminoglycans in Lung Cell Biology In recent years, the understanding of connective tissue structure and function of the extracellular matrix as a cell type-specific environment, consisting of various components including glycosaminoglycans, has improved. Glycosaminoglycans are major components of basement membranes. Beside heparan sulfate proteoglycans, such as perlecan, and chondroitin sulfate proteoglycans, basement membranes consist mainly of collagen type IV, laminin, and entactin, forming a complex interactive network (21,28,54,65–67). These membranes represent a specialized extracellular matrix that fulfills multiple functions: acting as an interface and physical barrier for inflammatory cells; separating different cell populations; and serving as an anchor and binding site for various cell types. However, it should be noted that the glycosaminoglycans and proteoglycans in the basement membrane are subjected to a continuous turnover during development; for example, chondroitin sulfates are prominently expressed in prenatal and early postnatal stages, and diminish progressively with advancing development of the lung (54). In an animal model, it has been shown that collagen type IV, laminin, and entactin are detectable from day 14 of gestation on, and become progressively prominent with time, while the proteoglycan perlecan is abundant in adult lung (24,54). Therefore, it can be assumed that glycosaminoglycans and proteoglycans are essentially involved in the regulation of embryogenesis and organ development (53,68,69).

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For a long time it has been postulated that glycosaminoglycans together with proteoglycans function only as regulators of the viscosity of the extracellular matrix of tissues (5,7–10,15,18,26,30,31) including the lung (67,70). In recent years, this view has been revised as most glycosaminoglycans and proteoglycans can bind growth factors and modulate their activity (9,23,27,38,39). It has been shown that glycosaminoglycans protect growth factors from degradation, and it is the interaction between negatively charged glycosaminoglycan chains and growth factors that determines differentiation of cells during development and maintenance of tissue organization (23). Glycosaminoglycans also participate in signaling regulating cell adhesion, and partake in many developmental and pathological processes, including tumorigenesis and wound repair (2,10,27,53). In regard to tumor development glycosaminoglycans or their receptors are associated with tumor progression, enhanced invasion, and angiogenesis (71–73). Increasing evidence suggests that proteoglycans determine the localization of glycosaminoglycans, whereas the glycosaminoglycans directly regulate cell function (53). However, the role of glycosaminoglycans in the pathophysiology of the lung has been the subject of recent investigations.

III. Effect of Hypoxia on Glycosaminoglycans Hypoxia is associated with interstitial lung diseases including fibrosis (74–76), pulmonary hypertension (77,78), and aggressive tumor progression (47). However, the possible impact of hypoxia restricted to small parts of the tissue such as in early stages of wound repair or growing tumors has yet to be investigated (79). Characteristics of lung fibrosis include: proliferation of fibroblasts which synthesize and deposit extracellular matrix molecules in the lung interstitium including GAGs (80–82); overexpression of TGFβ isoforms (83–85); and development of hypoxia (86–88). In regard to fibrosis, hypoxia in certain parts of the lung and the resulting alterations in the local composition of the extracellular matrix including glycosaminoglycans has only been reported in a few studies. Figure 1 depicts a summary scheme of events occurring during lung fibrosis. In the human lung, fibroblasts represent a crucial cell type accounting for 40% of all lung cells, and are responsible for the synthesis of most components of the extracellular matrix (18,19). In culture, primary human lung fibroblasts synthesize hyaluronic acid, heparan sulfate, dermatan sulfate, and chondroitin sulfates; these glycosaminoglycans were found associated with the cell layer (cells together with extracellular matrix) and were also secreted into the culture medium (89). Hypoxia significantly upregulated the synthesis of total glycosaminoglycans, but it did not alter either their structure or their length. However, hypoxia significantly enhanced the secretion of heparan sulfate into the culture medium (89,90).


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Figure 1 Flow chart of events and known feedback mechanisms involved in the development of lung fibrosis. The sequence of the events may vary according to the initiation of proliferative response or hypoxia.

More interestingly, hypoxia distinctly modulated the growth factor–induced synthesis of specific glycosaminoglycans. Hypoxia enhanced the TGFβ-induced total glycosaminoglycan synthesis. Interestingly, hypoxia enhanced: 1. The TGFβ-induced secretion of hyaluronic acid and dermatan and chondroitin sulfates; this effect was most prominent for TGFβ 3 . 2. The TGFβ 2-induced synthesis of dermatan and heparan sulfates associated with the cell layer (90). The increased secretion and/or deposition of dermatan and chondroitin sulfate subpopulations observed following hypoxia/TGFβ treatment may reflect the influence of these glycosaminoglycans to different ends in fibrosis. In the ECM, dermatan sulfate bound to the small proteoglycan decorin is associated with collagen fibers (91), and it may provide additional strength by assisting in the orientation of these fibers. Chondroitin sulfate chains are more diverse in function. They constitute parts of both small and large proteoglycans, such as biglycan and versican, respectively, that may be important in epithelial cell proliferation (92) and

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in hyaluronic acid–mediated fibroblast aggregation and cell movement (93). It has also been reported that chondroitin sulfate increases cell proliferation (94) and may thus be associated to the pathophysiological manifestation of increased cell proliferation in lung fibrosis. The effects of chondroitin sulfate on cell behavior may be depended on the core protein to which it is linked. A summary of the correlation between the length and the opposing effects of the three abovedescribed glycosaminoglycans on cell proliferation is provided in Figure 2. It has also been shown that hypoxia increased the PDGF-induced heparan sulfate associated with the cell layer and the PDGF-dependent secretion of hyaluronic acid into the cell culture medium of primary human lung fibroblasts (89). Some of the characteristics of fibrotic lung injury include the proliferation (80) and activation (77,80–82) of fibroblasts and deposition of glycosaminoglycans in the lung interstitium. Thus hypoxia-induced expression of glycosaminoglycans, mainly of heparan sulfate, may contribute to the development of secondary pulmonary hypertension (80). In the context of pulmonary hypertension it has been documented that a physical factor such as the pulmonary interstitial pressure is capable of modulating the extracellular matrix structure of the lung. Using a rabbit model, a significant effect of hypoxia on the composition of pulmonary interstitial proteoglycans was reported (95). Compared to normoxic tissue, hypoxia caused a weakening

Figure 2 Some of the glycosaminoglycans stimulated by hypoxia may be regarded as an attempt of the organ/organism to counteract the pro-proliferative action of growth factors or cytokines released. At least for three glycosaminoglycans, in vitro data suggest an antiproliferative action that correlates with their length.


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of the noncovalent bonds linking proteoglycans to other extracellular matrix components. Gel filtration chromatography showed an increased fragmentation of chondroitin sulfate and heparan sulfate proteoglycans during hypoxia, resulting in a loss of the architecture of the basement membrane (95). In another study, employing a rat model, treatment of lung parenchymal strips with specific glycosaminoglycan-degrading enzymes, such as chondroitinase ABC, heparitinase I, and hyaluronidase, modulated the mechanical properties of the tissue. Chondroitinase and heparitinase I caused a significant increase in hysteresis, but no changes in the resistance or the dynamic elasticity, as compared to control strips (70). Using cultures of primary human pulmonary vascular smooth muscle cells, we have shown that PDGF induces the synthesis and secretion of a specific form of hyaluronic acid with a molecular mass of 340 kDa (96). This specific hyaluronic acid inhibits cell proliferation, while it enhances cell migration involving de novo synthesis of MMP-2 (97). The described effect of the 340-kDa hyaluronic acid is similar to the effect of heparins with different length on cell proliferation of pulmonary vascular smooth muscle cells (98). Large heparin molecules are used in therapy for pulmonary hypertension where they downregulate the proliferation of vascular smooth muscle cells and therefore reduce vascular remodeling and subsequently pulmonary hypertension (98–100). Thus, it is possible that the synthesis of specific glycosaminoglycans may be regarded as an autoregulatory mechanism via which cells counteract the pro-proliferative signals of cytokines and growth factors. Such studies underline the importance of glycosaminoglycans on the structure and function of the extracellular matrix in the lung. Regarding the role of hypoxia on the development of pulmonary hypertension, it is interesting that hypoxia inhibited the PDGF-induced secretion of hyaluronic acid in pulmonary vascular smooth muscle cells (89). Hypoxia-associated pulmonary hypertension is characterized by expression of growth factors such as PDGF and interleukin-6 that both enhance the proliferation of vascular smooth muscle cells and eventually lead to hypertrophy (77,100,101). The enhanced vascular smooth muscle cell proliferation consequent to the hypoxia-induced expression of growth factors correlates with the hypoxia-induced reduction in the synthesis of antiproliferative molecules such as hyaluronic acid. While the cellular signaling pathway by which specific growth factors regulate the de novo synthesis of specific glycosaminoglycans has been documented, the pathway involved in hypoxia-induced glycosaminoglycan synthesis remains unclear. However, since hypoxia is known to stimulate the synthesis of cytokines and growth factors, including interleukins, PDGF, and TGF, its effect on glycosaminoglycan synthesis may be mediated via these factors. For example, stimulation of lung fibroblasts by TGFβ 1 or irradiation-evoked lung fibrosis in rats was shown to inhibit the activity of the rHYAL2 isoform of hyaluronidase and upregulate the activity of the rHAS2 isoform of hyaluronic acid synthase (51). It has

344


yet to be elucidated whether epimerases or sulfotransferases that are involved in the differential synthesis of dermatan and chondroitin sulfate are also affected by hypoxia (102). Changes in the ratio of the glycosaminoglycan composition in the extracellular matrix of the lung can also be controlled via their internalization as shown for hyaluronic acid under hypoxic culture conditions or in the presence of TGFβ in lung fibroblasts (81). Therefore, it remains to be clarified if the increased secretion and deposition of hyaluronic acid in response to hypoxia is partially masked by the increased internalization of this glycosaminoglycans by human lung fibroblasts.

IV. Glycosaminoglycans as New Therapeutic Agents or Targets As discussed above, glycosaminoglycans have a great potential to be used as therapeutic agents in lung diseases since they modulate essential cell functions. Heparin is regularly used as an anticoagulant (104–106), and hyaluronic acid is used in the treatment of interstitial cystitis (107,108) and in inflammatory/ degenerative joint and cartilage diseases (109–111). Heparan sulfate proteoglycans have been used to control metastasis formation (47). Regarding lung diseases, inhaled heparin is the only glycosaminoglycan that has been used in animal models for the therapy of pulmonary hypertension (98,103). The results of such studies remain controversial. Experiments in vitro may depend on the cell type or the animal model used. In therapy, their success or their failure may largely be determined by the way these components are administered and produced. For example, most of the glycosaminoglycans used therapeutically originate from animal tissue and may contain more or fewer impurities which may contribute to the reported side effects (112–114). Unfortunately, we are far from understanding the mechanisms controlling the length of glycosaminoglycans which appears to be crucial for their biological function, and we cannot intervene on their de novo synthesis and composition. Better knowledge and understanding of the role of glycosaminoglycans in the context of the extracellular matrix together with their biomechanical properties for the affected organ offer an opportunity to provide the basis for the development of new glycosaminoglycan-based therapeutic drugs.

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17 Integrins, Proteoglycans, and Lung Disease

JIAHUA XU Rensselaer Polytechnic Institute Troy, New York, U.S.A.

I.

Introduction

The lungs are ingeniously constructed to carry out their cardinal function—the exchange of gases between inspired air and the blood. The gas exchange region of the lung is composed of respiratory bronchioles, the alveolar ducts, the alveolar air space, and the alveolar walls. The alveolar walls are composed of the capillary endothelium, the alveolar epithelium, and the intervening extracellular matrix (ECM) organized into the basement membranes of the two cell layers and the interstitial tissue. The lung ECM components include collagens, elastins, proteoglycans, and noncollagenous glycoproteins, such as laminin and fibronectin. Collagens and elastins together constitute over 60% of the dry weight of the lung. In addition to supporting tissue architecture, ECM components regulate cell migration, proliferation, differentiation, and morphology by binding to cell surface receptors, predominantly integrins. These functions are essential in morphogenesis during lung development and pathogenesis of lung diseases. Integrins concomitantly recognize the structure of the surrounding matrix and mediate signals to the cell interior, leading to alteration in cell function. The understanding of integrin regulation will undoubtedly lead to the comprehension of the pathogenesis of lung diseases and provide therapeutic targets. This chapter will discuss 351

352

Xu

the role of integrins in several forms of lung diseases with a focus on the molecular mechanisms.

II. Cellular Function of Integrins A. Integrin Family

An appreciation of the role of integrins in lung diseases requires a basic understanding of integrin function. Integrins are heterodimers composed of a larger (α) and a smaller (β) subunit. Both contain a large extracellular domain, a transmembrane region, and a relatively small cytoplasmic domain (Fig. 1). The extracellular domains of the two subunits associate noncovalently to form a functional receptor. The ligand-binding region has been localized to the N-terminal portions of the α and β subunits (1). The N-terminal region of the α subunit is composed of seven repeats of about 60 amino acids each (2). Within the third repeat in some integrins is an inserted I-domain of approximately 200 amino acids. Eight different β subunits and 18 various α subunits are the source for 24 heterodimers identified thus far (Table 1). The β1 , α v , and β2 , are three most common subunits with 12, five, and four partners, respectively. Eighteen of 24 integrin receptors recognize and bind to extracellular matrix proteins while the rest are counterreceptors for cell surface proteins. Multiple integrins can bind to the same ligand. For example, there are eight integrin receptors known to bind to fibronectin. Similarly, a single integrin receptor can have several ligands. One such integrin is α 2 β1 , a collagen receptor that also binds laminin-1, tenascin, and a snake venom called jararhagin. The broad recognition specificity by both ligands and integrin receptors results in the functional overlapping. Integrins are thought to bind their respective ligands through short linear sequences that seem to contain acidic residue. The best-known example of the integrin-binding motifs in ligands is arginine-glycine-aspartic acid (RGD), originally found in fibronectin (3). This binding motif has now been identified in a wide range of extracellular matrix molecules, virus proteins, cytokines, and growth factors (4–60). In addition to the RGD sequence, other integrin recognition sites have also been identified, including the non-RGD site in fibronectin and the triple-helical motifs in type I collagen, recognized by α 4 integrins (α 4 β1 and α 4 β7 ) and α 2 β1 , respectively (7,8). B. Integrin Signal Transduction

The importance of integrins cannot be overstated. Initially recognized as “glue” that binds cells to their surroundings, integrins transduce extracellular signals to control cell morphology, growth, movement, apoptosis, and gene expression.

Integrins, Proteoglycans, and Lung Disease

353

Figure 1 Diagrammatic representation of integrin structure. (A) Ligand-binding region of an α subunit is the N-terminal portion of the extracellular domain that is composed of seven repeats (I–VII). I-domain is inserted within the third repeat for some integrins. The conservative region in the β subunit is associated with ligand binding. An α subunit has a larger extracellular domain, whereas a β subunit has a slightly larger cytoplasmic domain. TM: transmembrane domain; Intra: intracellular tail. (B) Heterodimer of an Idomain-containing integrin.

Ligands for integrins include ECM proteins and its degradation products, neighboring cell membrane proteins, and early immediate gene products. Sequential events following the occupancy of an integrin by its ligand are receptor clustering in the plasma membrane, recruitment of cytoskeletal proteins (tensin, α-actinin, talin, vinculin, and focal adhesion kinase) to the focal adhesion site, and accumulation of signaling molecules such as Src, Src substrates, Ras, the mitogenactivated protein kinases, and more cytoskeletal proteins such as F-actin and paxillin (9).

354 Table 1

β1

β2

αv

β7 α 6 β4 αIIb β3 a

Xu Integrins and Their Ligands Subunits

Ligands

α1 α 2a α3 α4 α5 α6 α7 α8 α9 α10a α11a αDa αLa αMa αXa β1 β3

Collagen, laminin Collagen, laminin, tenascin Collagen, laminin Fibronectin, VCAM-1 Fibronectin Laminin Laminin Fibronectin, tenascin Tenascin, VCAM-1 Collagen Collagen ICAMs ICAMs ICAM-1, C3b, fibrinogen, factor X C3b, fibrinogen, lipopolysaccharide Fibronectin, vitronectin Vitronectin, fibrinogen, fibronectin, thrombospondin, osteopontin, collagen, von Willebrand factor Vitronectin Fibronectin, tenascin Vitronectin, fibronectin Fibronectin, VCAM-1, MadCAM-1 E-cadherin Laminin Vitronectin, fibrinogen, fibronectin, thrombospondin, von Willebrand factor

a

β5 β6 β8 α4 αEa

The subunit contains I-domain.

Focal adhesion kinase (FAK), a nonreceptor protein kinase, is among the first proteins that are activated by integrin-dependent cell adhesion. FAK is activated upon integrin clustering through a mechanism involving the cytoplasmic tail of the β subunit. The C-terminal domain of FAK contains the focal adhesion targeting (FAT) region and binding sites for both cytoskeletal proteins and signaling molecules. The biological role of FAK includes control of anoikis and cell motility (10,11). Because FAK activation also leads to the recruitment of Grb2/ Sos complex, components of the Ras signaling pathway, FAK is thought to participate in cell growth possibly through MAP kinases, downstream effectors of Ras GTPases (12,13).


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In addition to activating FAK and triggering FAK-dependent signal transduction, integrins also have distinct functions. While mechanisms underlying the specificity of integrin signaling remain unclear, two models have been reported. First, integrins can associate with transmembrane adaptor proteins like caveolin and the transmembrane-4 superfamily proteins (TM4SF) (14,15). A subset of integrins (α 1 β1 , α 5 β1 , α 6 β4 , and α v β3 ) promote cell proliferation by recruiting Shc and activating the Ras-ERK pathway using caveolin, as an adaptor, whereas α 3 β1 and α 6 β1 associate with TM4SF proteins to activate phosphatidylinositol 4-kinase (PI-4K) and possibly other signaling molecules. Second, the cytoplasmic domains of integrin α subunits, i.e., α 2 and α 6 , have been reported to interact with cellular components and induce the MAP kinase signaling pathway (16,17). An important feature of integrin-mediated signal transduction is its ability to initiate signal transduction independently or to exert influence on signal transduction by other extracellular molecules, most notably growth factors. Adhesion to ECM has been shown to modulate cell proliferation and gene expression induced by growth factors (18,19). Activation of mitogen-activated protein kinase (MAPK) by growth factor is a key event in controlled cell proliferation. It has been reported that integrin-mediated cell adhesion to ECM synergizes with growth factors to activate MAPK (13,18). This ECM–growth factor coordination has also been demonstrated by platelet-derived growth factor (PDGF)-induced expression of fibroblast integrins α 2 , α 3 , and α 5 in three-dimensional (3D) ECM models (20). PDGF, together with a 3D fibrin-fibronectin matrix bed, upregulates integrins α 3 and α 5 . When the ECM partner of PDGF is switched to a 3D collagen lattice, the PDGF upregulation of α 3 , and α 5 is attenuated, whereas that of α 2 , the collagen receptor integrin, is further enhanced. How ECM and growth factors conduct their crosstalk is an intensively pursued research area. Since growth factor and integrin signal transduction pathways overlap with one another, there are many potential converging points for the interaction to occur. Several mechanisms have been proposed to explain such interactions potentially related to lung injury. Expression of two integrin ligands, Cyr61 and osteopontin, can be induced by growth factors. They may represent growth factor intermediaries to initiate the integrin signaling process. Cyr61, a growth factor–induced immediate early gene, promotes cell adhesion, migration, and proliferation. A ligand for α v β3 , it encodes a secreted cysteine-rich and heparin-binding protein associated with the ECM or with the cell surface through integrins (21). A similar mechanism may be employed by angiotensin II (AII), a factor that modulates cardiac hypertrophy, fibroblast proliferation, and ECM production. AII induces the expression of osteopontin, a phosphoprotein that binds α v β1 , α v β3 , α v β5 , (22), α 4 β1 (23), and α 9 β1 (24) integrins and is involved in the vascular cell remodeling process (22,25,26). The ligation of osteopontin to β3 integrin rapidly

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increased NF-κB activity, which is crucial for osteopontin-mediated cell survival (27). Insulinlike growth factor-1 (IGF-1), on the other hand, might use preexisting protein factors as liaison between influx soluble factors and membrane integrins. IGF-1 and integrin α 5 β1 recognize IGF binding protein-1 (IGFBP-1), a cell membrane protein. The binding of IGF-1 to IGFBP-1 and the activation of α 5 β1 are both required for IGF-1 to stimulate wound healing (28). Another potential mechanism for the crosstalk to occur is integrin-mediated phosphorylation of growth factor receptors. For example, integrin α 6 β4 and ErbB2, a tyrosine protein kinase, form a complex (29). Integrin activation is believed to potentiate the growth factor action by enhancing autophosphorylation of receptors for growth factors such as PDGF and EGF (30,31). Inhibition of tyrosine phosphorylation abrogated FGF-2 induced capillarylike tube formation inside a collagen lattice (32). Increasing evidence also suggests that growth factor–and integrin-initiated signals could converge downstream of growth factor receptors. Shc, an adapter protein in growth factor signal transduction, has proven an essential component in integrin α 1 β1 signaling pathway by gene knockout studies (33). The integrin α 1-null mouse fibroblasts fail to recruit and activate Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation, leading to eventual growth deficiency on collagens. Therefore, integrin α 1 β1 and growth factors apparently converge at Shc to mediate cell growth. Proteoglycans, either in ECM environment or on cell surface, can crosstalk with integrins at various levels (Table 2). Decorin, a small leucine-rich proteoglycan that has been extensively studied in lung injury as a TGFβ inhibitor, binds a number of ECM components including fibronectin, thrombospondin, and several types of collagens (34–36). It competes against integrin-mediated cell binding to fibronectin and thrombospondin (37,38), inhibits endothelial cell migration and tubelike structure formation (39), and facilitates fibronectin matrix assembly (40). Unlike decorin as an antiadhesive proteoglycan, agrin, a basement membrane–associated heparan sulfate proteoglycan, can serve as a substrate for cell adhesion mediated by integrin α v β1 during embryogenesis (41). Similarly, perlecan, a basement membrane–associated heparan sulfate proteoglycan, promotes β1-integrin-mediated cell adhesion (42). Functional cooperation also exists between cell-surface heparan sulfate proteoglycans and integrins. For example, syndecan-2 and integrins α 3 β1, α 4 β1, α 5 β1, and α v β3 are sufficient in fibronectin matrix assembly (43). The formation of focal adhesion complex induced by fibronectin depends on integrins as primary receptors and syndecan-4 as a coreceptor (44,45). This joint signal transduction by integrins and syndecan-4 to induce assembly of focal adhesions and actin stress fibers requires the small GTPase Rho (44) (Fig. 2).

Integrins, Proteoglycans, and Lung Disease Table 2

Crosstalk Between Integrins and Proteoglycans

Proteoglycans Decorin

Biglycan HSPG

Syndecan-1 Syndecan-2

Syndecan-4 Glypican Agrin Perlecan

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Integrin connections

References

Inhibition of cell migration on fibronectin and type I collagen Stabilization of fibronectin assembly Inhibition of cell binding to fibronectin, thrombospondin, and type XIV collagen Induction of fibroblast MMP-1 expression in cooperation with vitronectin- or 120-kDa fibronectindependent Enhancement of endothelial cell migration Recognition of I-domain of integrin MAC-1 (CD11b/CD18) Positive regulation of α4 β1-dependent adhesion to fibronectin Cell invasion into epithelium Induction of stress fiber formation in cooperation with integrin α5 β1 Induction of filopodia by active cdc42Hs through heparan sulfate chains in the extracellular domain. Induction of fibronectin assembly Induction of focal adhesion formation Comediator of αvβ3-mediated endothelial cell adhesion to thrombin Integrin αv β1 mediated adhesion to agrin and modulate agrin signaling Positive regulation of β1 integrin-mediated cell adhesion

39,40,46 40 37,38,47 48

49 50 51–53 54 55 56 43 44 57 41 42

Abbreviations: HSPG, heparan sulfate proteoglycan

III. Integrins in Common Lung Diseases Common lung diseases of acute and chronic forms are often associated with altered cell proliferation, cell adhesion, cell migration, gene expression, and cell apoptosis. Integrins affect these cell activities in most physiological and pathological processes. The prominent role of integrins in the lung diseases or development has been increasingly recognized by various experimental approaches, such as knockout mice, functional blocking of antibodies, and targeted gene overexpression (Table 3). How integrins contribute to the pathogenesis of selective lung diseases will be discussed next.

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A. Pulmonary Fibrosis

Pulmonary fibrosis is a progressive and largely untreatable group of disorders characterized by excessive deposition of extracellular matrix proteins, proliferation of myofibroblastlike cells, and organ contraction. TGFβ, found in all tissue types, plays a central role in tissue fibrosis including pulmonary fibrosis by increasing ECM protein synthesis, decreasing metalloproteinase activity, and promoting transformation of myofibroblasts and contraction of interstitial connective tissue. However, TGFβ is secreted as a latent form that consists of mature TGFβ and latency-associated peptide (LAP) to form the small latent complex (SLC). The activation of latent TGFβ is believed to be a highly regulated process. Using in vivo or in vitro models of fibrotic responses after lung injury, a number of studies have reported altered expression of integrins such as α 8 β1 (69), α 6 β1 (70), α E β7 (71), α 5 β1 (72), and β2 and α 4 integrins (73). Evidence has shown that integrin α v β6 , along with plasmin, thrombospondin, and reactive oxygen species, can activate latent TGFβ. A mechanism has been proposed that α v β6 binds to LAP at RGD sequences, leading to a conformational change that allows the mature TGFβ to interact with its receptor on cell membrane (74). Consistent with this hypothesis, transgenic mice that lack the β6 subunit fail to develop pulmonary fibrosis after induction by bleomycin (67). One feature in tissue fibrosis is to promote the production of other cytokines, i.e., IL-1β, TNFα, and osteopontin (75,76). Osteopontin is a cytokine with mineral- and cell-binding properties that can regulate cell activities by binding to integrin receptors α v β3 , α v β1 , α v β5 (25,26), α 4 β1 (23), and α 9 β1 (24). It was strongly expressed in alveolar macrophages accumulating in the fibrotic area of the lung in a mouse model of pulmonary fibrosis by intratracheal instillation of bleomycin (77). The development of the fibrotic process was associated with an increase in the expression of osteopontin mRNA and protein. Induction of osteopontin by TGFβ occurs at the transcriptional level and requires the transcriptional activators Smad3 and Smad4 (78,79). A pro-proliferative and survival factor, osteopontin promotes PDGF-mediated proliferation of fibroblasts (77) and

Figure 2 Signaling cooperation between integrins and heparin sulfate proteoglycans. Fibronectin is bound by a cell at RGD and IIICS sites and by heparan sulfate (HS) proteoglycan at specific binding sites for heparan sulfate. Decorin competes against cells for binding to fibronectin. After ligand ligation, integrins and syndecan-4, a cell associatedHS proteoglycan, interact with cytoskeleton through their respective cytoplasmic domains and activate small GTPase Rho, leading to the assembly of focal adhesions and microfilament bundles. Integrins and syndecan-4 are colocalized at focal adhesion sites. The HS side chains of a HS proteoglycan present in extracellular matrix or on cell surface bind fibroblast growth factor (FGF). Darker lines in proteoglycan represent the core protein; thin lines are HS side chains. Abbreviations: FGFR, FGF receptor; PM, plasma membrane.

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Xu Integrin Function in Lung

Function in lung development or injury Integrin α3 Basement membrane organization and branching morphology during lung organogenesis Integrin α4 Airway hypersensitivity

Airway inflammation and hyperresponsiveness LFA-1 (CD11a or αL ) Neutrophil emigration Mac-1 (CD11b or αM ) Neutrophil adhesion to fibrinogen and degranulation Integrin β2 Sequestration and infiltration of polymorphonuclear leukocytes and vascular injury. Leukocyte adhesion to endothelial cells Neutrophil recruitment Integrin β6 TGFβ activation, positive role in fibrosis and pulmonary edema, and negative role in inflammatory responses.

Experimental models

References

α3-Null mice

58

Antibody administered to transgenic mice overexpressing IL-5 by the airway epithelium Antibody administered to ovalbumin-sensitized mice

59

60

LFA-1-null mice

61

Mac-1-deficient mice

61

Mice transiently overexpressing NIF in pulmonary microvascular endothelial cells challenged by E. coli or LPS Patient with leukocyte adhesion deficiency uPAR-/-mice in response to P. aeruginosa pneumonia

62,63

β6-Null mice

66–68

64 65

Abbreviations: IL-5, interleukin-5; NIF, neutrophil inhibitory factor; LPS, lipopolysaccharide; uPAR, urokinase plasminogen activator receptor; TGFβ, transforming growth factor β.

rescues MMP inhibitor-induced apoptosis of smooth muscle cells (80). Blocking of α v subunit or α vβ3 impaired the ability of osteopontin to mediate both cellular functions. It has been reported that a phosphorylation-dependent interaction between the amino-terminal portion of osteopontin and its integrin receptor stimulated the expression of the macrophage pro-inflammatory cytokine IL-12,


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whereas a phosphorylation-independent interaction with CD44, a hyaluronan receptor, inhibited expression of T-cell anti-inflammatory IL-10 (81). Increased ECM deposition is a prominent feature associated with TGFβ in tissue fibrosis (82). ECM proteins, such as type I collagen (83), fibronectin (84), and laminin (85,86), are induced by TGFβ. However, TGFβ regulation of integrin expression can be bidirectional, depending on cell types (87,88) or specific integrins (89). For example, TGFβ stimulates integrin expression in monocytes, but inhibits it in microvascular endothelial cells (87,88). In keratinocytes, TGFβ upregulates the expression of α 5 β1 , α v β5 , α 2 β1 , induces the de novo synthesis of α v β6 , but downregulates α 3 β1 (89). In fibroblasts, TGF-β can induce the expression of α 2 β1, a collagen receptor integrin that modulates TGFβ-induced contraction of a 3D collagen lattice (90). Integrins may also modulate TGFβ-stimulated ECM deposition. For example, activation of integrins β1 , α 4 β7 , or α 4 β1 stimulates the production of interferon-γ (91–94), an antifibrotic cytokine that antagonizes TGFβ-stimulated collagen deposition by activating transcription factor STAT-1 (95). Myofibroblasts, key effector cells in the development of the fibrotic response (96), are characterized by large bundles of actin-containing microfilaments disposed along the cytoplasmic face of the plasma membrane and the establishment of cell-cell and cell-matrix linkages (97). TGFβ is a major promoter of myofibroblast differentiation by inducing α-smooth muscle actin (98–100). The accumulation of α-smooth muscle actin requires the TGFβ1-induced deposition and polymerization of ED-A fibronectin, an isoform de novo expressed during tissue injury and fibrotic changes (101). It appears that both ED-A fibronectin and TGFβ1 are necessary for myofibroblast conversion. In vitro, TGFβ1 increases total fibronectin levels by preferentially promoting accumulation of ED-A fibronectin (102,103), on which cells adhere and migrate more actively than other splicing variants of fibronectin (104), probably because of the altered accessibility of the RGO motif of ED-A fibronectin to integrin α 5 β1 . It is hypothesized that ED-A fibronectin could transduce and/or synergize signals by TGFβ1. The integrins are involved in this process at two levels. They are receptors for fibronectin as well as essential components in the fibronectin matrix. The interaction between fibronectin and α 5 β1 initiates signal transduction pathways that overlap with growth factor signal transduction pathways and lead to many physiological processes (105,106). The fibronectin matrix assembly, the only fibronectin structure in which the ED-A domain can exert its permissive function of TGFβ activity (101), requires the activation of integrins α 3 β1 and α 4 β1 and the interaction between integrins and cytoskeletal proteins (107–109). Myofibroblasts express α-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. The emergence of the myofibroblast phenotype in tissues undergoing fibrosis and in wound healing

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is thought to be responsible for the increased tissue contractility. Contraction is commonly studied in vitro in a 3D collagen lattice populated with the cells of interest. Not surprisingly, the enhanced contractile capacity of cells with a myofibroblast phenotype isolated from the lung of bleomycin-treated rats is dependent on TGF-β (90,110,111). In addition to TGFβ fibrotic factors, i.e., PDGF (112) and osteopontin (22,113), have been found to induce the collagen gel contraction. In contrast, antifibrotic factors, such as interferon-γ and prostaglandins, inhibit the contraction of the collagen matrices (114). Integrins appear to participate in the collagen gel contraction mediated by these factors. For example, TGFβ induces collagen gel contraction by increasing cellular α 2 β1 level (90). Several laboratories have also found that PDGF-BB induced α 2 β1 in human dermal fibroblasts (115,116). Osteopontin, an integrin ligand, requires β3 integrin to promote gel contraction. The ligation of osteopontin to α v β3 integrin stimulates pp60c-src kinase activity (117) and NF-κB (27); Indeed, NF-κB activity is required for collagen gel contraction (118). Integrin α 2 β1 , a collagen receptor in fibroblasts, is the first among several integrins, i.e., α 1 β1 collagen receptor and α 6 β1 laminin receptor (119–122), identified, to mediate matrix contraction (123). The role of α 2 β1 in collagen gel contraction has been shown in a broad range of cell types (124–126). The cytoplasmic domain, but not the extracellular domain, of integrin α 2 subunit may contain information to regulate tissue contraction (123). This domain is responsible for α 2 β1-initiated signal transduction pathway leading to p38α MAP kinase activation (17). Inhibition of p38α MAP kinase attenuated the contraction of the collagen gel (127). Administration of a p38 MAP kinase inhibitor resulted in reduced fibrotic response in bleomycin-induced pulmonary fibrosis model in rats (128). B. Acute Lung Injury

The syndrome of acute lung injury is characterized by an elevated level of flooding of alveolar spaces with a protein-rich exudate that impairs pulmonary gas exchange leading to arterial hypoxemia, pulmonary hypertension, and respiratory failure (129). Animals induced by a variety of stimuli such as bleomycin, oleic acid, endotoxin, and hyperoxia provide experimental models for acute lung injury. Interleukin (IL)-1, known for its pro-inflammatory effects, is elevated in bronchoalveolar lavage (BAL) fluid and alveolar macrophages in patients with adult respiratory distress syndrome (ARDS) (130,131). In animal models, shorttime exposure to IL-1 by injection of recombinant IL-1 into rodent tracheas leads to development of acute alveolar leakage and neutrophil inflammation (132,133).


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Transient overexpression of IL-1β in rat lungs leads to acute lung injury (134). Control of IL-1β production may provide a valid target for therapeutic intervention in acute lung injury. Fibronectin and fibrin(ogen), two major components of the provisional matrix that is deposited at the site of injury, stimulate IL-1β production through their receptor integrins α 5 β1 and β2 subgroup, respectively (135,136). The regulation by both ECM proteins can occur at the level of transcription. A NK-κB regulatory element in the IL-1β gene promoter is necessary for fibrinogen action, whereas an AP-1-binding site mediates fibronectin-induced IL-1β production (137–139). After the activation by inflammatory mediators following local or systemic insults, circulating neutrophils sequester within pulmonary vasculature, then adhere to the endothelium, and eventually migrate out of the pulmonary capillaries into the lung parenchyma and air space. Integrins, particularly the β2 subgroup, play an important role in this process. There are four known β2 integrins: α L β2 (CD11a/CD18), α M β2 (CD11b/CD18), α D β2 , and α X β2 (CD11c/CD18). The ligands for β2 integrins include intercellular adhesion molecules (ICAMs) for α L β2 , α M β2 , and α D β2 ; the C3b component of complement Factor X and fibrinogen for α M β2 and α X β2 ; and lipopolysaccharide (LPS) for α X β2 . The interaction between β2 integrins and ICAMs is one of mechanisms that account for the neutrophil traffic in the lung, primarily in the steps of adhesion and migration, also termed emigration. Indeed, the increase in expression of β2 integrins has been observed in acute lung injury (140–142). Neutrophils release cytotoxic compounds while adherent to endothelium, epithelium, or interstitial ECM proteins, but not while suspended in the bloodstream. Adhesion apparently activates neutrophils, leading to an oxidative burst (143–145). Integrin-mediated signal transduction pathways are responsible for this activity. For example, stimulation of the respiratory burst in neutrophils by cytokines, such as TNFα and f-met-leu-phe is enhanced by adhesion to the ECM and blocked by integrin antibodies (146). Further study has shown that integrinmediated adhesion via α L β2 and α X β2 , but not α M β2 , triggers the respiratory burst in neutrophils (147). Bacterial LPA and TNFα, activators of p38 MAP kinase, induce the β2 integrin-dependent neutrophil adhesion (148). The cytoplasmic domain of the β2 subunit is believed to control this process by interacting with cytoskeletal and signaling proteins (149).

C. Chronic Obstructive Pulmonary Disease and Asthma

Asthma is among a group of disorders that are associated with airflow obstruction within the lung. These lung disorders are referred as chronic obstructive pulmonary disease (COPD). Two major components of asthma are chronic airway in-

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flammation and bronchial hyperresponsiveness. Asthma patients typically show the accumulation of mucus in the bronchial lumen, thickened basement membrane underlying the mucosal epithelium, and hypertrophy and hyperplasia of smooth muscle cells, accompanied by intense inflammation due to recruitment of eosinophils, macrophages, and other inflammatory cells. A number of studies have focused on the role of integrins in the recruitment of eosinophils which massively infiltrate the lung tissues and migrate through lung epithelium into the airways, leading to a 50- to 100-fold increase in the number of eosinophils relative to neutrophils in the bronchial mucosa in asthma. Several integrins have been implicated in the eosinophil accumulation at the site of inflammation. They include α 4 (77,150–153), α 4β1 (154,155), α 4β7 (156), β6 (155,157). Integrin α 4β1 (very late antigen-4, or VLA-4; CD49d/CD29), a fibronectin and VCAM-1 receptor predominantly expressed on lymphocytes, monocytes, and eosinophils, but not on neutrophils, is exclusively studied for its role in eosinophil accumulation. Eosinophils adhere to endothelial or epithelial cells through interaction between primarily α 4β1 and vascular adhesion molecule (VCAM)-1. The α 4β1-mediated eosinophil adhesion to VCAM-1 initiates a tyrosinedependent signaling pathway, leading to the generation of superoxide anions (158). The interaction between the α 4β1 of eosinophils and the CS-1 region of tissue fibronectin may trigger autocrine cytokine synthesis and release, thereby promoting cell survival and persistence within the tissues (159,160). The α 4β1mediated adhesion process both requires and modulates the function of other soluble inflammatory factors. For example, the eosinophil adhesion to ICAM-1 requires the presence of granulocyte macrophage colony-stimulating factor (GMCSF) (161). The adhesion between eosinophils and pulmonary microvascular endothelial cells through α 4β1 and VCAM-1 can be enhanced by TNFα and IL4 (162). On the other hand, blocking of α 4β1 function by antibody suppressed the airway hyperresponsiveness induced by IL-5, a cytokine important in the pathogenesis of asthma (163). In addition to α 4β1 , β2 integrins may be involved in the pathogenesis of asthma through (1) ICAM-2-dependent tissue infiltration of T-cells (163), (2) chemoattractant-stimulated adhesion or migration of eosinophils together with the α 4 integrins (164–166), and (3) the prolonged survival of eosinophilia (157). Antagonists of integrins have shown great promises in treating asthma in animal models (60). Reagents that target at α 4β1-mediated cell adhesion processes include Antigren R, an antibody against the α 4 subunit (167), and BIO-1211, a small peptide based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin (154,168). Other peptide inhibitors have also been developed based on the binding epitope of VCAM-1 or an RGD motif (169).


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stimulate the respiratory burst of human neutrophils. J Cell Biol 1992; 116:1007– 1017. Detmers PA, Zhou D, Polizzi E, Thieringer R, Hanlon WA, Vaidya S, Bansal V. Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrindependent neutrophil adhesion and the adhesion-dependent oxidative burst. J Immunol 1998; 161:1921–1929. Dedhar S, Hannigan GE. Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr Opin Cell Biol 1996; 8:657–669. Teixeira MM, Robinson MK, Shock A, Hellewell PG. alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation. Br J Pharmacol 2001; 132:596–604. Hisada T, Hellewell PG, Teixeira MM, Malm MG, Salmon M, Huang TJ, Chung KF. Alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice. Am J Respir Cell Mol Biol 1999; 20:992–1000. Patel KD. Mechanisms of selective leukocyte recruitment from whole blood on cytokine-activated endothelial cells under flow conditions. J Immunol 1999; 162: 6209–6216. Taylor BM, Kolbasa KP, Chin JE, Richards IM, Fleming WE, Griffin RL, Fidler SF, Sun FF. Roles of adhesion molecules ICAM-1 and alpha4 integrin in antigeninduced changes in microvascular permeability associated with lung inflammation in sensitized brown Norway rats. Am J Respir Cell Mol Biol 1997; 17;757–766. Abraham WM, Gill A, Ahmed A, Sielczak MW, Lauredo IT, Botinnikova Y, Lin KC, Pepinsky B, Leone DR, Lobb RR, Adams SP. A small-molecule, tight-binding inhibitor of the integrin alpha(4)beta(1) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep. Am J Respir Crit Care Med 2000; 162:603–611. McNulty CA, Symon FA, Wardlaw AJ. Characterization of the integrin and activation steps mediating human eosinophil and neutrophil adhesion to chronically inflamed airway endothelium. Am J Respir Cell Mol Biol 1999; 20:1251–1259. Walsh GM, Symon FA, Lazarovils AL, Wardlaw AJ. Integrin alpha 4 beta 7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin. Immunology 1996; 89:112–119. Chihara J, Kakazu T, Higashimoto I, Saito N, Honda K, Sannohe S, Kayaba H, Urayama O. Signaling through the beta2 integrin prolongs eosinophil survival. J Allergy Clin Immunol 2000; 106:S99–S103. Nagata M, Sedgwick JB, Bates ME, Kita H, Busse WW. Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation. J Immunol 1995; 155:2194–2202. Walsh GM, Symon FA, Wardlaw AJ. Human eosinophils preferentially survive on tissue fibronectin compared with plasma fibronectin. Clin Exp Allergy 1995; 25: 1128–1136. Higashimoto I, Chihara J, Kakazu T, Kawabata M, Nakajima S, Osame M. Regulation of eosinophil cell death by adhesion to fibronectin. Int Arch Allergy Immunol 1996; 111(suppl 1):66–69. Nagata M, Sedgwick JB, Kita H, Busse WW. Granulocyte macrophage colony-

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18 Heparin as a Potential Therapeutic Agent to Reverse Vascular Remodeling

HARI G. GARG and CHARLES A. HALES

ROBERT J. LINHARDT

Massachusetts General Hospital Harvard Medical School Boston, Massachusetts, U.S.A.

University of Iowa Iowa City, Iowa, U.S.A.

I.

Introduction

Vascular changes after chronic hypoxia are characterized by hyperplasia, hypertrophy, and migration of smooth muscle cells (SMCs) in the media of muscular and partially muscular pulmonary arteries (1,2). A number of factors are known to cause SMC migration, such as serum, platelet-derived growth factor (PDGF)BB (3,4), transforming growth factor-β (5), fibrinogen (6), oxidized low-density lipoprotein (7,8), and angiotensin II (9,10). Heparin, discovered nearly 80 years ago (11), is widely used as an anticoagulant drug. Besides its anticoagulation activity, heparin has a variety of other biological and biochemical activities that include the following: [1] regulation of lipid metabolism (12); [2] control of blood fluidity at the endothelial surface (13); [3] control of cell attachment to various proteins in extracellular matrix (ECM) (14–16); [4] binding with acidic and basic fibroblast growth factors (17,18); [5] binding to interleukin-3 and granulocyte-macrophage colony stimulating factor (19,20); and [6] inhibition of serotonin induced pulmonary artery smooth muscle cell hypertrophy (21). Heparin stimulates endothelial cell growth (22), whereas it inhibits the proliferation of renal mesangial cells (23), rat cervical 377

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epithelial cells (24), transformed cell lines (25–27), and systemic and pulmonary artery smooth muscle cells (28,29). Of the types of biological activities just mentioned, anticoagulation has been extensively discussed in several reviews (30– 37). Other biological activities have recently been reviewed (38–46). Regulation of vascular cellular smooth muscle proliferation by heparin was reviewed in 1989 (47), 1994 (48), and 2000 (49). These reviews specifically discuss the domains in heparin responsible for its antiproliferative activity on aortic vascular smooth muscle cells. Briefly, they suggested: 1. 2.

3. 4. 5. 6.

The anticoagulant and antiproliferative properties of heparin reside in different heparin domains. The 3-O-sulfo group on the internal glucosamine residue of a chemically synthesized pentasaccharide (Fig. 1) (50) is critical for growth inhibitory capacity of the pentasaccharide (51). A dodecasaccharide sequence in heparin contains the full antiproliferative activity (52). A 2-O-sulfo group in the glucuronic acid residues of heparin is not essential for antiproliferative activity (53). N-acetylation of N-desulfonated glucosamine residues does not seem to restore the antiproliferative activity (54). Both O-sulfo and N-sulfo groups are important for antiproliferative activity, but the relationship between the extent of N-desulfonation and the inhibition of cell proliferation is not straightforward (54).

Because vascular remodeling with smooth muscle cell hypertrophy and hyperplasia contribute to the high pulmonary vascular resistance seen in primary as well as secondary pulmonary hypertension, interest continues in heparin as a possible therapeutic agent to reverse vascular remodeling. Efforts have been made to establish the domain within the heparin polysaccharide, which is related to the inhibition of growth of pulmonary artery smooth muscle cell (PASMC). In studies aimed at understanding heparin’s structure-activity relationship (SAR), a

Figure 1 Antithrombin-binding, anticoagulant pentasaccharide demonstrating a structure critical for growth inhibition. Where X ⫽ H or SO3⫺ and Y ⫽ SO3⫺ or Ac. The central residue has a unique 3–O–sulfo group.

Heparin as a Potential Therapeutic Agent Table 1

General Composition of Different Glycosaminoglycans

Glycosaminoglycan

Saccharide backbone

Hyaluronan Chondroitin sulfate Dermatan sulfate

→4)-β-d-GlcA(1→3)-β-d-GlcN(1→ →4)-β-d-GlcA(1→3)-β-d-GalN(1→ →4)-α-l-IdoA(1→3)-β-d-GalN(1→ [→4)-β-d-GlcA(1→] →4)-β-d-GlcA)(1→4)-α-d-GlcN(1→ [→4)-α-l-IdoA(1→] →4)-α-l-IdoA(1→4)-α-d-GlcN(1→ [→4)-β-d-GlcA(1→]

Heparan sulfate Heparin

379

N-acetyl 1 1 1

SO 3⫺ 0 1 1

⬍1

0–2

⬍⬍1

2–3

GlcA ⫽ glucuronic acid; IdoA ⫽ iduronic acid; GlcN ⫽ glucosamine; GalN ⫽ galactosamine; residue shown in brackets is minor component.

variety of methods including chemical modification, fractionation, and enzymatic and chemical degradation have been employed (55,56).

II. Heparin Structure A. Arrangement of Sugars in Glycosaminoglycan Chains

Heparin is a member of a class of acidic polysaccharides called glycosaminoglycans (GAGs) (Table 1) (57). Heparin consists of alternating residues of uronic acid (either α-l-iduronic acid [major] or β-d-glucuronic acid [minor]) and hexosamine (α-d-glucosamine) connected through 1→4-glycosidic linkages and covalently bound to serine residues of the serglycin core protein. Heparin has Osulfo, N-sulfo, and N-acetyl substituents that are usually distributed in a heterogeneous array along the GAG chains. Heparan sulfate is a structurally related GAG, having a reduced content of O- and N-sulfo groups and iduronic acid (Table 1). B. Protein Core

The protein cores of heparan sulfate are diverse and heterogeneous, varying in size from 20 to 150 kDa and appear to share only the capacity to bear GAG chains. Repetitive serine-glycine sequences are found in the protein core of the proteoglycan carrying the heparin chain. The GAG chains are connected to the protein core through a tetrasaccharide linkage region (Fig. 2) (57). Heparin helicity results in a secondary structural pattern or coiling of the GAG chains (58) (Fig. 3). Heparin’s secondary structure, in conjunction with its chirality (i.e., configuration of the carbon atoms at the asymmetric centers), inherent in the constituent monosaccharides, makes heparin a uniquely complex macromolecule.

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Figure 2 The biosynthesis and structure of the heparin proteoglycan. The linkage region tetrasaccharide as well as key intermediate disaccharide structures formed during heparin biosynthesis are shown in boxes.

III. Low-Molecular-Weight Heparins Low-molecular-weight (LMW) heparins, currently used in treatment of acute proximal deep-vein thrombosis, are obtained by the controlled enzymatic or chemical depolymerization of heparin (56). In the past decade several LMW heparins (Table 2) have been prepared and approved by the U.S. Food and Drug Administration. These LMW heparins, approximately one-third the size of heparin, show improved pharmacokinetic and pharmacodynamic profiles.

Figure 3 The structure of a heparin dodecasaccharide sequence obtained from NMR (58), studies show the helicity of the heparin polymer.

Heparin as a Potential Therapeutic Agent Table 2 Trade name(s)

381

Low-Molecular-Weight Heparins Approved name(s)

Fluxum

Parnaparin sodium

Fragmin Fraxiparin Logiparin Lovenox Normiflo

Dalteparin sodium Nadroparin sodium Tinzaprin sodium Enoxaparin sodium Ardeparin sodium

Sandoparin

Certiparin sodium

Manufacturer(s) Opocrin S.p.a., Alfa Wasserman Pharmacia Upjohn Sanofi Recherche Novo Nordisk Aventis Wyeth-Ayerst Research, Pharmacia, Hepar Sandoz AG

Method(s) of production Peroxidolysis Deaminative cleavage Deaminative cleavage Enzymatic β-elimination Chemical β-elimination Peroxidolysis Deaminative cleavage

LMW heparins directly inhibit the intrinsic factor Xa activity complex (intrinsic tenase) but have no effect on the pro-thrombic activity (pro-thrombinase). In order to understand the role of sulfo group on these activities, LMW heparin was N-desulfonated and hypersulfonated separately. N-desulfonation of LMW heparin reduced its affinity for antithrombin. In contrast, hypersulfonation enhanced both the intrinsic tenase and pro-thrombic inhibitory activities. Hence, the biological activity of these heparin derivatives showed that they act as potent antithrombin-independent inhibitors of coagulation by attenuating intrinsic tenase and depending on the sulfonation may also inhibit prothrombinase (59). IV. Differences in the Structure of Heparin and Heparan Sulfate Heparin is uniquely found in the intracellular granules of certain mast cells, while heparan sulfate is ubiquitously distributed in the extracellular environment. Heparin and heparan sulfate originate from the same biosynthetic precursor, N-acetylheparosan (Fig. 2). Following the initial assembly of the N-acetylheparosan polymer from monosaccharide precursors, biosynthesis proceeds much further for heparin than for heparan sulfate (60–64). These additional biosynthetic steps for heparin result in a glycosaminoglycan with a higher content of N-sulfo groups, iduronate, and O-sulfo groups than heparan sulfate (65). Both heparin and heparan sulfate demonstrate substantial sequence heterogeneity (Fig. 4). Two carbon atoms on each glucosamine residue (positions 3 and 6) and one carbon on each uronic acid residue (position 2′) can contain an O-sulfo group. One carbon (position 2) on the glucosamine residue can contain either an N-acetyl or N-sulfo group, and the uronic acid residue can be isomeric (position 5) giving rise to either glucuronic or iduronic acid. Forty-eight different

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Figure 4 The possible 48 disaccharide sequences that can be found in heparin and heparan sulfate are shown. Many, but not all, of these sequences have been reported to date (From Refs. 66 and 67).

disaccharide structures can result from the combination of the different monosaccharide residues in heparin and heparan sulfate GAG chains (Fig. 4). V.

Heparin and Pulmonary Hypertension

Heparin is released by endothelial cells, and hypoxia increases this release (68). Heparin thus has the potential to regulate pulmonary vascular growth and remodeling. The effect of heparin on pulmonary hypertension and the associated vascular remodeling has been reviewed by us (69). We have shown in a mouse model of chronic hypoxia, that heparin inhibited the medial smooth muscle increase in vessels associated with terminal bronchioles, reduced right ventricular systolic pressure, and partially prevented the increase in medial thickness of intracinar vessels after 26 days of hypoxia (70). Heparin had no effect on the hematocrit and was effective at low doses that did not prolong the partial thromboplastin

Heparin as a Potential Therapeutic Agent

(a)

383

(b)

Figure 5 Light micrograph showing: (A) Distal pulmonary artery with two discernible elastic laminae and a thick media from a hypoxic control animal. The artery is adjacent to a terminal bronchiole. (B) Distal pulmonary artery also with two elastic laminae from a heparin-treated hypoxic animal. Here the media is significantly thinner. (Elastin stain; original magnification: ⫻ 125.) (From Ref. 72.)

time. Heparin did not block the rise in right ventricular systolic pressure after acute hypoxia, indicating that heparin prevented vascular remodeling through a mechanism that did not involve blockade of hypoxic vasoconstriction. Subsequently we showed in a guinea pig model of chronic hypoxia pulmonary hypertension in which we could measure cardiac output (71), that certain commercial heparin preparations given by continuous subcutaneous infusion resulted in 50% reduction in medial thickness of alveolar duct vessels (Fig. 5) and completely prevented the medial smooth muscle increase in vessels associated with terminal bronchioles (Fig. 6) (72). Cardiac output was unaffected by the heparin. Moreover, we found that fully established hypoxic pulmonary hypertension in the guinea pig was substantially reversed by heparin (73) and that heparin administration by aerosol was effective (74). We have also shown that different heparin lots even from the same company vary in their ability to inhibit SMC proliferation and hypertrophy (21) and that this variation correlates with the ability of these heparins to prevent hypoxic pulmonary hypertension (29). Rats were said to be resistant to heparin, but we found that a strongly antiproliferative heparin was effective in rats (75).

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(a)

(b)

Figure 6 Light micrograph showing: (A) Distal pulmonary artery accompanying an aveolar duct from a hypoxic control animal. Two elastic laminae are seen, separated by a thickened media. (B) Distal pulmonary artery accompanying an alveolar duct from a heparin-treated hypoxic animal. There is scant smooth muscle present in between the two elastic laminae. (Elastic stain; original magnification: ⫻ 500.) (From Ref. 72.)

VI. Antiproliferative Activity of Heparin and Its Derivatives A. Mechanisms Contributing to Heparin Inhibition of Smooth Muscle Cell Growth

Heparin is a potent inhibitor of smooth muscle cell (SMC) proliferation (29,76– 78), and this is true of both anticoagulant and nonanticoagulant forms (79). Although much attention has been focused on factors that stimulate SMC proliferation (80), very little is known about the mechanisms maintaining these cells in a quiescent state, or about the reestablishment of a quiescent state after their proliferative response has been initiated. Circulating heparin binds to endothelial cells and is taken up by the reticuloendothelial system where it enters a cellular pool to be released at a later stage (81). Heparin also binds to specific binding sites on smooth muscle cells and is internalized (82). Some antiproliferative effects are mediated by this specific binding, although it is not clear whether internalization is essential. Heparin blocks the cell cycle at either the G 0 /G 1 transition point (83) or at mid to late G 1 progression (82–85), and may inhibit such cellular intermediate processes as pro-


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Table 3 Properties of Heparins from Different Manufacturers Hexosamine e

Upjohn Elkins-Sinn Choay

USP a (U/mg)

Antiproliferative activity b

Protein content c

Amino acid d

GlcN

GalN

140 180

48 12 0

1.56 0.17 1.57

DTSEGAYFKH SGMYFKH DT(tr)S(tr)EGMFKHR

26.3 24.4 4.38

none 0.23 none

a

USP units of anticoagulant activity from the manufacturer. Percent inhibition of bovine pulmonary artery smooth muscle cell growth in vitro at a concentration of 1.0 µg/mL. c Percent of the total heparin (w/w). d Amino acids present in the core protein of heparin. e Percent w/w. b

tein kinase C activation, c-fos and c-myc induction (86,87), activator-protein-1 (AP-1)/fos-jun binding activity and posttransitional modification of jun B (88– 90). Heparin has also been shown to selectively block the protein kinase C pathway of mitogenic signaling (91) and the phosphorylation of mitogen-activated protein kinase (MAPK) (92). We have demonstrated that pulmonary artery smooth muscle cell (PASMC) mitogens such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) act through the Na⫹ /H⫹ antiporter, by stimulating a one-for-one exchange of extracellular Na⫹ for intracellular H⫹, to cause intracellular alkalinization, a permissive first step for cell division (93). Further, we have demonstrated that antiproliferative heparins block Na⫹ /H⫹ exchange in a manner directly related to antiproliferative activity (94). B. Structure-Activity Relationship (SAR)

To understand the SAR of the heparin polysaccharide, we compared the antiproliferative activity of three commercially available heparins. These preparations were from Upjohn, Elkins-Sinn, and Choay Pharmaceuticals. The growth inhibition activity of these heparins on pulmonary artery smooth muscle cells varied and was in the order: Upjohn ⬎ Elkins-Sinn ⬎ Choay, respectively (94). The properties of these heparin preparations are summarized in Table 3. C. Influence of Molecular Weight, Protein Core, and Heparin GAG Chains on Antiproliferative Activity

SAR studies carried out by preparing discrete sizes of antiproliferative heparin fragments by chemical modification of heparin show that dodecasaccharide and

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larger fragments had maximal antiproliferative activity (52,95). Further, Tiozzo and coworkers (96) demonstrated that the reduction of the molecular weight of heparin is associated with a progressive reduction of the antiproliferative activity. These studies were based on chemically modified heparin. In recent years, we assessed the influence of molecular weight (MW), protein core, and GAG chain of native heparin on PASMC proliferation (96). We fractionated highly potent Upjohn heparin by dissolving it in water and dialyzing it against water without chemical depolymerization. We then lyophilized both dialyzate, having a LMW (⬍3.5 kDa) and retentate having a high molecular weight (HMW) (⬎3.5 kDa). The residual core peptide of the Upjohn heparin was isolated by exhaustive treatment with heparin lyase I and III (97). GAG chains of the Upjohn heparin were liberated with alkaline borohydride treatment (98). No appreciable difference on the growth inhibition of PASMC between LMW and HMW heparin fractions was found. The protein core showed no antiproliferative activity. The GAG chains showed a similar inhibition on the growth of PASMC as that of parent heparin. These data suggest that the antiproliferative properties of heparin reside in the GAG chain and not in core protein. These data also suggest that both HMW and LMW heparin fractions have sufficient N- and O-sulfo groups in their carbohydrate residues necessary for the antiproliferative effect. D. 3-O-Sulfonation of Glucosamine Residue Is Not Critical for Antiproliferative Activity in Full-Length Heparin

Three commercially available heparins were degraded with heparin lyase I and III to evaluate their overall content of 3-O-sulfo group containing glucosamine residues to see if 3-O-sulfo group content correlated the antiproliferative effect of the three heparins. These enzymes are unable to degrade intermediate heparin oligosaccharides containing 3-O-sulfo groups in a glucosamine residue into disaccharide units. Instead, tetrasaccharides containing 3-O-sulfo groups were formed (99,100). Thus, the content of heparin lyase resistant tetrasaccharides in the digest correlated with the content of 3-O-sulfo groups. The oligosaccharide profiles of the three different pharmaceutical heparins after digestion with heparin lyase resistant I and III demonstrated that the most potent heparin (Upjohn) contained the least amount of heparin lyase resistant tetrasaccharide and hence the least amount of 3-O-sulfo groups on glucosamine residues. These results suggest that the presence of a 3-O-sulfo group in pharmaceutical heparin was not an essential requirement for antiproliferative activity, as previously reported (51) based on data derived from the synthetic pentasaccharide (50). The unsaturated disaccharides liberated after heparin lyase treatment of these three heparins were analyzed by us, and results are presented in Table 4 (97). We showed that the most potent Upjohn heparin preparation had the

Heparin as a Potential Therapeutic Agent Table 4

Disaccharide Composition of Heparins from Different Manufacturers

Disaccharide 1 2 3 4 5 6 7 8

387

X6

Y2

X 2′

Upjohn

Elkins-Sinn

Choay

H H SO 3 H SO 3 H SO 3 SO 3

Ac SO 3⫺ Ac Ac SO 3 SO 3 Ac SO 3

H H H SO 3 II SO 3 SO 3 SO 3

0.9 0.3 — — 5.4 4.8 0.4 86.8

3.9 2.0 3.9 1.7 11.5 6.3 1.5 66.3

14.5 — — — — 18.0 1.5 66.0

Disaccharides were released by treating exhaustively with heparin lyase I and III. Results are expressed as percent of total disaccharides released.

largest amount of a highly sulfated unsaturated disaccharide (Table 4, disaccharide 8). E. Effect of Sulfonation of Polysaccharides on Antiproliferative Activity

The effect of N- and O-linked sulfo groups on glucosamine and uronic acid sugar residues in heparin on antiproliferative activity has been studied in several laboratories. Tiozzo and coworkers (54) modified heparin to produce N-desulfo and Odesulfo heparin derivatives and found that the 2-O-sulfo group in heparin was important for antiproliferative properties (54). Wright and coworkers (53), on the other hand, reported that the 2-O-sulfo group in heparin was not essential for antiproliferative activity. To clarify the role of N- and O-sulfo groups for PASMC growth inhibition, we prepared fully O-sulfonated heparin, heparan, chondroitin sulfate, dermatan sulfate, and hyaluronan using sulfur trioxide (Fig. 7) (101,102). N-sulfonated acharan sulfate was also prepared by N-deacetylation and N-sulfonation of acharan sulfate (Fig. 7) (103,104). All these derivatives were analyzed for PASMC antiproliferative activity (Fig. 8) (105). 1. Heparin and Heparan Sulfate

Fully sulfonated heparin (Fig. 7) did not produce any greater growth inhibition of PASMC than did the parent heparin, indicating that native heparin already has

388

Figure 7 cans.

Garg et al.

Major and variable sequences of original and fully sulfated glycosaminogly-


389

Figure 8 Effect of various polysaccharides on bovine pulmonary artery smooth muscle cells grown in media containing 10% fetal calf serum and either original (solid bars) or fully sulfonated glycosaminoglycan (striped bars) or acharan sulfate (solid bar) or N-sulfoacharan sulfate (striped bar). Samples are standardized to growth in media containing 10% serum without polysaccharide. ⫹ represents a significant reduction in cell growth as compared to the standard (p ⬍ 0.05); ⫹⫹ represents a significant increase in cell growth as compared to the standard (p ⬍ 0.05); a represents a significant reduction in cell growth as compared to standard ( p ⬎ 0.05); b represents a significant reduction in cell growth as compared to both the standard and the native polysaccharide ( p ⬎ 0.05); c represents a significant reduction in cell growth as compared to native acharan sulfate ( p ⬎ 0.05), but not to the standard (from reference 105, with permission).

the full complement of N- and O-sulfo groups necessary to produce maximum antiproliferative activity (Fig. 8) (105). However, fully sulfonated heparan sulfate containing both N-acetyl and N-sulfo substituents suppressed the growth of PASMC to a greater degree than the parent heparan sulfate, showing that heparan sulfate has insufficient O-sulfo groups for full antiproliferative potency. 2. Hyaluronan

The antiproliferative activity of native hyaluronan (Fig. 8) became strikingly significant after sulfonation and equaled that of native heparin (105). Since hyaluronan, like chondroitin and dermatan sulfates, has a 1→3 linkage between uronic

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acid and hexosamine residues, whereas heparin and heparan sulfate have a 1→4 linkage, the linkages between its uronic acid and hexosamine residues (Fig. 7) do not seem to be critical for antiproliferative activity. 3.

Acharan Sulfate

Neither acharan sulfate nor N-sulfoacharan sulfate had any antiproliferative activity. This result demonstrates that the presence of O-sulfo and N-sulfo groups on a glycosamine-iduronate backbone (Fig. 7) alone is insufficient to produce an antiproliferative activity (105). Indeed, the spatial positioning of these sulfo groups appears to play a major role in activity. 4.

Chondroitin and Dermatan Sulfates

Full sulfonation of chondroitin and dermatan sulfates (Fig. 7) reversed their proliferative effect on PASMC and produced an antiproliferative effect similar to heparin (Fig. 8) (105). Both chondroitin and dermatan sulfate have a variable sequence with a low content of O-sulfo groups in their hexosamine residues. These data suggest that the presence of an enhanced level of O-sulfo groups on both hexosamine and uronate residues is necessary for antiproliferative activity. Since both chondroitin and dermatan sulfate GAGs contain N-acetylgalactosamine residues only, the above data also suggest that N-sulfoglucosamine residues in heparin can be replaced by a N-acetylgalactosamine residue. Further, since in chondroitin and dermatan sulfates all the hexosamine sugar residues contain N-acetyl groups, N-sulfo-substituted basic sugar residues appear not to be critical for antiproliferative activity. F. Anticoagulant Activity

The anticoagulant activity of fully sulfonated heparin was significantly reduced in comparison to native heparin’s anticoagulant activity. All the other sulfonated GAGs showed very little anticoagulation activity (105). This demonstrates that the structural determinants of heparin for its anticoagulant and antiproliferative activities are unrelated. G. Minimum Oligosaccharide Size Requirement of Heparin GAG Chain

Previous studies on the size requirement for heparin’s antiproliferative effect have shown that a tetrasaccharide was inactive. The smallest-size oligosaccharide that had some activity was a pentasaccharide (51). The dodecadisaccharide had the same antiproliferative activity as native heparin (53). We have found that a heparin oligosaccharide containing seven residues each of glucosamine and uronic


391

acid, i.e., a tetradecasaccharide (14-mer), is the minimum size oligosaccharide that is essential for antiproliferative potency as of native heparin (106). These differences of size requirement may be due to different cell types.

VII. Summary and Conclusion In summary, the preceding studies on the effects of heparin and its derivatives on PASMC antiproliferative properties show: 1. 3-O-sulfo group substitution of glucosamine residues is not critical in whole heparin for antiproliferative activity. 2. Molecular weight of a given heparin (over the range examined) does not effect its potency as an antiproliferative agent. 3. Antiproliferative properties of heparin reside in the GAG chain, and not in the core protein. 4. A certain number of O-sulfo groups of heparin is essential for the full antiproliferative effect of heparin. 5. N-sulfo group on hexosamine residues is not critical for antiproliferative activity. 6. Hexosamine residues can be either glucosamine or galactosamine. 7. The anomeric linkage between uronic acid and hexosamine residues is not critical for antiproliferative activity. 8. Commercially available heparins have a varying degree of antiproliferative activity on PASMC. 9. Antiproliferative and anticoagulant activities reside in different domains of heparin. 10. The minimal oligosaccharide size requirements for antiproliferative activity is 14-mer. The biosynthesis of heparin chains is initiated by the formation of N-acetylheparosan, a (→4) β-d-GlcA (1→4) α-d-GlcNAc (1→) n polymer (Fig. 2) that is subsequently modified. The stepwise modification reactions are generally incomplete, in the sense that only a fraction of the potential substrate residues are utilized at each step. These processes therefore lead to sequence heterogeneity of heparin (Fig. 4). Functional properties of heparin and other proteoglycans depend heavily on their ability to bind receptors. Heparin binds with receptors in a specific manner. By virtue of this property, heparin possesses different types of biological activities which derive from different structural domains of the molecule. An increase in antiproliferative activity of fully sulfonated heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronan shows a potential for the development of one of these derivatives as a therapeutic agent for treatment of vascular remodeling in the near future.

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Garg et al. Acknowledgment

This chapter is based on an invited review article published by us in Am J Physiol Lung Cell Mol Physiol (49) with permission.

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AUTHOR INDEX

Italic numbers give the page on which the complete reference is listed.

A Abbaszade I, 8, 17 Abbey DE, 242, 260 Abdel Wahab N, 307, 321 Abdelaziz MM, 326, 332 Aberdam D, 361, 370 Abraham WM, 91, 106, 108, 117, 123, 364, 375, 376 Abrahamson DR, 324, 329 Abramson O, 361, 371 Abuelo D, 307, 321 Achur RN, 176, 187 Adachi M, 75, 95 Adachi Y, 259, 267 Adams DH, 86, 104, 340, 348 Adams DO, 82, 101 Adams JL, 362, 373 Adams SP, 364, 375 Adamson IY, 25, 26, 32, 117, 130 Addicks K, 11, 20 Adelberg S, 120, 132 Adelmann-Grill BC, 361, 370 Adelroth E, 220, 228, 237, 240 Adler KB, 195, 208 Adzick NS, 27, 33, 80, 81, 100 Afford SC, 325, 331

Ahlen K, 362, 372 Ahlstrom C, 305, 318 Ahmed A, 364, 375 Ahrens T, 30, 34, 79, 88, 99, 117, 129 Aiello VD, 47, 53 Aitken K, 116, 120, 129 Akatsuka M, 75, 82, 93 Akesson A, 181, 189 Akhurst JR, 180, 189 Akiyama SK, 201, 210, 243, 253, 261, 266, 303, 316 Aklhalil A, 176, 187 Al Jamal R, 55, 58, 59, 60, 61, 64, 66, 67, 69, 71, 340, 348 Alaish SM, 81, 100 Alam CA, 108, 123 Albert RK, 357, 368 Albertini R, 147, 156, 158, 165, 167, 305, 319 Alder S, 378, 393 Alessandrini A, 40, 50, 52 Alitalo T, 214, 230 Allan DA, 344, 350 Allard JD, 86, 103 Allen A, 269, 286 Allen CL, 206, 212 Allen JD, 377, 393

399

400 Allen SJ, 23, 24, 31 Alliel PM, 299, 311 Almeida M, 355, 356, 366 Alon R, 253, 266, 357, 368 Alpers CE, 41, 48, 52, 295, 309 Altraja A, 220, 237 Amanuma K, 84, 102 Amara FM, 80, 100 Amento E, 159, 167 Amuro Y, 75, 95 Anand SS, 378, 394 Ananyeva NM, 114, 127 Andeoli T, 378, 395 Anderson JAM, 379, 395 Anderson K, 284, 289 Anderson MP, 283, 288 Ando T, 75, 82, 93 Andreasen PA, 218, 234 Andres JL, 299, 312 Andrew M, 174, 185 Angel P, 218, 234 Angello JC, 111, 126, 201, 209, 303, 306, 317, 320 Anglani F, 174, 186 Annila A, 305, 319 Anselmetti D, 50, 53 Anthonisen N, 242, 260 Anthony DC, 180, 189, 363, 374 Antonini A, 356, 366 Antonsson P, 10, 19, 214, 229, 258, 267 Aoki T, 79, 91, 99 Arai H, 244, 262, 280, 288 Arai M, 307, 321 Arap W, 299, 312 Arata J, 9, 18 Arfsten A, 352, 365 Aries SP, 324, 330 Arikava-Hirasawa E, 11, 12, 20 Aritomi T, 110, 126 Arizpe H, 82, 101, 120, 132 Arkonac BM, 78, 97, 299, 312 Armand G, 91, 93, 106, 108, 123, 145, 164 Armaud G, 339, 348 Armstrong RM, 75, 93, 295, 311

Author Index Arner E, 8, 17 Arnold F, 27, 30, 33, 83, 102, 306, 320 Arnold K, 305, 318 Arnott S, 336, 346 Arroyo-Yanguas Y, 174, 186 Artis DR, 364, 376 Aruffo A, 77, 78, 97 Arvilommi AM, 12, 21 Asa D, 254, 266 Asai K, 377, 392 Asakura S, 174, 185 Asch A, 363, 374 Aschauer H, 27, 33 Ashizawa N, 355, 362, 366, 372 Ashkar SS, 342, 349, 361, 370 Asman I, 137, 138, 141 Aspberg A, 171, 183, 200, 202, 209, 210, 213, 228 Asplund T, 75, 95 Assman V, 79, 98, 99, 116, 117, 129 Assoian RK, 86, 104, 218, 233, 355, 366 Asundi V, 295, 310 Asundi VK, 214, 229, 300, 314 Asuwa N, 75, 94 Aszodi A, 10, 11, 19, 20, 173, 184 Ateeq HS, 364, 376 Atha DH, 377, 392 Attisano L, 14, 21 Au YPT, 385, 397 Aubert JD, 223, 238 Aubin JE, 359, 369 Auf der Meyer HA, 357, 368 Auger F, 244, 262 August EM, 75, 95 August JT, 41, 50, 52 Augustine Calabro AJ, 76, 96 Augustine ML, 24, 28, 31, 32, 110, 126 Austen KF, 15, 21 Austen L, 75, 76, 79, 93, 99 Austin J, 8, 17 Autio I, 377, 392 Aviezer D, 306, 320 Avioli LV, 175, 187 Aviram M, 305, 319 Avraham S, 15, 21

Author Index

401

Axelsson I, 295, 310 Azumi N, 41, 50, 52, 137, 141

B Babyak MA, 254, 266 Bachhuber BG, 205, 212 Bachofen H, 57, 66, 69, 72, 144, 164 Bachrach NM, 63, 71 Backer V, 221, 237, 238 Backstrom G, 381, 395 Badimon JJ, 307, 320 Baggio B, 174, 186 Bagli DJ, 75, 76, 79, 80, 81, 93, 113, 116, 120, 126, 129 Bai H, 307, 321 Bai S, 359, 370 Bai TR, 200, 209, 221, 223, 237, 238 Baird A, 252, 266 Bajorath J, 15, 21, 78, 97 Baker CC, 224, 238 Baker JR, 338, 347 Baldi A, 173, 174, 185, 304, 317 Ballantyne CM, 357, 368 Balmain A, 180, 189 Balmes J, 220, 236 Balsamo J, 202, 210 Balzar S, 226, 239 Banderschueren B, 41, 50, 52 Bandtlow CE, 202, 210 Banerjee SD, 113, 127 Banerji S, 114, 127 Banhart RL, 385, 397 Bansal V, 363, 375 Bansil R, 269, 285, 286 Banyard J, 75, 94 Bao C, 27, 30, 31, 33, 35, 75, 82, 93, 108, 120, 122, 124, 125, 132 Bar-Shavit R, 357, 368 Barabasi AL, 57, 70 Barbanti M, 378, 395 Barbee RA, 221, 237 Barbero-Marcial M, 47, 53 Barcellos-Hoff MH, 86, 104 Barchan K, 174, 185

Bardelli A, 115, 128 Baribault H, 10, 18, 172, 184, 216, 232, 247, 265, 300, 315 Baris YI, 136, 140 Barnes MJ, 352, 365 Barquin N, 359, 369 Barral D, 12, 20 Barret C, 114, 127 Barrett BH, 304, 318 Barritault D, 174, 186 Barritt D, 304, 317 Barry FP, 8, 18, 292, 308 Bartolazzi A, 78, 97 Bartold PM, 75, 94, 336, 345 Barton RW, 118, 120, 131 Basbaum CB, 270, 277, 286, 287, 336, 346 Bashey RI, 108, 123 Bashkin P, 377, 393 Basset F, 201, 209 Basset G, 162, 167 Bassols A, 218, 234, 243, 261 Basson CT, 307, 321 Bast RC, 246, 263 Bateman J, 172, 184, 216, 232 Bates JH, 66, 72 Bates ME, 364, 375 Batey R, 244, 262 Battifora H, 136, 140 Bauer M, 172, 184, 356, 357, 367, 368 Bauvois B, 361, 370 Baybayan P, 14, 21 Bayless KJ, 355, 366 Baylin SB, 41, 50, 52 Bayliss LE, 57, 69 Bayliss MT, 5, 9, 17, 18, 171, 183, 213, 228 Baynes JW, 108, 124 Beales MP, 10, 19, 172, 184 Beasley R, 220, 236 Beaudet A, 283, 288 Beaudet AL, 357, 368 Beaumont B, 295, 309 Beavan L, 216, 232, 248, 265 Becerril C, 247, 264

402 Becherer JD, 246, 263 Beck M, 214, 230 Beck-Schimmer B, 27, 31, 33, 109, 125 Becker AE, 50, 53 Beckett P, 246, 263 Becklake MR, 242, 260 Beckman IG, 79, 98 Bedossa P, 204, 211 Beebe DCX, 83, 102 Beeler DL, 378, 395 Begib R, 340, 349 Begleiter A, 87, 105 Beighton P, 12, 20 Bel EH, 228, 240 Belch AR, 80, 100, 115, 117, 118, 128, 129 Bell CE, 300, 313 Bell JI, 77, 78, 97 Bell MV, 77, 97 Bellia V, 224, 226, 239 Beloucif S, 363, 374 Belting M, 300, 313, 337, 338, 347 Beltran L, 327, 332 Bendall L, 200, 209 Bengali ZH, 241, 260 Benitz WE, 382, 396 Benot FP, 340, 348 Bensadoun ES, 27, 32, 41, 52, 56, 61, 69, 70, 108, 123, 178, 188, 196, 197, 201, 202, 208, 222, 238, 326, 331 Benstrup KE, 344, 350 Bentz H, 224, 239, 295, 311 Beran M, 300, 314 Berditchevski F, 355, 362, 365, 373 Berenson G, 336, 346 Berenson GS, 304, 318 Bereznay O, 86, 105, 219, 235 Bereznay OH, 203, 204, 211 Berg RA, 258, 267 Bergenfeldt M, 357, 369 Berger JT, 65, 72, 282, 288 Berger M, 326, 332 Bergh J, 26, 32, 87, 105, 113, 118, 120, 127, 131 Berisini M, 364, 376 Berman B, 304, 317

Author Index Bernaudin JF, 324, 330 Bernfield M, 13, 20, 163, 168, 169, 171, 183, 253, 266, 294, 300, 305, 309, 314, 319, 340, 349, 378, 382, 394, 396 Berry L, 174, 185 Bertin K, 151, 153, 166 Bertolami CN, 78, 81, 98, 100 Bertolesi GE, 378, 393 Bertolotto A, 284, 289 Berton G, 363, 374 Bertorelli G, 177, 188, 221, 228, 237, 240 Bertrand P, 84, 103 Betail G, 220, 236 Bhaskar KR, 269, 270, 271, 274, 278, 281, 284, 285, 286, 287, 288, 289 Bhavanadan VP, 344, 350 Bianchini P, 386, 397 Bianco P, 10, 19, 171, 172, 183, 184, 221, 238, 258, 267, 295, 309 Bidanset DJ, 217, 233, 303, 316 Bierhaus A, 88, 105, 119, 120, 132 Biesterfeldt J, 24, 28, 32, 76, 96, 110, 126 Bieth JG, 324, 330 Bihl M, 343, 350, 361, 371 Bilton D, 323, 329 Bin Z, 215, 230, 327, 333 Bini A, 25, 26, 32 Binns RM, 88, 105, 119, 120, 132 Birk DE, 172, 184 Birkedal-Hansen H, 246, 263 Birkenmeier T, 359, 369 Birkenmeier TM, 243, 261 Birkhead JR, 306, 320 Birnby LM, 340, 349 Bisacia F, 108, 124 Bischoff J, 305, 319 Bishop PN, 335, 345 Bisignani GJ, 81, 101 Bissell DM, 362, 373 Bitterman P, 117, 130 Bitterman PB, 120, 132 Bittner K, 338, 347 Bizzarri C, 363, 374

Author Index Bjermer L, 85, 103, 117, 130, 281, 288 Black JL, 113, 127 Black WC, 191, 207 Blackburn B, 364, 376 Blaschke E, 145, 164 Blasi F, 218, 234 Blessing E, 248, 265, 295, 309 Blieden T, 243, 244, 261, 262 Blieden TM, 244, 262 Blijham GH, 306, 320 Bliven ML, 64, 71 Bloch W, 11, 20 Blochberger TC, 295, 310 Block LH, 75, 94, 109, 125, 180, 189, 342, 343, 349, 350 Blomgren B, 176, 187 Bloomsfield F, 377, 393 Boat AC, 282, 288 Boat TF, 282, 283, 288 Boatman ES, 61, 70 Bochaton-Piallat ML, 361, 371 Bochnowicz S, 362, 373 Bockus DE, 49, 53 Boddeke HG, 337, 346 Boden SD, 63, 71 Boehm T, 306, 320 Boerwinkle E, 307, 321 Boittin M, 218, 234 Boivin GP, 217, 233 Boldt J, 194, 208 Bolender RP, 47, 53 Boles BK, 359, 369 Bonadio J, 10, 19, 172, 184, 258, 267 Bondjers G, 305, 318, 319 Bonner JC, 248, 265 Bonnet FJ, 299, 311 Bonsignore G, 224, 226, 239 Bonventre JP, 385, 397 Borchers MT, 357, 368 Border WA, 10, 19, 172, 173, 180, 184, 185, 218, 219, 235, 251, 252, 265, 266, 335, 345 Bordin L, 174, 186 Boren J, 305, 318 Borg TK, 362, 373 Bornstein P, 218, 234

403 Borrios R, 147, 159, 166 Borsi L, 361, 371 Bosi F, 151, 153, 166 Bosken CH, 221, 237 Boskey A, 10, 19, 172, 184, 258, 267 Bost F, 3, 16 Botinnikova Y, 364, 375 Bottomley GS, 295, 310 Bouchard T, 299, 312 Boucher RC, 283, 288 Boucher Y, 306, 320, 356, 367 Bouck N, 217, 233 Boudier C, 324, 330 Boudreau N, 75, 94 Boulet LP, 177, 181, 188, 220, 221, 236, 237, 244, 262 Bourdon MA, 202, 210, 295, 309 Bourdoulous S, 200, 209 Bourguignon GJ, 114, 127 Bourguignon LY, 114, 127 Bourguignon LYW, 78, 97, 114, 128 Bourin MC, 337, 347, 378, 393 Bousquet J, 224, 226, 239 Boutet M, 177, 188, 220, 221, 236, 237, 244, 262 Boutin C, 135, 138, 140, 142 Bowden DH, 25, 26, 32, 117, 130 Bowe MA, 173, 185 Bowen WR, 50, 53 Bowie AG, 30, 31, 35 Boxer LA, 363, 374 Boyce DE, 120, 133 Boyce ST, 344, 350 Boyer CM, 246, 263 Boyle J, 75, 94 Boynow JA, 282, 288 Bradding P, 364, 376 Bradley JD, 269, 285 Brahimi-Horn MC, 277, 287 Bramley AM, 200, 208 Brandan E, 218, 228, 235, 240 Brandstetter RD, 382, 396 Brandt E, 326, 332 Brandt R, 137, 138, 141 Brant RF, 344, 350 Branton MH, 361, 370

404 Brass DM, 205, 212 Brauker JH, 163, 168 Braun J, 324, 330 Braun K, 300, 315 Braun M, 204, 212 Braun RK, 87, 105, 173, 180, 185, 219, 236, 295, 309, 359, 369 Braunwald E, 82, 102 Bray BA, 26, 32, 56, 69, 87, 105, 108, 110, 111, 113, 118, 123 Breen EC, 65, 72 Breenberg AH, 326, 331 Brees DK, 10, 19, 173, 185, 219, 235 Brehm-Gibson T, 24, 28, 32, 76, 96, 110, 126 Brennan FR, 29, 34 Brennan JE, 224, 239 Brennan MJ, 214, 229 Bretscher A, 114, 127 Bretschneider E, 204, 212 Breuer R, 181, 190 Breuss JM, 359, 369 Brew K, 159, 167 Brewster CEP, 204, 212 Briggs DM, 174, 186 Briggs WH, 175, 187 Brigham KL, 160, 167 Brinck J, 76, 96, 110, 113, 126, 127 Bringas P, 147, 165, 219, 236 Briol A, 115, 128 Briskin MJ, 76, 96, 110, 126 Brisse J, 225, 226, 239 Brochard L, 147, 159, 166 Brody AR, 205, 212, 248, 265 Brody BR, 118, 131 Brody JS, 61, 71, 146, 147, 165 Broekelmann T, 173, 185, 215, 231 Broekelmann TJ, 202, 203, 211, 244, 262 Brooks B, 174, 186 Brosnan JT, 336, 345 Broszat M, 218, 235 Brouwer A, 362, 373 Brown CB, 115, 118, 128 Brown EL, 176, 177, 187 Brown JC, 356, 367

Author Index Brown JR, 108, 123 Brown KE, 384, 397 Brown LA, 118, 131, 202, 210, 357, 369 Brown LJ, 361, 372 Brown LV, 113, 126 Brown MJ, 162, 168 Brown N, 28, 33 Brown R, 57, 70, 281, 288 Brown SJ, 171, 183 Brown TA, 299, 312 Brownfield CM, 363, 374 Bruce MC, 246, 263 Bruckner P, 218, 235 Bruckner-Tuderman L, 341, 349 Brun J, 220, 236 Brun-Buisson C, 147, 159, 166 Brunn GJ, 11, 20 Bruystens AM, 300, 313 Bryan-Rhadfi J, 181, 190 Buchberg AM, 214, 229 Bucht SA, 137, 138, 141 Buczek-Thomas JA, 181, 189 Buddecke E, 214, 230 Buee L, 307, 321 Buermann CW, 327, 333 Bull HB, 57, 70 Bullard D, 357, 368 Bullard KM, 81, 100 Burdick MD, 27, 30, 31, 33, 35, 75, 82, 93, 108, 120, 125, 132 Burg MA, 299, 304, 312, 317, 356, 367 Burke AK, 27, 32, 41, 52, 56, 61, 69, 70, 108, 123, 178, 188, 196, 197, 201, 202, 208, 222, 238, 326, 331 Burke-Gaffney A, 364, 376 Burn T, 8, 17 Burnett D, 86, 104, 246, 263, 324, 325, 330, 331 Burnier JP, 364, 376 Burri PH, 85, 103 Burridge K, 353, 356, 361, 365, 366, 372 Burrows B, 242, 260 Busch SJ, 2, 16, 335, 345, 385, 397 Buschmann MD, 63, 71

Author Index

405

Bush HL, 299, 312 Buskens C, 146, 165 Buskens CA, 176, 188 Buskens CAF, 11, 20 Busse WW, 364, 375, 376 Busutti RW, 361, 371 Butler G, 284, 289 Butler GS, 200, 209 Butler WT, 359, 369 Buxbaum RE, 68, 72 Byers HR, 78, 98

C Cabrera RC, 81, 100 Cadene M, 324, 330 Cahalon L, 253, 266 Cahill RNP, 136, 140 Calabretta B, 173, 174, 185 Calabro A, 24, 28, 32, 110, 126 Calandra S, 378, 386, 395, 397 Calastrini C, 339, 348 Calderon M, 228, 240, 326, 332 Caleb BJ, 385, 397 Camejo G, 304, 305, 317, 318 Camenisch I, 340, 348 Camenisch TD, 30, 34, 76, 96, 110, 126 Cameron L, 220, 237 Camp RL, 29, 34, 78, 97 Campbell AM, 352, 365 Campbell EJ, 325, 331 Campbell MH, 176, 188 Camper SA, 79, 99 Campisi J, 385, 397 Canemisch TD, 24, 28, 32 Caniggia I, 64, 71, 175, 177, 187 Cannizzaro LA, 9, 18, 214, 215, 230, 307, 321 Canny G, 324, 330 Canonico A, 160, 167 Cantin AM, 194, 208 Cantlay AM, 255, 267 Cantor H, 342, 349, 361, 370 Cantor JO, 91, 92, 93, 106, 108, 123, 145, 164, 339, 348 Cao A, 14, 21

Cao G, 79, 84, 99, 114, 115, 117, 118, 127 Cao L, 201, 210 Cao X, 359, 370 Capeau J, 277, 287 Capehart AA, 28, 34, 201, 210 Capola I, 387, 389, 390, 391, 398 Cappelli C, 148, 152, 166 Cardell BS, 220, 236 Cardin AD, 2, 16, 335, 345, 385, 397 Cardoso WV, 181, 189, 244, 262 Carey DJ, 300, 301, 313, 314, 315 Carlin S, 113, 127 Carlson DM, 387, 397 Carlstedt I, 269, 278, 286, 288 Carraway MS, 280, 288 Carre PC, 120, 132 Carroll H, 11, 19 Carroll JN, 221, 237 Carter BJ, 378, 393 Carter WG, 299, 312 Cartier A, 177, 188, 221, 237 Carver W, 362, 373 Cary LA, 353, 354, 365 Cary NR, 295, 310 Casalini A, 228, 240 Casaroli-Marano RP, 357, 368 Cass D, 357, 369 Cass DL, 80, 100 Cassiman JJ, 243, 261, 300, 313, 314 Castel S, 357, 368 Castellani P, 361, 371 Castellot JJ, 378, 384, 385, 394, 395, 396, 397 Castile RG, 57, 70 Castro AC, 364, 376 Casu B, 378, 393 Caterson B, 8, 17, 327, 333, 336, 345 Cattini PA, 116, 129 Cattolico L, 12, 20 Catzavelos C, 29, 34, 79, 98 Cauberghs M, 66, 67, 72 Cavari S, 378, 393 Cavazza A, 136, 140 Cawley JC, 115, 118, 128 Cawston T, 327, 332

406 Celli BR, 61, 70 Ceol M, 174, 186 Cepinskas G, 324, 329 Cerreta JM, 91, 93, 106, 108, 123, 145, 164, 339, 348 Cetta G, 172, 184, 216, 232 Ceulemans H, 300, 313 Chada K, 258, 267 Chaffee E, 136, 141 Chahinian AP, 137, 138, 139, 141, 142 Chaisson ML, 356, 366 Chait A, 304, 318 Chakir J, 220, 236, 244, 262 Chakravarti S, 11, 19, 20, 172, 184, 361, 370 Chakrin LW, 278, 288 Chamba A, 325, 331 Chambers CB, 344, 350 Chambers RC, 181, 190, 259, 267 Chamlian A, 225, 226, 239 Champion B, 246, 263 Chan BM, 362, 373 Chan KN, 326, 332, 364, 376 Chan SCH, 324, 330 Chandler DB, 86, 103 Chanez P, 224, 226, 239 Chang IM, 387, 398 Chang JF, 353, 354, 365 Chang KH, 352, 365 Chang MC, 116, 129 Chang RJ, 295, 311 Chantler E, 269, 285 Chapman HA, 206, 212, 258, 267 Charo IF, 352, 365 Chartrain N, 246, 264 Cheifetz S, 218, 233, 299, 312 Chellaiah M, 362, 372 Chen ES, 247, 264 Chen EY, 14, 21 Chen H, 203, 211, 307, 321 Chen J, 355, 366 Chen JK, 84, 102 Chen LL, 364, 376 Chen Q, 353, 365 Chen WT, 243, 261 Chen WY, 80, 100

Author Index Chen X, 114, 127 Chen XC, 4, 16 Chen XN, 10, 19, 176, 188 Chen YQ, 175, 187, 215, 230, 244, 262 Chen YW, 114, 128 Cheng F, 174, 186, 214, 229 Cheng GC, 362, 373 Cheng JF, 6, 17 Cheng PW, 282, 283, 288 Cheng SH, 283, 288 Cheng SL, 175, 187 Cheresh DA, 355, 362, 366, 372 Chermousov M, 300, 314 Cherr GN, 27, 32 Chervoneva I, 172, 184 Chetta A, 177, 188, 221, 228, 237, 240 Cheung HK, 355, 366 Cheung WF, 115, 128 Chevalier S, 299, 312 Chi EY, 357, 368 Chi-Rosso G, 362, 373 Chiang L, 117, 130 Chiappara G, 224, 226, 239 Chida K, 244, 262 Chihara J, 364, 375 Chin BY, 27, 31, 33, 75, 82, 93, 108, 124 Chin JE, 364, 375 Chin MT, 78, 98, 299, 312 Chiquet-Ehrismann R, 202, 210, 218, 234 Chisholm RA, 118, 131 Chiu ES, 81, 100 Cho HG, 362, 373 Cho HJ, 217, 232 Cho O, 362, 373 Cho SH, 221, 237 Cho SK, 327, 332 Cho YJ, 221, 237 Choay J, 378, 394, 395 Choi AM, 27, 31, 33, 108, 122, 124 Choi AMK, 75, 82, 93 Choi DC, 221, 237 Choi HU, 9, 18, 214, 217, 229, 233, 301, 306, 316, 319

Author Index Choi JO, 223, 238 Chojkier M, 204, 211 Chollet-Martin S, 363, 374 Chong LT, 364, 376 Chopra RK, 214, 229 Chou P, 359, 369 Christiansen SC, 91, 106 Christie PE, 357, 368 Christina H, 340, 348 Christodoulou M, 246, 263 Christofferson R, 83, 84, 102 Chrzanowska-Wodnicka M, 361, 372 Chu CS, 324, 330 Chu HW, 220, 226, 237, 239 Chu SJ, 357, 368 Chui H, 364, 376 Chun LE, 55, 58, 69 Chun MW, 387, 398 Chung JJ, 108, 125 Chung K, 78, 98 Chung KF, 364, 375 Church FC, 174, 185, 301, 316 Churchill M, 246, 263 Churg A, 258, 267 Ciccolella DE, 181, 190 Cichy J, 324, 330 Cidre LL, 378, 393 Cindhuchao N, 391, 398 Cingi MR, 378, 386, 395, 397 Cippitelli M, 361, 371 Cisar LA, 377, 392 Cissel DS, 224, 238 Ciura S, 47, 53 Civitelli R, 363, 374 Cizmeci-Smith G, 300, 301, 314, 315 Claesson-Welsh L, 83, 84, 102 Clancy R, 244, 262, 323, 329 Clark AF, 342, 349 Clark CH, 336, 346 Clark K, 364, 376 Clark RA, 355, 361, 362, 366, 371, 372, 373 Clark-Lewis I, 200, 209, 246, 263 Clary C, 75, 76, 93 Clasper S, 114, 127 Clausell N, 75, 94

407 Clay D, 84, 103 Clements J, 246, 263 Clemmons DR, 356, 366 Clezardin P, 217, 233, 356, 357, 367 Cliff WJ, 204, 211 Clintron C, 217, 232 Cloutier JM, 246, 264 Clowes A, 300, 315 Clowes AW, 47, 53, 202, 211, 303, 307, 316, 321, 327, 332, 378, 385, 393, 397 Clowes MM, 307, 321 Coakley RJ, 324, 330 Cocharane CG, 91, 106 Cochran DP, 90, 106 Cockwell P, 340, 348 Coers W, 66, 67, 72, 181, 189, 248, 251, 255, 265, 267, 326, 332 Cohen I, 9, 11, 18, 20 Cohen IK, 81, 100, 214, 215, 230 Cohen IR, 11, 20, 378, 393 Cohen RM, 307, 321 Coito AJ, 361, 371 Coker RJ, 340, 349 Coker RK, 223, 238 Colby TV, 136, 140, 202, 203, 211 Cole GJ, 49, 53 Cole PJ, 323, 329 Coleman JW, 174, 186 Coles SJ, 274, 287 Collins JV, 220, 236 Collis L, 115, 118, 128 Cologlio PM, 77, 97, 115, 128 Comper WD, 136, 140 Condic ML, 362, 372 Congiu T, 40, 50, 52 Connell MG, 344, 350 Conner GE, 79, 91, 99, 106 Conner KJ, 300, 314 Connolly KM, 246, 263 Conrad GW, 171, 175, 184, 187, 295, 310, 311, 339, 348 Consigli S, 299, 312 Converse RL, 11, 19 Conway JG, 246, 263 Cook HT, 224, 239

408 Coomans C, 300, 313 Cooney CL, 338, 347 Cooper DL, 15, 21 Cooper DM, 220, 236 Cooper JA, 118, 120, 130 Cooper MD, 284, 289 Copp AJ, 303, 316 Corbi AL, 352, 365 Cornelis FB, 77, 97 Cornuet PK, 4, 16, 214, 229, 295, 310 Corpuz LM, 295, 310 Corpuz M, 295, 311 Corry D, 357, 369 Corson JM, 136, 138, 141, 142 Cosio MG, 242, 261 Cosnigli GF, 221, 237 Costabel U, 117, 130 Costanzo MR, 295, 310 Costell M, 11, 20 Coster L, 181, 189, 344, 350 Cottell DC, 324, 329 Couch L, 244, 262 Couchman JR, 11, 12, 13, 20, 21, 79, 98, 146, 165, 248, 265, 300, 303, 313, 316, 338, 347, 356, 367, 377, 393 Coulden RA, 323, 329 Coulet M, 220, 236 Coulson AH, 242, 260 Coultas DB, 191, 207 Coulter SN, 6, 17 Courty J, 174, 186 Cowan K, 295, 300, 310, 314 Cowan KN, 360, 370 Cowman M, 27, 31, 33, 75, 82, 93, 120, 132 Cowman MK, 91, 106 Cox G, 120, 133, 340, 348 Coxson HO, 251, 265 Coyne E, 336, 346 Crainie M, 80, 100 Cranfill K, 283, 288 Crausman RS, 194, 207 Crawford HC, 246, 263 Crawford SE, 217, 233 Creeth JM, 269, 286

Author Index Crescenzi M, 356, 366 Crippes Trask B, 215, 231 Cripps A, 244, 262 Cripps V, 79, 99 Crombleholme TM, 81, 100 Crone C, 162, 167 Crosby J, 357, 368 Croteau D, 41, 50, 52 Crouch E, 26, 32, 178, 188, 194, 208, 244, 262, 340, 348 Crouch EC, 146, 147, 165 Cruaud C, 12, 20 Cruz TF, 115, 128 Crystal RG, 120, 132, 147, 158, 159, 165, 167, 192, 194, 202, 203, 207, 208, 211, 324, 330 Cs-Szabo’ G, 10, 19 Csaky KG, 180, 189, 227, 239, 340, 349 Cuff CA, 114, 127 Culav EM, 336, 346 Cullen B, 361, 372 Cully Z, 221, 238 Culp LA, 217, 233, 306, 319, 377, 392 Culty M, 24, 31, 85, 103, 119, 132 Cupp M, 377, 392 Curpen GD, 108, 116, 124 Currie DC, 323, 329 Cutroneo KR, 86, 104 Cutz E, 220, 236 Cybulsky AV, 356, 366 Cyr MD, 356, 366 Cysyk RL, 75, 95

D D’Amato DA, 118, 120, 131 D’Arcy EM, 147, 158, 165, 247, 264 D’Armiento J, 247, 258, 264, 267 D’Ortho M, 147, 159, 166 Dahl IMS, 138, 139, 141, 142 Dahlberg CGW, 385, 397 Dahlin KJ, 299, 312 Dalal BI, 223, 238 Dalal SS, 247, 258, 264, 267 Dalen KT, 2, 4, 16, 336, 346

Author Index Dalhoff K, 324, 330 Dall P, 79, 99 Dalton M, 362, 372 Dalton SL, 87, 105, 118, 131, 203, 211, 217, 233, 357, 369 Damen CA, 306, 320 Damiani PJ, 81, 100 Dammaer U, 50, 53 Damsky CH, 301, 315, 357, 368 Daniel PF, 273, 287 Danielpour D, 118, 132 Danielson KG, 9, 10, 18, 170, 172, 176, 183, 184, 214, 215, 216, 230, 232, 247, 265, 300, 315 Dano K, 218, 234 Darby I, 205, 212 Das I, 269, 286 Dasch J, 295, 311 Daum GU, 385, 397 David G, 41, 50, 52, 146, 165, 243, 261, 300, 304, 306, 313, 314, 317, 320, 357, 368, 378, 394 Davidson AH, 246, 263 Davidson JM, 218, 234, 244, 262, 336, 345 Davies CL, 306, 320, 356, 367 Davies M, 108, 124, 324, 329 Davies RJ, 228, 240, 278, 288, 326, 332 Davis GE, 355, 366 Davoine CS, 12, 20 Day AA, 214, 229, 278, 287 Day AJ, 76, 78, 96, 97 Day C, 352, 365 De Biasi M, 108, 124 De Boer M, 357, 369 De Cat B, 300, 313 De Luca A, 173, 174, 185, 304, 317 De Luca G, 147, 153, 155, 156, 158, 163, 165, 167, 168, 305, 319, 342, 343, 349, 357, 368 De Luca M, 361, 370 De Marcillac GD, 324, 330 De Mattei M, 339, 348 de Navasquez S, 220, 236 De Silva JL, 323, 329 De Vries E, 364, 376

409 De Waal RMW, 299, 300, 312, 313 Deakin JA, 174, 186 DeAngelis PL, 76, 96, 109, 125 Debruyne H, 174, 186 DeCarlo M, 151, 166 Decock B, 300, 313 Dedhar S, 363, 375 Deed R, 108, 113, 123, 124 DeGrendele HC, 29, 34, 82, 101, 120, 132 Deguine V, 27, 33 Dehio C, 357, 368 Deisenhofer J, 216, 232 Dejmek A, 137, 139, 141 Dejter SW, 214, 229 Dekhuijzen PN, 177, 181, 188, 252, 266 Del Borrello E, 339, 348 Del Donno M, 177, 188, 221, 228, 237, 240 Del Fabbro M, 152, 153, 154, 155, 161, 162, 163, 166, 167, 168, 342, 343, 349, 357, 368 Delacourt C, 147, 159, 166 Delacourte A, 307, 321 Delage J, 220, 236 Delbe J, 174, 186 DelClaux C, 147, 159, 166 DeLisser H, 79, 80, 82, 84, 87, 88, 99, 110, 117, 119, 122, 126 DeLisser HM, 114, 115, 117, 118, 127, 326, 331 Dell A, 387, 397 Delmas P, 217, 233, 356, 367 Delmas PD, 357, 367 Delorme MA, 174, 185 Delpech B, 84, 103, 138, 141 DeMarzo N, 220, 236 Demedts M, 41, 50, 52 Demoor-Fossard M, 215, 218, 230, 234 Dempsey LA, 11, 20 Demsey E, 159, 167 Dencoff JE, 26, 32 Deneffe G, 41, 50, 52 Denfeld RW, 79, 88, 99 Denhardt DT, 361, 370 Denhez F, 306, 320, 356, 367

410 Dent CM, 327, 333 Desmouliere A, 205, 212, 361, 371 Desrochers C, 244, 262 Detels R, 242, 260 Detmar M, 174, 186, 306, 320 Detmers PA, 363, 375 Deudon E, 277, 287 Devalia JL, 228, 240, 326, 332 Dexter JM, 377, 393 Dey CR, 227, 239 Dhand R, 61, 70 Dhillon DP, 323, 329 Di Corleto PE, 205, 212 Di Girolamo N, 246, 263 Di Liberto M, 86, 104 Di Stefano S, 356, 366 Diamond JR, 378, 394 Diamond MS, 357, 368 Diarra-Mehrpour M, 3, 16 DiBattista JA, 243, 262 Dichgans J, 181, 190 Dickinson R, 78, 97 Dickinson RB, 79, 88, 98 Diegelmann RF, 81, 100 Dieterich W, 172, 184, 356, 357, 367, 368 Dietz HC, 307, 321 Dijkman H, 146, 165 Dijkman JH, 324, 330 Dinakarpadian D, 159, 167 Dingemans KP, 50, 53 DiPietro LA, 253, 266 Dirksen A, 221, 237 DiStefano A, 220, 236 Dittmar T, 299, 312 Dixon AK, 118, 131 Djabari Z, 361, 370 Djukanovic R, 204, 212, 226, 239 Do YS, 355, 362, 366, 372 Dobrowoska G, 385, 397 Docherty AJPY, 218, 234 Doctrow S, 377, 393 Dodd CM, 216, 231, 339, 347 Dodge GR, 11, 20, 197, 208 Doege K, 4, 16 Doege KJ, 6, 17

Author Index Doggett NA, 28, 33 Dohlnikoff M, 147, 165 Dohrman A, 277, 287 Doi T, 377, 393 Dolan M, 4, 16 Dolan MC, 176, 177, 187 Dolhnikoff M, 10, 19, 56, 69, 170, 176, 183, 223, 238, 247, 265 Dominguez P, 10, 19, 172, 184, 258, 267 Domowicz M, 339, 348 Doneva T, 50, 53 Dong S, 49, 53 Donovan MJ, 357, 368 Doody J, 180, 189 Doran P, 117, 130 Doring G, 324, 325, 330, 331 Dorschner RA, 174, 186, 306, 320, 336, 346 Dotzlaw H, 175, 187 Doughtery GJ, 15, 21 Dours-Zimmermann MT, 199, 208, 341, 349 Dowan K, 295, 310 Dowd CJ, 338, 347 Downey R, 247, 264 Downward J, 299, 312 Drake CJ, 306, 320 Drazen JM, 221, 237, 270, 284, 286, 289 Dreher KL, 214, 229, 295, 310 Dreshaj I, 57, 70 Dreyfuss D, 64, 72 Drummond AH, 246, 263 Du Bois RM, 223, 238, 299, 312, 324, 330, 340, 349 Du HK, 383, 396 Dubaybo BA, 218, 234 Dube J, 220, 236, 244, 262 Dubey RK, 377, 392 Duchaussoy PI, 378, 394 Duddle JM, 228, 240 Dudhia J, 9, 18 Dufresne A, 340, 349 Dugas M, 177, 188, 221, 237 Duggan M, 281, 288

Author Index

411

Dumitrescu L, 181, 190 Duncan KL, 75, 95 Dunlevy JR, 10, 19, 294, 309 Dunsmore SE, 244, 262, 325, 331, 336, 345 Durand KTH, 194, 207 Durmowicz A, 159, 167 Durr J, 300, 313 Durussel L, 174, 186, 217, 233, 357, 367 Dustin ML, 82, 101 Dvorak HF, 202, 210 Dyne KM, 172, 184, 216, 232 Dzau VJ, 42, 47, 52

E Eastmond NC, 174, 186 Eaton MW, 363, 374 Ebaid M, 47, 53 Ebara S, 6, 17 Ebihara T, 61, 62, 66, 70, 72, 113, 127, 178, 188, 326, 332 Echtermeyer F, 306, 320, 356, 367 Eckes B, 175, 187 Edelman ER, 301, 315, 378, 394 Edelstein C, 304, 318 Edgren G, 300, 313 Edward M, 75, 94 Edwards DR, 218, 234 Edwards IJ, 304, 306, 318, 319 Edwards JE, 47, 53 Eger W, 78, 98 Eguchi G, 295, 309 Ehnis T, 172, 184, 356, 357, 367, 368 Ehrlich BE, 337, 347 Ehrlich HP, 81, 101 Ehualaeshet T, 87, 105 Eichstetter I, 9, 10, 18, 19, 214, 215, 230 Eickelberg O, 341, 343, 349, 350, 361, 371 Eidelman DH, 242, 261 Eidelman Y, 81, 100 Eijan AM, 378, 393 Eisen AZ, 362, 373

Eisentein R, 136, 140 Eisinger M, 306, 320 Ek B, 82, 101 Ekfors T, 295, 310 Eklund A, 85, 90, 103, 145, 164 Ekman G, 174, 185 Elbaz D, 253, 266 Elefteriou F, 173, 179, 184, 215, 231 Elenius K, 299, 306, 311, 320 Elfsberg K, 304, 317 Elias E, 86, 104 Elias JA, 258, 267, 340, 348 Eller J, 323, 329 Ellingsworth LR, 224, 238, 239 Elliot SJ, 377, 393 Elliott G, 344, 350 Elliott J, 221, 237 Elliott M, 220, 236 Ellis DL, 344, 350 Ellis I, 75, 94 Ellis IR, 75, 94 Elmer WA, 63, 71 Emilie D, 224, 239 End P, 248, 265 Endo T, 218, 233 Eng B, 378, 393 Engel J, 248, 265 Engstrom-Laurent A, 85, 103, 117, 130, 136, 140 Enleu GL, 242, 260 Ensadoun A, 377, 392 Entman ML, 357, 368 Entwistle J, 76, 79, 80, 96, 99, 100, 115, 128 Erickson DR, 344, 350 Erickson HP, 202, 210 Ericksson S, 254, 266 Eriksson E, 174, 186, 306, 320, 336, 346 Eriksson L, 75, 85, 95 Eriksson S, 136, 140, 242, 261 Erikstein B, 139, 142 Erle DJ, 357, 361, 369, 371 Erlinger R, 49, 53 Ernst P, 220, 237 Ertl RF, 259, 267

412

Author Index

Esko JD, 4, 16, 304, 317, 377, 393 Eskra JD, 64, 71 Esparza-Lopez J, 14, 21 Esterbauer H, 305, 319 Estess P, 29, 34, 82, 101, 120, 132 Etcubanas E, 118, 131 Ethell IM, 202, 210 Evanko SP, 64, 67, 71, 72, 76, 96, 111, 115, 117, 126, 128, 176, 177, 187, 201, 209, 295, 303, 310, 317 Evans A, 299, 312 Evans DM, 300, 314 Evans JN, 195, 208, 361, 370 Evans RA, 324, 329 Everts KB, 327, 332 Exposito JY, 173, 179, 184, 215, 231 Eyre DR, 55, 58, 69 Ezura Y, 172, 184

F Faassen AE, 79, 98 Fabbri LM, 220, 236, 242, 261 Facchin S, 174, 186 Fackre DS, 214, 229 Fager G, 304, 317 Fairbrother WJ, 364, 376 Faivre V, 363, 374 Fakuchi Y, 359, 370 Falanga V, 86, 104, 218, 233 Falcioni R, 356, 366 Falcone DJ, 299, 312 Falconer C, 176, 187 Falk P, 108, 124 Faller B, 324, 330 Fallon JR, 173, 185 Fallon JT, 359, 369 Fan X, 27, 31, 33 Fanburg BL, 377, 393 Farber C, 363, 374 Farber JM, 30, 31, 35, 108, 125 Fareed J, 336, 346, 387, 398 Farese RV, 357, 369 Farmer S, 357, 368 Farndale RW, 352, 365 Farquhar MG, 146, 165

Fassler R, 10, 11, 19, 20, 173, 184 Fastermann D, 301, 315 Fath MA, 324, 330 Fattah D, 364, 376 Fattal M, 224, 239 Fauci AS, 86, 104, 218, 233 Faure MP, 246, 264 Fausto N, 356, 366 Fawell SE, 362, 373 Fazzio A, 9, 18, 214, 215, 230 Fee F, 180, 189 Fehrenbach H, 113, 127, 223, 238 Feinberg RN, 83, 102 Fells GA, 194, 208 Fellstrom B, 82, 102 Feng Y, 175, 187 Fenton MJ, 363, 374 Ferenc A, 340, 348 Ferguson M, 41, 48, 52, 295, 309 Ferguson MWJ, 81, 100, 206, 212 Fernandez CA, 246, 263 Fernandez JA, 174, 185, 301, 316 Fernig DG, 174, 186, 344, 350 Ferrando R, 220, 236 Ferrans VJ, 158, 167, 192, 201, 207, 209, 324, 330 Ferrara TB, 40, 49, 52 Ferrari P, 27, 33 Ferrick DA, 359, 369 Ferrone S, 299, 312 Feusi E, 109, 125 Feyzi E, 304, 317 Fiaux GW, 181, 189, 245, 263 Fidler SF, 364, 375 Fieber C, 79, 98, 99, 116, 117, 129 Fiegel V, 117, 130 Figarella C, 278, 287 Figueroa JE, 304, 318 Filardo E, 299, 312 Filardo EJ, 362, 372 Filiat M, 327, 333 Filion MC, 75, 93 Filipic M, 246, 263 Filliat M, 270, 286 Filmus J, 29, 34, 79, 98, 300, 313 Finaly GA, 247, 264

Author Index Fingleton B, 246, 263 Finkbeiner WE, 270, 277, 286, 287, 336, 346 Finlay GA, 147, 158, 165, 245, 247, 263, 264 Fischer DC, 75, 94, 202, 211 Fischer JW, 300, 307, 315, 321, 327, 332, 356, 367 Fischer S, 6, 17 Fischer SG, 63, 71 Fish JE, 327, 332 Fishback N, 158, 167, 246, 264 Fishbein MC, 82, 102 Fisher EA, 305, 318 Fisher LW, 9, 10, 18, 19, 171, 172, 180, 183, 184, 189, 214, 215, 216, 221, 229, 230, 231, 232, 238, 258, 267, 278, 287, 295, 301, 309, 315, 327, 333 Fisher MJ, 301, 315 Fite D, 11, 20 Fitzgerald C, 362, 372 Fitzgerald G, 364, 376 Fitzgerald KA, 30, 31, 35 Fitzgerald ML, 13, 20, 169, 171, 183 Fitzgerald MX, 147, 165, 245, 247, 263, 264, 324, 329 Flad HD, 326, 332 Flanders KC, 86, 104, 203, 204, 211, 224, 238, 239 Flannery CR, 8, 17, 327, 333, 336, 345 Fleischmajer R, 216, 231 Fleming MV, 158, 167, 246, 264 Fleming WE, 364, 375 Flipsen JTM, 66, 67, 72, 181, 189, 248, 251, 265 Flipsen TM, 326, 332 Floerchinger CS, 26, 32 Flower CD, 323, 329 Flynn E, 306, 320 Fok K, 224, 239 Folkman J, 83, 102, 306, 319, 320, 377, 393 Folkvord JM, 362, 372 Fong S, 364, 376 Fontaine B, 12, 20

413 Fontenot JD, 304, 318 Foo ML, 136, 140 Forabosco A, 14, 21 Foresi A, 177, 188, 221, 228, 237, 240 Forino M, 174, 186 Forsberg LS, 270, 286 Forsberg N, 82, 101 Forster MJ, 337, 346, 379, 395 Forstner JF, 269, 270, 285, 286 Forteza R, 79, 91, 99, 106, 108, 117, 123 Fosang AJ, 6, 17, 144, 164, 247, 264, 292, 308, 378, 394 Foster LC, 78, 97, 98, 299, 312 Fouret P, 324, 330 Foweraker JE, 323, 329 Fox J, 117, 130 Fox RB, 40, 49, 52 Frachon I, 224, 239 Fraisse L, 27, 33 Franchina M, 339, 348 Frank CB, 64, 71, 344, 350 Frank R, 361, 370 Fransson LA, 75, 93, 171, 174, 183, 186, 203, 211, 214, 229, 243, 261, 300, 313, 336, 337, 338, 345, 347 Frantz ID, 57, 70 Franzblau C, 181, 190 Fraser JR, 24, 26, 32, 56, 60, 69, 74, 92, 336, 345 Fraser JRE, 2, 5, 16, 17, 136, 140, 336, 346 Fraser P, 307, 321 Fraser RG, 197, 201, 208 Fraser RS, 197, 201, 208 Frati L, 361, 371 Frebourg T, 138, 141 Fredberg JJ, 57, 70 Fredenburgh JC, 379, 395 Freemantle C, 108, 123 Freemont AJ, 108, 113, 123, 124 Freissler E, 357, 368 Freund RM, 81, 100 Freundlich B, 340, 348 Frew AJ, 226, 239 Frewin MB, 253, 254, 266

414

Author Index

Fridman R, 246, 263 Fries KM, 243, 244, 261, 262 Frisch SM, 353, 365 Fritze LMS, 336, 346, 377, 392 Frost GI, 27, 33, 145, 163, 164, 338, 347 Fu Z, 65, 72 Fujimoto N, 243, 246, 262, 264 Fujino S, 174, 185 Fukai N, 306, 320 Fuki IV, 305, 318 Fukuda T, 361, 372 Fukuda Y, 147, 166, 201, 209 Fukui Y, 27, 33 Fukushima D, 227, 239, 246, 263 Fulcher CA, 301, 315 Fuller GM, 25, 26, 32, 75, 93 Fuller JA, 357, 369 Fulmer JD, 192, 207 Fulop C, 109, 126 Fumagalli L, 363, 374 Funaki C, 377, 392 Funderburgh JL, 4, 11, 16, 19, 171, 175, 183, 187, 295, 310, 311, 339, 348 Funderburgh ML, 171, 175, 183, 187, 295, 310, 311, 339, 348 Fung YC, 56, 69 Furch LT, 202, 210 Furdon P, 246, 263 Furlonger C, 29, 34, 79, 98 Fushimi T, 324, 330 Fuster V, 307, 320, 378, 394

G Gaarde W, 87, 105, 173, 180, 185, 219, 236 Gabbiani G, 204, 205, 211, 212, 361, 371 Gadek JE, 192, 207 Gadson PF, 362, 372 Gaffney J, 27, 31, 33, 108, 114, 124, 127, 306, 320 Gagliano A, 284, 289 Gaiano RD, 356, 366

Gailit J, 363, 374 Galabert C, 270, 286, 327, 333 Galanaud P, 224, 239 Galera P, 215, 230 Galis ZS, 159, 167 Gallagher JT, 3, 13, 16, 21, 174, 186, 273, 287, 300, 314, 377, 381, 393, 395 Gallatin WM, 79, 98 Gallo RL, 174, 186, 294, 306, 309, 320, 336, 346, 378, 394 Galloway WA, 246, 263 Galt T, 173, 180, 185, 219, 236 Gambaro G, 174, 186, 338, 347 Gandley RE, 58, 70 Ganguly S, 15, 21 Gao M, 201, 209 Garat C, 87, 105, 203, 211 Garbes P, 300, 314 Garcia R, 357, 368 Gardiner JE, 301, 315 Gardner DL, 82, 101 Gardner HA, 356, 367 Gares SL, 79, 99, 115, 118, 128 Garg HG, 37, 38, 52, 378, 379, 386, 387, 389, 390, 394, 395, 397, 398 Garik P, 269, 285 Garrone R, 173, 179, 184, 215, 231 Gartner MC, 15, 21 Gaspari AA, 244, 262 Gassmann M, 340, 348 Gasvik K, 305, 319 Gauldie J, 47, 49, 53, 120, 133, 147, 165, 173, 180, 181, 185, 189, 219, 227, 236, 239, 244, 253, 262, 266, 340, 348, 349, 363, 374 Gauntlett R, 364, 376 Gaxiola M, 247, 264 Ge Q, 113, 127 Gearing AJ, 246, 263 Gebbinck JW, 79, 98 Geesin JC, 361, 372 Gehron Robey P, 10, 19 Geinoz A, 361, 371 Geiser T, 118, 131, 357, 369 Genereux GP, 197, 201, 208

Author Index Gengrinovitch S, 304, 317 George P, 323, 329 Gerard C, 284, 289 Gerdin B, 82, 102, 110, 126, 204, 211 Gerth U, 77, 97 Gerwin BI, 218, 234 Gerwin N, 29, 34, 79, 98 Ghahary A, 339, 347 Gharee-Kermani M, 362, 372 Ghezzo H, 242, 261 Ghosp P, 295, 310 Giachelli CM, 355, 356, 366 Giancotti FG, 355, 356, 365, 367 Giandomenico A, 26, 32, 87, 105 Giannetti A, 361, 370 Gibaldi M, 378, 394 Gibbons GH, 42, 47, 52 Gibbs AR, 136, 140 Gibbs DF, 327, 332 Gibson RL, 90, 106 Giessauf A, 305, 319 Gilbert R, 246, 263 Gill A, 364, 375 Gillesie DJ, 136, 140 Gilliam AC, 181, 189 Gillitzer R, 79, 98 Gillooly M, 181, 189, 245, 263 Ginsberg MH, 201, 210, 352, 361, 365, 372 Ginsburg M, 247, 264 Ginzburg Y, 357, 368 Giordano A, 173, 174, 185, 304, 317 Giri SN, 86, 87, 103, 104, 105, 173, 180, 185, 219, 235, 236, 359, 369 Gismondi A, 361, 371 Gittenberger-De Groot AC, 362, 373 Gittes GK, 27, 33 Gladson CL, 300, 313 Glancy DL, 304, 318 Glant TT, 29, 34 Glaser CB, 295, 311 Glass GM, 57, 70 Gleisner JM, 61, 70 Glimcher MJ, 342, 349, 361, 370 Glossl J, 214, 221, 230, 238, 247, 264, 306, 319

415 Glusa E, 204, 212 Gmachl M, 27, 32 Gobbi P, 40, 50, 52 Godden JL, 75, 94 Godfrey RWA, 220, 228, 237, 240 Goenen MJH, 306, 320 Goerke J, 88, 89, 105 Goetinck P, 306, 320 Goetinck PF, 356, 367 Gohring W, 304, 317, 356, 367 Gold LI, 201, 209, 223, 224, 238, 243, 261, 295, 310 Goldberg IJ, 304, 318 Goldstein RH, 61, 70 Goldstein S, 10, 19, 172, 184, 258, 267 Goldstein SL, 357, 368 Gomel V, 82, 102 Gonano C, 148, 150, 152, 166 Gong D, 269, 286 Gong JH, 200, 209, 246, 263 Gonzalez-Amaro R, 82, 101 Gordon D, 195, 208 Gordon JL, 246, 263 Gordon MY, 377, 393 Goretzki L, 304, 317 Gosiewska A, 361, 372 Gosset P, 225, 226, 228, 239, 240 Goswani A, 340, 349 Gotoda T, 78, 97 Gotte M, 13, 20, 169, 171, 183 Gottlicher M, 79, 99, 116, 129 Gottlieb DJ, 181, 190 Gottschalk A, 344, 350 Gotwals PJ, 87, 105, 118, 131, 219, 235, 357, 362, 369, 373 Gougerot-Pocidalo MA, 363, 374 Goumans MJ, 226, 239 Govindraj P, 12, 20 Gowda DC, 176, 187 Gradowski JF, 300, 314 Graf K, 355, 362, 366, 372 Graham FL, 180, 189, 227, 239, 340, 349 Graham H, 10, 18, 172, 184, 216, 232, 247, 265, 300, 315 Graham LD, 303, 316

416 Grako KA, 299, 304, 312, 317 Granes F, 357, 368 Granger DN, 152, 166 Grant DS, 247, 265 Grassel S, 11, 20, 378, 393 Greally P, 324, 330 Greaves J, 137, 139, 141 Greaves MF, 377, 393 Green MR, 138, 142 Green SJ, 24, 31, 42, 52, 77, 97 Greenberg AH, 75, 79, 80, 82, 86, 87, 88, 94, 99, 104, 105, 108, 110, 115, 116, 117, 118, 119, 122, 124, 126, 129, 132, 203, 204, 211, 219, 235 Greenfield B, 78, 97 Greenspan DS, 9, 18 Gregory RJ, 283, 288 Greilling H, 75, 94 Grey AM, 75, 94 Griffight MJ, 357, 369 Griffin B, 336, 346 Griffin JH, 174, 185, 301, 315, 316 Griffin RL, 364, 375 Griffioen AW, 306, 320 Griffiths MJ, 86, 87, 103, 105, 118, 131, 357, 369 Griffiths MJD, 203, 211, 217, 233 Grimaud JA, 304, 317 Grimm PC, 79, 80, 82, 87, 88, 99, 110, 117, 119, 122, 126, 326, 331 Grimminger F, 340, 349 Grimshaw J, 335, 344 Griswold DE, 362, 373 Grodzinksy AJ, 63, 71 Groenewegen G, 306, 320 Groffen AJ, 146, 165, 176, 188 Groffen AJA, 11, 20 Groneck P, 90, 106 Groning A, 219, 235 Gross I, 163, 168 Gross P, 254, 266 Grotendorst GR, 202, 203, 211, 377, 392 Grover J, 10, 19, 64, 71, 172, 175, 176, 184, 187, 188 Gruber A, 301, 316

Author Index Grubman SA, 270, 286 Grundboec-Jusco J, 273, 287 Guan JL, 353, 354, 365 Gudewicz PW, 253, 254, 266 Guillemin B, 359, 370 Guimaraes A, 47, 53 Guimaraes I, 47, 53 Guimond S, 292, 308, 378, 394 Gulbins E, 181, 190 Gunay NS, 379, 387, 395, 398 Gunnar Sedin E, 23, 24, 31 Guntherodt HJ, 50, 53 Guo BP, 176, 177, 187 Guo D, 307, 321 Guo YS, 379, 395 Gustafson S, 82, 101 Gustafsson E, 11, 20 Gutierrez G, 159, 160, 167 Gutierrez P, 295, 309 Gutierrez PS, 41, 48, 52 Gutierrez-Ramos JC, 29, 34, 79, 98 Gyetko MR, 357, 369

H Haber E, 78, 97, 299, 312 Habuchi O, 339, 348 Hada T, 75, 95 Haggerty JG, 15, 21 Hagihara K, 202, 210 Hagmar B, 137, 141 Haigh M, 216, 231 Hair GA, 63, 71 Hakala JK, 305, 319 Hakama M, 136, 140 Hakim S, 79, 99 Hakkert BC, 79, 98 Hakkinen L, 120, 132 Hales CA, 343, 344, 350, 377, 378, 382, 385, 386, 387, 389, 390, 391, 393, 394, 396, 397, 398 Halfter W, 49, 53 Hall CL, 76, 79, 80, 96, 97, 99, 100, 108, 110, 113, 115, 117, 118, 124, 128 Hallen A, 381, 395

Author Index Hallgren R, 26, 32, 82, 85, 87, 90, 102, 103, 105, 110, 117, 118, 120, 126, 130, 131, 132, 281, 288 Halliday JL, 220, 237 Halper J, 378, 393 Halperin JL, 378, 394 Halpert I, 202, 210 Hamaguchi M, 2, 16, 24, 28, 32, 76, 95, 110, 126, 337, 346 Hamann D, 357, 369 Hamasaki Y, 76, 95, 109, 110, 126 Hamel L, 299, 312 Hamid Q, 50, 53, 68, 72, 177, 188, 220, 223, 237, 238, 326, 331, 361, 371 Hamida BC, 12, 20 Hamilton JA, 269, 286 Hamilton S, 228, 240 Hamilton TA, 82, 101 Hammar SP, 49, 53 Hammond CE, 364, 376 Hammouda H, 12, 20 Hampson F, 244, 262 Hampson IN, 27, 30, 33, 83, 102, 306, 320 Hanahan D, 83, 102 Hangumaran S, 115, 128 Hanlon WA, 363, 375 Hannigan GE, 363, 375 Hansson HE, 136, 141 Hanyu T, 272, 287 Harding K, 120, 133 Hardingham TE, 6, 9, 17, 18, 144, 164, 247, 264, 292, 308, 378, 394 Hardwick C, 79, 99 Harf A, 147, 159, 166 Harlan JM, 357, 368 Harler MB, 355, 365 Harms D, 117, 130 Haro H, 246, 263 Haroske G, 113, 127 Harper JR, 87, 105, 173, 180, 185, 219, 235, 236 Harrach B, 10, 19, 247, 248, 264, 265, 306, 319 Harris CC, 218, 234 Harrison DJ, 254, 255, 267

417 Harrison JH, 118, 120, 131 Harrison MR, 81, 100 Harrison RE, 75, 76, 79, 80, 81, 93, 100, 113, 116, 126, 129, 216, 232 Hart CE, 362, 372 Hart DA, 64, 71 Hart IR, 79, 98, 99, 116, 117, 129 Hart J, 120, 133 Hart MH, 79, 98 Hartmann D, 228, 240 Hascall VC, 2, 4, 6, 16, 17, 28, 33, 55, 56, 68, 74, 75, 76, 81, 92, 94, 95, 96, 109, 125, 126, 144, 145, 146, 163, 164, 214, 229, 272, 284, 287, 288, 325, 331, 378, 394 Haschek WM, 194, 208 Hasham S, 307, 321 Hashimoto M, 51, 53 Hasilik A, 219, 235 Hasoun PM, 343, 350 Hassell J, 4, 16 Hassell JR, 4, 10, 11, 12, 16, 19, 20, 146, 165, 171, 172, 183, 184, 214, 229, 247, 265, 294, 295, 309, 310 Hassoun PM, 383, 396 Hastie AT, 327, 332 Hastings G, 114, 127 Hathaway M, 86, 104 Haubeck HD, 75, 94 Haudenschild CC, 115, 128 Hausser H, 5, 15, 17, 21, 175, 187, 213, 215, 216, 219, 228, 230, 231, 232, 235, 248, 265, 304, 318, 327, 333, 338, 347 Haustein UF, 361, 370 Hautamaki RD, 247, 264 Hautanen A, 218, 234 Hautmann MB, 203, 211 Haxhiu Poskurica B, 162, 163, 168 Hay DW, 362, 373 Hay J, 25, 26, 32, 118, 120, 130 Hay JG, 324, 330 Hayashi S, 223, 238 Hayashi T, 158, 167, 246, 264, 377, 392 Hayes JA, 219, 235

418 Hayes JP, 245, 263 Hayman EG, 300, 314 Haynes BF, 75, 95 Hazlett LD, 269, 285 He J, 114, 127 Heard BE, 138, 141 Heath D, 47, 53 Heath EC, 324, 330 Heath JK, 218, 234 Hecht D, 306, 320 Heck LW, 324, 329 Heckmann M, 361, 370 Hedborn E, 10, 19, 172, 184, 217, 232 Hedgecock C, 117, 130 Hedin U, 303, 316, 385, 397 Hedlof E, 137, 138, 141 Heegaard AM, 9, 10, 18, 19, 171, 172, 183, 184, 258, 267 Heidemann SR, 68, 72 Heine UI, 86, 104, 218, 233 Heinegard D, 10, 19, 56, 66, 69, 118, 131, 153, 167, 170, 171, 172, 173, 176, 180, 183, 184, 188, 200, 202, 209, 210, 213, 214, 216, 217, 218, 223, 228, 229, 232, 235, 238, 247, 251, 258, 264, 265, 267, 295, 300, 306, 310, 315, 319 Heinegard DK, 378, 394 Heinel LA, 253, 254, 266 Heino J, 352, 355, 361, 365, 366, 371 Heinrikson RL, 254, 266 Heldin CH, 75, 88, 93, 304, 317 Heldin P, 27, 33, 75, 76, 79, 83, 84, 87, 88, 93, 95, 96, 99, 102, 105, 110, 117, 118, 119, 126, 130, 145, 163, 164, 338, 340, 347, 349 Helft G, 307, 320 Heller RA, 86, 103 Hellewell PG, 364, 375, 376 Helliwell SM, 323, 329 Hellstrom S, 82, 102 Hellwig SMM, 306, 320 Hemdon JE, 138, 142 Hemken C, 118, 132, 340, 348 Hemler ME, 355, 362, 365, 373 Hemple SL, 343, 350

Author Index Henderson DJ, 303, 316 Henderson K, 362, 373 Henderson WR, 357, 368 Heneka M, 181, 190 Henke C, 117, 130, 201, 209 Henley C, 307, 320 Hennies J, 30, 34 Henson PM, 31, 35, 75, 82, 93, 120, 133 Hentati F, 12, 20 Herdman M, 228, 240 Heredia A, 243, 261 Heremans A, 243, 261 Hermas J, 344, 350 Hermonen J, 352, 365 Hernandez-Rodriguez NA, 223, 238 Hernas J, 170, 183 Herndon M, 174, 186, 306, 320, 336, 346 Hernnas J, 56, 66, 69, 118, 131, 176, 188, 223, 238 Heroutl T, 174, 186 Herring TM, 214, 229 Herrlich P, 28, 31, 34, 35, 76, 77, 78, 79, 88, 96, 97, 98, 99, 116, 117, 129, 340, 348 Herrmann K, 361, 370 Hershenson MB, 61, 71, 90, 106 Hershkoviz R, 253, 266 Heunks L, 252, 266 Hickerson WL, 82, 102 Hidebrand KA, 344, 350 Hierck BP, 362, 373 Higashimoto I, 364, 375 Higashino K, 75, 95 Higuchi ML, 47, 53 Hilal N, 50, 53 Hildebrand A, 10, 19, 172, 173, 180, 184, 218, 235, 252, 266, 335, 345 Hildebrand P, 361, 371 Hildebrandt J, 57, 69 Hileman E, 381, 395 Hileman RE, 324, 330 Hill SL, 325, 331 Hiller-Hitchcock, 175, 187 Hilliam C, 221, 237

Author Index Hilliard KA, 326, 332 Hinek A, 300, 315 Hines E, 357, 368 Hinkes M, 378, 394 Hinkes MT, 294, 309 Hirakawa S, 9, 18 Hirakawa YS, 78, 98 Hiramatsu A, 227, 239, 246, 263 Hiromi M, 201, 209 Hiromi N, 280, 288 Hirose J, 68, 72, 174, 186, 200, 201, 209 Hirose M, 68, 72, 174, 186 Hirschberg CB, 381, 395 Hirschel BJ, 204, 211 Hirsh J, 378, 379, 394, 395 Hisada T, 364, 375 Hiyama K, 118, 131 Hjerpe A, 75, 95, 137, 139, 141, 307, 321, 339, 347 Ho CKY, 270, 286 Ho CS, 326, 332 Ho PL, 326, 332 Hoare K, 79, 99 Hocking AM, 9, 18, 213, 214, 228, 230 Hocking DC, 361, 372 Hodge-Dufour J, 30, 31, 35, 108, 125 Hodgkin JE, 242, 260 Hodson ME, 138, 141 Hodson WA, 61, 71, 85, 90, 103, 106, 146, 165 Hoessli DC, 115, 128 Hof PR, 307, 321 Hofbauer E, 180, 189 Hoffman S, 202, 210 Hoffmeister K, 75, 94 Hoffren AM, 352, 365 Hofmann M, 78, 79, 97, 98, 99, 116, 117, 129 Hofsteenge J, 218, 234 Hogg JC, 27, 32, 41, 52, 56, 61, 69, 70, 108, 123, 178, 188, 196, 197, 201, 202, 208, 220, 221, 222, 223, 236, 237, 238, 242, 251, 260, 265, 326, 331 Hohn P, 79, 99

419 Holgate ST, 179, 188, 204, 212, 220, 221, 226, 236, 238, 239 Holland JF, 137, 138, 139, 141 Hollander W, 304, 317 Hollenbaugh D, 78, 97 Hollinger DA, 219, 235 Hollmann J, 214, 230 Holmdahl L, 108, 124 Holmes DF, 10, 18, 172, 184, 216, 232, 247, 265, 300, 315 Holstege A, 204, 211 Homer RJ, 258, 267 Homma M, 228, 239 Honda A, 75, 94 Honda K, 76, 95, 364, 375 Honeyman T, 385, 397 Honma M, 225, 226, 239 Hoogewerf AJ, 254, 266, 377, 392 Hook M, 79, 99, 176, 177, 187, 214, 217, 229, 233, 336, 345, 377, 381, 393, 395 Hopensteudt D, 387, 398 Hoppe W, 214, 215, 230 Hopwood JJ, 5, 17 Horchar T, 4, 16 Horio T, 377, 392 Horley KJ, 295, 310 Hormann H, 247, 264, 306, 319 Horton MR, 27, 30, 31, 33, 35, 75, 82, 93, 108, 113, 124, 125, 269, 286 Horwitz AF, 201, 210 Horwitz MI, 327, 333 Hoshi H, 225, 226, 228, 239 Hoshino M, 221, 237 Hou G, 79, 80, 82, 87, 88, 99, 110, 117, 119, 122, 126, 326, 331 Hou XM, 117, 118, 119, 122, 130 Houghlum K, 204, 211 Houghton SJ, 323, 329 Hounsell EF, 344, 350 Houston JC, 220, 236 Howard EW, 361, 371 Howard JJ, 284, 288 Howarth PH, 179, 188, 204, 212, 220, 221, 226, 236, 238, 239 Howell WH, 254, 266, 377, 392

420 Howells N, 79, 99, 116, 129 Hoyle GW, 205, 212 Hoyt DG, 25, 26, 32, 118, 120, 131, 219, 235 Hrousis CA, 221, 237 Hruska KA, 362, 372 Hsiung SH, 364, 376 Hsu W, 56, 60, 69 Hsueh WA, 355, 362, 366, 372 Huang C, 115, 128 Huang J, 50, 53, 68, 72, 177, 188, 223, 238, 326, 331 Huang TJ, 364, 375 Huang X, 86, 87, 103, 105, 118, 131, 217, 233, 357, 369 Hubbard RC, 194, 208, 324, 330 Huber HL, 220, 236 Huber LA, 30, 31, 35, 115, 128 Huber R, 14, 21 Hubmayr RD, 68, 72 Hudson LD, 194, 208 Huggins GS, 78, 98, 299, 312 Hughes CE, 8, 17, 327, 333, 336, 345 Hughes R, 57, 69 Hughes-Benzie RM, 14, 21 Huhtala P, 301, 315, 357, 368 Huitema S, 66, 67, 72, 181, 189, 248, 251, 265, 326, 332 Hull RD, 344, 350 Hullinger TG, 359, 370 Hulmes DJ, 181, 189, 245, 263 Humphries DE, 15, 21 Humphries MJ, 357, 368 Hung MC, 114, 128 Hung X, 203, 211 Hunninghake GW, 26, 32, 117, 118, 130, 132, 192, 207, 244, 262, 340, 343, 348, 350 Hunt DA, 86, 104 Hunziker E, 11, 20 Hunziker EB, 63, 71 Hurt-Camejo E, 304, 305, 317, 318, 319 Hurta RAR, 75, 79, 80, 88, 94, 115, 129 Husain M, 47, 53 Hutchinson NI, 246, 264

Author Index Huttenlocher A, 301, 315, 357, 368 Hutton WC, 63, 71 Huttunen P, 352, 365 Hybertson BM, 362, 373 Hyde DM, 86, 87, 103, 105, 173, 180, 181, 185, 189, 219, 235, 236, 244, 262 Hyman R, 28, 34, 77, 97 Hynes RO, 217, 233, 356, 367 Hyon S, 63, 71 Hypia T, 352, 365

I Ibbott GS, 113, 126 Ienaga H, 359, 370 Ignotz RA, 218, 233 Iguchi A, 356, 367 Iida J, 202, 210 Iida N, 114, 128 Ijima H, 284, 289 Ijuin C, 76, 95 Ikeda AK, 201, 209 Ikeda M, 377, 392 Ikegami M, 85, 90, 103 Ikegaya K, 10, 19, 173, 185, 219, 235 Ikenda T, 272, 287 Imagawa M, 2, 16, 24, 28, 32, 76, 95, 110, 126, 337, 346 Imai E, 10, 19, 173, 185, 219, 235 Imai K, 227, 239, 246, 247, 263, 264 Imai Y, 75, 94 Imamura Y, 9, 18 Imanari T, 387, 389, 390, 398 Inazawa J, 28, 34 Ingber DE, 63, 68, 71, 72, 306, 319 Ingenito EP, 57, 70 Ingram RH, 57, 70 Inki P, 120, 132 Innerarity TL, 305, 318 Inoue S, 247, 265 Inoue T, 78, 98 Inoue Y, 75, 94 Iocono JA, 81, 101 Iozzo RV, 5, 8, 9, 10, 11, 17, 18, 19, 20, 55, 56, 57, 59, 69, 145, 146, 165,

Author Index 169, 172, 173, 174, 175, 176, 177, 179, 183, 184, 185, 187, 199, 208, 213, 214, 215, 216, 217, 228, 229, 230, 231, 233, 247, 265, 292, 294, 300, 301, 303, 304, 305, 307, 308, 315, 316, 317, 318, 321, 335, 345, 378, 393 Ip M, 325, 331 Ip MSM, 324, 326, 330, 332 Ireland H, 301, 316 Irish P, 86, 105, 219, 235 Irle C, 204, 211 Iruela-Arispe M, 202, 210, 306, 319, 320 Iruela-Arispe ML, 327, 332 Irving LB, 244, 262 Isacke CM, 340, 348 Isaka Y, 10, 19, 173, 185, 219, 235 Isakoff SJ, 355, 365 Isaksson A, 174, 186 Isemura M, 272, 287 Ish-Shalom D, 76, 77, 96 Ishibashi M, 110, 126 Ishihara H, 6, 17 Ishihara M, 357, 368, 381, 395 Ishii H, 300, 313 Ishioka S, 118, 131 Ishizaki M, 147, 166 Isner JM, 248, 265, 295, 307, 309, 320 Isogai Z, 202, 210 Isoyama S, 228, 239 Israel E, 57, 70 Israel-Biet D, 117, 130 Issekutz AC, 364, 376 Issekutz TB, 361, 364, 371, 376 Itano N, 2, 16, 24, 27, 28, 32, 33, 75, 76, 95, 110, 126, 337, 346 Itin P, 299, 312 Ito K, 8, 18, 200, 209 Ito M, 244, 262, 280, 288 Itoh F, 226, 239 Itoh S, 226, 239 Ivarrson M, 204, 211 Ivaska J, 352, 355, 365, 366 Iverius PH, 336, 346 Ivins JK, 294, 309

421 Iwamatsu A, 357, 368 Iwasaki T, 30, 31, 35 Izquierdo JL, 242, 261 Izumi T, 244, 262

J Jaakkola O, 377, 392 Jack RM, 219, 235 Jackson CJ, 306, 319 Jackson DG, 77, 78, 97, 114, 127, 364, 376 Jackson EK, 377, 392 Jackson JC, 90, 106 Jackson RL, 2, 16, 335, 345, 385, 397 Jackson SK, 243, 261 Jacobelli J, 361, 371 Jacobs L, 216, 231 Jacobson A, 76, 96, 110, 126 Jacquinet JC, 378, 394 Jaenisch R, 357, 368 Jaffe A, 357, 369 Jagidar J, 340, 349 Jahn M, 108, 124 Jain RK, 306, 320, 356, 367 Jakowlew S, 86, 104 Jalkanen M, 12, 21, 120, 132, 253, 266, 299, 300, 306, 311, 314, 320 Jalkanen S, 253, 266 James AL, 221, 237 Janoff A, 254, 267 Janssen HMJ, 245, 263 Janssens SP, 343, 344, 350, 378, 393 Jansson M, 361, 370 Jarvelainen HT, 202, 211, 295, 299, 303, 306, 310, 311, 316, 319, 357, 368 Jasani B, 136, 140 Jasmin C, 84, 103 Jefferson DM, 270, 286 Jeffery PK, 220, 223, 228, 236, 237, 238, 240, 270, 286, 340, 349 Jeitner TM, 363, 374 Jenesen G, 221, 237 Jenkins KL, 306, 319 Jenkinson D, 117, 129

422 Jensen JI, 344, 350 Jensen K, 202, 210 Jensen T, 307, 321 Jepsen KJ, 11, 19 Jessurun J, 117, 130, 201, 209 Jesudason EC, 344, 350 Jetha N, 82, 102 Jhali N, 228, 240 Jiang D, 82, 102 Jimenez SA, 108, 123, 218, 234 Jingbo A, 79, 99 Jo YY, 387, 398 Jobe AH, 85, 90, 103, 106 Jochmans K, 174, 186 Johansson C, 29, 34 Johansson O, 176, 187 Johansson S, 336, 345, 377, 393 Johansson SA, 220, 228, 237, 240 Johnson BJ, 176, 177, 187 Johnson D, 117, 130, 201, 209 Johnson KJ, 327, 332, 363, 374 Johnson MD, 223, 238 Johnson MS, 352, 365 Johnson PR, 113, 127 Johnson TL, 214, 229 Johnsson H, 75, 85, 90, 95, 103, 110, 126 Johnston PW, 220, 237 Johnstone SR, 299, 312 Jolles P, 299, 311 Jonas M, 357, 368 Jones PL, 360, 370 Jones R, 270, 286 Jones RC, 357, 368 Jones S, 364, 376 Jonsson M, 300, 313, 337, 347 Jonzon A, 23, 24, 31 Jorcano JL, 29, 34, 81, 101 Jordana G, 244, 262 Jordana M, 120, 133, 225, 226, 239, 244, 262, 340, 348 Jordon SE, 228, 240 Joritka M, 299, 311 Joseph PAM, 386, 397 Joseph PM, 343, 350, 377, 378, 385, 387, 389, 390, 393, 397, 398

Author Index Juergens U, 91, 106 Jules-Elysee K, 86, 103 Juliano RL, 353, 365 Jullien P, 217, 233 Jung KY, 223, 238 Just W, 9, 18, 171, 183, 214, 215, 230 Justice P, 357, 368 Juul SE, 61, 71, 85, 90, 103, 106, 146, 165

K Kaakkola P, 12, 21 Kabbash L, 363, 374 Kaczka DW, 57, 70 Kadler KE, 10, 18, 172, 184, 216, 232, 247, 265, 300, 315 Kaftan E, 337, 347 Kagan E, 41, 50, 52, 137, 141 Kahari VM, 218, 234, 355, 366 Kainulainen V, 299, 311 Kakazu T, 364, 375 Kakizoe T, 78, 97 Kalie N, 79, 80, 82, 87, 88, 99 Kalkunte S, 364, 376 Kamata S, 27, 33 Kaminski N, 86, 87, 103, 105, 118, 131, 203, 211, 357, 369 Kamm RD, 221, 237 Kampe M, 220, 237 Kanai Y, 78, 97 Kanamori M, 6, 17 Kanda S, 83, 84, 102, 356, 367 Kaneda Y, 10, 19, 173, 185, 219, 235 Kang AH, 86, 104, 105, 118, 120, 131, 219, 235, 361, 370 Kano H, 377, 392 Kao CH, 84, 102 Kao CWC, 11, 19 Kao WW, 175, 187 Kao WWY, 11, 19 Kaplan ED, 295, 309 Kaplan M, 305, 319 Kapoor R, 11, 20 Kapyla J, 352, 365 Karakhanova S, 31, 35

Author Index Karakiulakis G, 75, 94, 109, 125, 340, 341, 342, 343, 349, 350 Karamanos NK, 75, 95, 307, 321, 339, 347 Karmiol S, 362, 372 Karnovsky MJ, 378, 384, 385, 393, 394, 395, 397 Karp S, 246, 263 Karvinen S, 76, 95, 109, 126 Kaschak M, 254, 266 Kashem MA, 324, 330 Kashgarian M, 361, 371 Kasper M, 88, 105, 113, 119, 120, 127, 132, 223, 238 Kass ME, 224, 238 Kassner PD, 362, 373 Kataoka S, 75, 82, 93 Kato H, 174, 185, 361, 371 Kato M, 294, 305, 309, 319, 378, 394 Katoh K, 357, 368 Kauffman HF, 66, 67, 72, 181, 189, 248, 251, 255, 265, 267, 326, 332 Kaufmann R, 204, 212 Kaverina I, 30, 31, 35, 115, 128 Kawabata M, 364, 375 Kawaguchi Y, 6, 17 Kawakatsu H, 87, 105, 203, 211, 217, 233, 357, 369 Kawamoto M, 259, 267 Kawano K, 201, 209 Kawashima H, 68, 72, 174, 186, 200, 201, 209 Kawatani A, 244, 262 Kay AB, 220, 236 Kay CJ, 10, 19, 172, 184 Kaya G, 29, 34, 81, 101 Kayaba H, 364, 375 Keefe DH, 57, 70 Keeley F, 300, 315 Keene DR, 172, 179, 184, 216, 217, 231, 233 Kehrl JH, 218, 233 Keivens VM, 361, 372 Kelaita D, 353, 365 Keller S, 91, 92, 106 Kelley J, 361, 370

423 Kelly GS, 344, 350 Kelly S, 148, 152, 166 Kemp A, 203, 204, 211 Kenagy RD, 202, 210, 327, 332 Kendall T, 357, 369 Kennedy JS, 117, 129 Kennedy SP, 361, 372 Kent PW, 273, 287 Keogan MT, 323, 329 Kerhl JH, 86, 104 Kermarrec N, 363, 374 Kern DG, 194, 207 Kernacki KA, 269, 285 Kerr JS, 254, 267 Keski-Oja J, 86, 104, 218, 234, 252, 266 Ketter S, 27, 32 Khair OA, 326, 332 Khairullina A, 172, 184, 217, 232 Khaldoyanidi S, 31, 35 Khalil N, 80, 81, 82, 86, 87, 100, 104, 105, 110, 117, 118, 119, 122, 126, 132, 203, 204, 211, 219, 235, 326, 331 Khatri IA, 270, 286 Khoo KH, 387, 397 Kiani CG, 201, 210 Kidao S, 246, 263 Kiefer F, 29, 34, 79, 98 Kielisch H, 340, 349 Kikuchi T, 30, 31, 35 Kilshaw PF, 359, 369 Kim JH, 29, 34 Kim KC, 270, 286 Kim SJ, 86, 104 Kim WD, 242, 261 Kim YJ, 63, 71 Kim YS, 387, 398 Kim YY, 221, 237 Kim-Park HY, 385, 397 Kimata K, 2, 8, 16, 17, 18, 27, 28, 33, 75, 76, 95, 110, 126, 200, 201, 202, 209, 210, 295, 303, 307, 309, 316, 321, 337, 338, 346, 347 Kimble R, 363, 374 Kimoto S, 175, 187

424 Kimura A, 273, 287 Kimura JH, 214, 229 Kimura T, 6, 17 Kincade PW, 28, 34 Kinch MS, 353, 365 Kindig T, 29, 34 Kindy MS, 384, 397 King GM, 25, 26, 32 King SR, 82, 102 King T, 120, 132 King TE, 194, 208 Kinsella MG, 61, 71, 79, 85, 90, 99, 103, 106, 120, 132, 146, 165, 248, 265, 295, 300, 303, 306, 307, 310, 315, 316, 319, 321, 356, 357, 367, 368 Kireeva ML, 355, 366 Kirkland SC, 362, 373 Kita H, 364, 375 Kitagawa H, 335, 336, 345 Kitagawa Y, 200, 209 Kitaichi M, 147, 166, 244, 262 Kittaka M, 301, 315 Kivilouto R, 136, 140 Kjellen K, 378, 394 Kjellen L, 292, 308, 336, 345, 381, 395 Klagsbrun M, 246, 252, 263, 266, 304, 317, 377, 393 Klareskog L, 82, 102 Klass CM, 356, 367 Klech H, 117, 130 Klein DJ, 79, 98, 307, 321 Kleinerman J, 241, 260 Kleinman HK, 214, 229, 247, 265, 377, 392 Klemperer P, 135, 140 Klewer SE, 24, 28, 32, 76, 96, 110, 126 Klewes L, 79, 99 Klezovitch O, 304, 318 Klier FG, 300, 313 Kneussel MP, 340, 348 Knight CG, 352, 365 Knighton D, 117, 130 Knudson CB, 27, 32, 76, 77, 78, 96, 98, 111, 126

Author Index Knudson W, 27, 32, 76, 77, 96 Kobayashi DK, 247, 264 Kobayashi H, 75, 93 Kobayashi S, 218, 234 Kobe B, 216, 232 Kobzik L, 242, 261 Kocher O, 361, 372 Kock R, 75, 94 Kodaka H, 272, 287 Kodama Y, 219, 235 Koehler E, 361, 371 Koessler KK, 220, 236 Kohno M, 377, 392 Kohyama T, 362, 372 Koivisto L, 355, 361, 366, 371 Kojima T, 300, 314 Kokenyesi R, 294, 309, 378, 394 Kokkonen JO, 300, 314 Kolb M, 47, 49, 53, 173, 180, 185, 189, 219, 236, 253, 266, 363, 374 Kolbasa KP, 364, 375 Koli K, 218, 234 Kolsch E, 295, 311 Kolset SO, 299, 300, 312, 314 Kon S, 359, 370 Kondiaah P, 86, 104 Kondo H, 78, 97 Kondo K, 78, 97 Konishi T, 174, 185 Konno K, 244, 262, 280, 288 Konttiken YT, 147, 166, 247, 264 Koo M, 244, 262 Koob TJ, 55, 58, 69, 70, 216, 232 Kopp JB, 361, 370 Korang K, 175, 187, 215, 230 Korchak HM, 324, 330 Korenberg JR, 10, 19, 176, 188 Kornovski BS, 91, 106 Korthy AL, 219, 235 Kosaka S, 300, 313 Koshikawa T, 377, 392 Koshino T, 284, 289 Kosinka D, 254, 266 Koslowski R, 223, 238 Koss MN, 158, 167, 246, 264

Author Index Kosuge T, 78, 97 Koteliansky VE, 87, 105, 118, 131, 219, 235, 357, 362, 369, 373 Kourembanas S, 340, 349 Kovach NL, 202, 210 Kovacs EJ, 253, 266 Kovanen PT, 300, 304, 305, 314, 318, 319 Koyama N, 303, 316, 377, 392 Koyama T, 78, 98 Koza M, 336, 346 Kraayenbrink M, 278, 288 Kradin RL, 382, 383, 396 Krakoff IH, 118, 131 Kramps JA, 324, 330 Kraneveld AD, 364, 376 Kredentser J, 91, 106 Kreidberg JA, 357, 368 Kreil G, 27, 32, 33 Kremmidiotis G, 15, 21 Kresse H, 5, 10, 15, 17, 18, 19, 21, 172, 173, 175, 184, 185, 187, 213, 214, 215, 216, 217, 218, 219, 221, 228, 230, 232, 233, 235, 238, 247, 248, 264, 265, 295, 301, 304, 311, 315, 318, 327, 333, 338, 347, 356, 367 Krieg T, 175, 187 Kristensen P, 218, 234 Krueger RCJ, 90, 106 Krufka A, 304, 317 Krull NB, 175, 179, 186 Krummel TM, 81, 101 Krumwiede M, 117, 130, 201, 209 Krupinski J, 27, 31, 33, 108, 114, 124, 127, 306, 320 Krusius T, 171, 183, 214, 229, 230, 278, 287, 295, 309 Krutzsch HC, 203, 211 Kryceve-Martinerie C, 217, 233 Ksander GA, 75, 93 Kuang SQ, 307, 321 Kubalak S, 24, 28, 32, 76, 96, 110, 126 Kublin CL, 217, 232 Kubota T, 359, 369

425 Kudoh S, 147, 166 Kuettner KE, 78, 98 Kugelman LC, 15, 21 Kuhn C, 61, 70, 194, 200, 207, 208, 209, 244, 262 Kuhn KM, 305, 318 Kuijpers TW, 79, 98, 357, 369 Kulkarni A, 361, 372 Kulkarni AB, 10, 19, 172, 184, 258, 267 Kulseth MA, 299, 312 Kumar NM, 359, 369 Kumar P, 108, 113, 124 Kumar S, 27, 30, 31, 33, 83, 84, 102, 103, 108, 113, 114, 123, 124, 127, 306, 320 Kundig T, 79, 98 Kunkel SL, 86, 104 Kunz G, 301, 316 Kuopio T, 295, 310 Kupfer A, 82, 101 Kupiec-Weglinski JW, 361, 371 Kupper TS, 362, 373 Kureger RC, 61, 71 Kuroda K, 109, 110, 126 Kuroda T, 361, 371 Kurokawa Y, 147, 166, 247, 264 Kurpakus WM, 269, 285 Kuruda K, 76, 95 Kusano Y, 357, 368 Kuschak TI, 80, 100 Kusche M, 381, 395 Kuwano K, 221, 237 Kuyper CMA, 245, 263 Kuzuya F, 377, 392 Kuzuya M, 356, 367, 377, 392 Kvietys PR, 324, 329 Kwaspen F, 300, 313 Kyriakoulis K, 301, 316

L La Riviere G, 79, 98 La Rocca AM, 224, 226, 239 Laato M, 306, 320

426 Labargy F, 147, 159, 166 LaBrenz SR, 214, 229 Laderoute MP, 79, 98 Lafitte JJ, 270, 286, 327, 333 Lafuma C, 147, 159, 166, 228, 240 Lai-Fook SJ, 113, 126 Laiho M, 218, 234 Laine J, 295, 310 Laitinen LA, 220, 237 Laitinene A, 220, 237 Lake FR, 31, 35, 75, 82, 93, 120, 133 Lalaguna F, 304, 318 Laliberte R, 220, 236 Lam SC, 355, 366 Lam WK, 325, 326, 331, 332 Lamande SR, 172, 184, 217, 232 LaMantia C, 11, 19 Lamb D, 181, 189, 245, 255, 263, 267, 269, 271, 286, 287 Lambeau G, 356, 367 Lamblin G, 228, 240, 270, 286, 327, 333 Lamm WJ, 357, 368 Lammers JWJ, 252, 266 Lammi M, 299, 311 Lammi MJ, 109, 126 LaMont JT, 269, 285, 286 Lamonte C, 118, 131 Lamoureux P, 68, 72 Land IM, 340, 348 Lander AD, 56, 59, 69, 292, 294, 300, 303, 305, 308, 309, 313, 316, 319 Landschulz KT, 175, 187 Lane DA, 301, 316 Lane WS, 306, 320 Lang MR, 181, 189, 245, 263 Lange LA, 80, 100, 108, 110, 113, 115, 116, 117, 118, 124, 128 Lange P, 221, 237 Langer C, 304, 318 Langford JK, 56, 59, 69, 303, 316 Languino LR, 219, 235 Lara SL, 306, 307, 320, 321 Larjava H, 120, 132, 218, 234 Lark MV, 159, 167, 246, 264 Laros CD, 245, 263

Author Index Larson RS, 352, 365 Larsson E, 82, 102 Larsson S, 136, 140 Larsson T, 171, 183 Laskin DL, 86, 104 Lass JH, 11, 19 Lassila R, 300, 314 Laterra J, 377, 392 Lau LF, 355, 366 Laudanna C, 363, 374 Lauder I, 325, 331 Lauffenburger DA, 201, 210 Lauredo I, 91, 106, 108, 117, 123 Lauredo IT, 364, 375 Laurell CB, 254, 266 Laurent C, 82, 102 Laurent G, 25, 26, 32, 118, 120, 130 Laurent GJ, 118, 131, 181, 190, 223, 238, 259, 267, 340, 348, 349 Laurent TC, 2, 5, 16, 17, 24, 26, 31, 32, 56, 60, 69, 74, 75, 82, 88, 92, 93, 95, 102, 110, 117, 126, 130, 136, 138, 140, 141, 336, 345, 346 Laurent UB, 56, 60, 69, 336, 345 Laurent UBG, 2, 5, 16, 136, 140 Laurie GW, 146, 147, 165 Lauweryns JM, 66, 67, 72 Lavender JP, 323, 329 Laviolette M, 177, 188, 220, 221, 236, 237, 244, 262 Law MR, 138, 141 Lawler J, 217, 233, 356, 367 Lawrence DA, 217, 233 Lazaar AL, 78, 97 Lazarovils AL, 364, 375 Lazo JS, 25, 26, 32, 118, 120, 131, 219, 235 Le AH, 12, 20 Le Baron R, 307, 320 Le Bousse-Kerdiles MC, 84, 103 LeBaron RG, 67, 72, 199, 208, 217, 233, 303, 316, 377, 393 Leblond CP, 247, 265 LeBoeuf RD, 75, 93 Lebowitz MD, 242, 260 Lebrini A, 361, 371

Author Index LeBrun L, 387, 398 Lechner JF, 218, 234 Ledbetter S, 11, 20 Ledbetter SR, 254, 266 Leder P, 304, 317 Lederman I, 378, 394 Ledger P, 84, 102 Lee GK, 221, 237 Lee HM, 223, 238 Lee I, 305, 318 Lee JC, 362, 373 Lee JJ, 357, 368 Lee JY, 30, 34, 75, 93, 108, 113, 119, 125 Lee NA, 357, 368 Lee RT, 175, 187, 362, 373 Lee SH, 223, 238 Lee SL, 377, 393 Lee TC, 325, 331, 340, 349, 359, 370 Lee VS, 41, 50, 52 Lee YM, 362, 373 Lees VC, 84, 102 Legrand Y, 84, 103 Lehmann-Horn F, 12, 20 Leigh MW, 282, 288 Leith DE, 57, 70 Lemaire F, 147, 159, 166 Lemire JM, 202, 211, 295, 300, 309, 315, 327, 332 Lemjabbar H, 228, 240 Lenas P, 2, 16, 24, 28, 32, 76, 95, 110, 126, 337, 346 Lenot B, 224, 239 Lentsch AB, 363, 374 Leone DR, 364, 375, 376 Leone JW, 254, 266 Lepperdinger G, 27, 33, 145, 163, 164, 338, 347 Lerebours G, 138, 141 Lesley J, 28, 34, 77, 97 Leslie KO, 195, 208 Letarte M, 299, 312 Lethias C, 173, 179, 184, 215, 231 Letourneau PC, 79, 88, 98, 362, 372 Leung DYM, 220, 237 Leung TC, 202, 210

427 Lever R, 181, 190 Levesque MC, 75, 95 Levi E, 246, 263 Levi-Schaffer F, 244, 262 Levin-Jacobsen AM, 138, 141 Levine D, 359, 369 Levine EM, 377, 393 Levine JM, 338, 347 Levine RA, 385, 397 Levison H, 220, 236 Levy L, 387, 398 Lewandowska K, 217, 233, 306, 319 Lewis CS, 327, 332 Leygue E, 175, 187 Li H, 202, 210 Li J, 64, 72 Li L, 359, 370 Li MW, 27, 32, 91, 106 Li W, 214, 229 Li X, 116, 129, 220, 236 Li Y, 27, 33, 84, 103, 145, 163, 164, 338, 347 Liang J, 82, 102 Liao F, 30, 31, 35, 108, 125 Liau G, 114, 127 Liaw L, 355, 366 Libby P, 159, 167, 175, 187 Lider O, 253, 266 Lidholt K, 335, 336, 345, 378, 381, 394, 395 Lieb T, 79, 91, 99, 106 Liebecq C, 214, 229 Lilien J, 202, 210 Lilja K, 136, 140 Lim HH, 223, 238 Limper AH, 202, 203, 211 Lin H, 295, 310 Lin HY, 299, 312 Lin KC, 364, 375, 376 Lin TH, 353, 365 Lin X, 356, 366 Lincecum J, 13, 20, 169, 171, 183 Lind T, 381, 395 Lindahl U, 15, 21, 292, 304, 308, 317, 337, 347, 378, 381, 393, 394, 395 Lindblad S, 82, 102

428 Linden M, 220, 237 Lindholt K, 378, 394 Lindner V, 362, 373 Lindstedt G, 137, 141 Lindstedt K, 300, 314 Lindzvist U, 137, 139, 141 Line BR, 192, 207 Ling LE, 355, 362, 366, 373 Linhardt RJ, 324, 330, 379, 381, 387, 389, 390, 391, 395, 398 Linnoila RI, 41, 50, 52 Liotta LA, 86, 104, 158, 167, 218, 233, 246, 264 Liou TG, 325, 331 Liszio C, 10, 18, 215, 216, 230, 232, 327, 333 Little CB, 8, 17, 327, 333, 336, 345 Little CD, 306, 320 Little SA, 58, 70 Litwack ED, 56, 59, 69, 300, 303, 313, 316 Liu B, 56, 69 Liu C, 76, 96 Liu CY, 11, 19, 175, 187 Liu D, 362, 373 Liu J, 9, 10, 18, 64, 71, 115, 128, 175, 177, 187, 218, 234, 300, 314 Liu JY, 118, 131, 205, 212 Liu M, 64, 71, 91, 92, 106, 108, 123, 175, 177, 187 Liu MJ, 76, 95 Liu P, 79, 80, 82, 87, 88, 99, 110, 117, 119, 122, 126, 326, 331 Liu RQ, 8, 17 Liu W, 56, 59, 69, 303, 316 Liu X, 386, 397 Liu XD, 362, 372 Lloyd A, 246, 263 Lloyd DA, 344, 350 Lloyd IJ, 336, 346 Lo SK, 357, 369 Lobb RR, 364, 375 Lode H, 323, 329 Lodish HF, 299, 312 Loftis AY, 25, 26, 32 Loftus JC, 201, 210, 352, 365

Author Index Lohi J, 218, 234 Lohmeyer J, 340, 349 Lokeshwar VB, 108, 114, 115, 117, 118, 119, 124, 127, 344, 350 Lomazov IR, 305, 318 Long CJ, 175, 187 Longaker MT, 27, 33, 81, 100 Longas MO, 216, 231 Longati P, 115, 128 Longenecker G, 10, 19, 172, 184, 258, 267 Longley RL, 356, 367 Looney RJ, 243, 244, 261, 262 Lopes AAB, 47, 53 Lopes EA, 47, 53 Lopez F, 304, 305, 318 Lopez-Casillas F, 14, 21 Lopez-Vidriero MT, 269, 286 Lorenzo P, 27, 33, 171, 183, 213, 228 Lories V, 300, 313 Lorimer S, 220, 237 Lormeau JC, 378, 394, 395 Lortat-Jacob H, 304, 317 Loschmann P, 181, 190 Lose E, 378, 394 Lose EJ, 294, 309 Losty PD, 344, 350 Loushin C, 248, 265, 295, 309 Lovvorn HN, 81, 100 Low RB, 195, 208 Lowell CA, 363, 374 Lowenstein CJ, 27, 31, 33, 75, 82, 93, 108, 113, 124, 125 Lu CJ, 117, 118, 119, 122, 130 Lu H, 84, 103, 357, 368 Luca G, 153, 161, 162, 167 Lucey EC, 61, 70, 158, 167 Lucher TF, 377, 392 Ludwig M, 50, 53, 177, 188, 223, 238, 326, 331, 332, 340, 348 Ludwig MS, 10, 19, 55, 56, 57, 58, 59, 60, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 113, 127, 147, 165, 170, 176, 178, 183, 188, 223, 238, 247, 265 Lui J, 146, 165 Lui M, 145, 164

Author Index

429

Lundberg E, 324, 330 Lundgren R, 85, 103, 117, 130, 281, 288 Lundmark K, 303, 316 Lung lR, 218, 234 Lunn WS, 328, 333 Luo J, 305, 319 Lurie S, 86, 104, 118, 120, 131, 361, 370 Lusis AJ, 307, 320 Lustig F, 304, 317 Lutchen KR, 57, 70 Luukonen M, 76, 81, 96 Lwebuga-Mukasa JS, 359, 369 Lympany PA, 340, 349 Lynn BD, 116, 117, 129 Lynn WS, 108, 123, 270, 280, 286, 288 Lyon M, 13, 21, 174, 186 Lyon NB, 37, 38, 52, 379, 395

M Ma B, 258, 267 Ma YS, 246, 263 Mabilat C, 84, 103 Mabry M, 41, 50, 52 MacCallum D, 76, 81, 96 MacDonald ED, 216, 231 Mackarel AJ, 324, 329 MacKenzie A, 14, 21 MacKie RM, 75, 94 Mackool RJ, 27, 33 Maclean D, 82, 102 MacNaul KI, 246, 264 MacNee W, 254, 267 Madden J, 226, 239 Madison R, 254, 266 Madri JA, 361, 372 Madsden Cort S, 203, 211 Maeda A, 118, 131 Maeda K, 295, 309, 359, 370 Maeda M, 359, 370 Maeda T, 361, 372 Maesterelli P, 220, 236, 242, 261 Magie AR, 242, 260 Magnan A, 224, 225, 226, 239

Magnuson T, 11, 19 Magny M-C, 10, 18 Mah AF, 344, 350 Mai BH, 79, 88, 99 Maibach HI, 75, 93 Mailleau C, 277, 287 Mainiero F, 355, 361, 365, 371 Maino G, 204, 211 Majerus PW, 300, 313 Majima T, 64, 71 Majno G, 204, 211 Majumdar S, 220, 237 Mak TW, 29, 34, 79, 98 Malava L, 217, 233, 356, 367 Maldonado V, 147, 159, 166 Mali M, 12, 21 Malik AB, 357, 369 Malinowski NM, 75, 95 Malizia G, 202, 211 Malm MG, 364, 375 Malmstrom A, 56, 66, 69, 75, 93, 94, 118, 131, 170, 174, 175, 176, 183, 185, 186, 187, 188, 203, 211, 214, 223, 229, 238, 243, 258, 261, 267, 344, 350 Malo JL, 177, 188, 221, 237 Manabe R, 361, 372 Mandl I, 119, 132 Mangeat P, 114, 127 Mangoura D, 339, 348 Mani K, 300, 313, 337, 338, 347 Maniscalco WM, 176, 188, 223, 238 Mann DM, 173, 174, 185, 218, 234, 251, 265, 304, 317 Mann K, 11, 20 Mann MM, 171, 183, 295, 310, 339, 348 Mano M, 385, 397 Manolitsas ND, 228, 240 Mant MJ, 80, 100, 115, 117, 118, 128, 129 Maoz M, 357, 368 Mapp CE, 220, 236, 242, 261 Marcantonio EE, 355, 365 Marchi E, 378, 395 Marchisio PC, 361, 370

430 Marchuk LL, 64, 71 Marconi A, 361, 370 Marcum JA, 301, 315, 377, 392 Marek VW, 113, 126 Margetts PJ, 47, 49, 53, 173, 180, 185, 189, 219, 236, 253, 266, 363, 374 Margolis RK, 338, 347 Margolis RU, 338, 347 Margulis A, 27, 32, 76, 96 Mariani E, 148, 152, 166 Mariani TJ, 178, 188 Marineli W, 117, 130 Mark MP, 338, 347 Markwald RR, 28, 34, 201, 210 Maroko PR, 82, 102 Maroudas A, 5, 17 Mars WM, 300, 314 Marshall BC, 224, 238 Marshall JF, 79, 99, 116, 117, 129 Marshall RP, 118, 131 Martel-Pelletier J, 243, 246, 262, 264 Martensson G, 135, 136, 137, 139, 140, 141, 142 Martin CJ, 61, 70 Martin GA, 385, 397 Martin GR, 146, 147, 165, 202, 203, 211, 377, 392 Martin JG, 361, 371 Martin JP, 3, 16 Martin PT, 356, 367 Martin RJ, 220, 237 Martin TR, 362, 373 Martinet N, 117, 130 Martinet Y, 117, 130, 202, 203, 211 Martinussen HJ, 82, 102 Maruyama I, 300, 313 Maruyama M, 324, 330 Maruyama T, 387, 398 Marynen P, 146, 165, 300, 313, 314 Masellis-Smith A, 117, 129 Maslen K, 6, 17 Mason DP, 300, 315, 356, 367 Mason RJ, 113, 117, 127, 130, 200, 209 Mason RM, 307, 321 Massague J, 14, 21, 180, 189, 217, 218, 233, 234, 299, 312

Author Index Massey FJ, 242, 260 Masta S, 243, 261 Masterson JB, 147, 158, 165, 247, 264 Mastrangelo AM, 363, 374 Masuda J, 307, 321 Masuda T, 324, 330 Mathay MA, 203, 211 Matheke ML, 244, 262 Mathews MC, 295, 311 Mathieson JM, 280, 288 Mathieu-Costello O, 65, 72 Matijevic-Aleksic N, 300, 313 Matozaki T, 115, 128 Matrisian LM, 246, 263 Matsuda H, 307, 321 Matsui H, 6, 17 Matsumoto T, 219, 235 Matsumura Y, 78, 97 Matthay MA, 87, 105, 118, 131, 357, 362, 369, 373 Matzura D, 295, 310 Maurel P, 295, 309, 338, 347 Mauviel A, 175, 187, 215, 230 May AJ, 57, 69 Mayne R, 172, 179, 184, 216, 231 McAnulty J, 340, 349 McAnulty RJ, 118, 131, 223, 238, 340, 348 McAulay A, 228, 240 McBride OW, 214, 230 McCaffrey TA, 299, 312 McCallion RL, 81, 100 McCarter JH, 220, 236 McCarthy C, 271, 287 McCarthy JB, 79, 88, 98, 202, 210 McCarthy L, 117, 130 McCartney-Francis N, 86, 104 McCluskey P, 246, 263 McCormick JR, 219, 235 McCormick LL, 181, 189 McCoshen J, 91, 106 McCourt PA, 108, 124 McCourt PAG, 82, 101 McCulloch CA, 246, 263 McDonald JA, 2, 16, 24, 28, 30, 31, 32, 34, 76, 79, 95, 96, 99, 110, 126, 194,

Author Index 202, 203, 207, 208, 211, 243, 244, 261, 262, 337, 346, 361, 372 McElvaney NG, 147, 158, 159, 165, 167, 324, 330 McEver RP, 305, 319 McFall AJ, 303, 316 McGeehan GM, 246, 263 McGowan SE, 85, 103, 218, 234, 243, 261, 327, 333 McGuffee LJ, 58, 70 McKee CM, 27, 30, 31, 33, 35, 75, 82, 93, 108, 113, 120, 124, 125, 132 McKeown-Longo PJ, 361, 372 McLaughlin MK, 58, 70 McLeskey SW, 336, 346 McMahon KJ, 247, 264 McManus BM, 295, 310 McNamer R, 218, 234 McNulty CA, 364, 375 McPherson JD, 79, 99, 307, 321 McPherson JM, 75, 93, 362, 372 McQuibban GA, 200, 209, 246, 263 McQuillan CI, 172, 184, 278, 287 McQuillan DJ, 9, 10, 18, 19, 172, 174, 179, 184, 185, 213, 214, 216, 228, 230, 231, 304, 317 McQuillan JJ, 243, 261 McReynolds RA, 219, 235 McSharry C, 244, 262 McTavish AJ, 356, 366 Meade JB, 301, 316 Mecham RP, 173, 178, 185, 188, 215, 231, 300, 315 Meckler Y, 278, 287 Meehan WP, 355, 366 Meek LM, 6, 17 Meijne AML, 202, 210 Meininger GA, 355, 366 Meisler N, 86, 104 Melching LI, 218, 234 Melchiori A, 361, 370 Melder RJ, 306, 320, 356, 367 Melendez J, 147, 159, 166 Melia S, 281, 288 Melnick M, 77, 97 Melo F, 228, 240

431 Melrose J, 295, 303, 310, 316 Menard O, 117, 130 Menasche M, 27, 33 Mendis DB, 173, 185 Mendoza V, 14, 21 Meneguzzi G, 361, 370 Mercer EW, 173, 174, 185 Mercurio AM, 355, 365 Merendino A, 224, 226, 239 Mergey M, 277, 287 Merle B, 174, 186, 217, 233, 356, 357, 367 Merrilees MJ, 75, 94, 295, 300, 309, 315, 336, 346 Merten MD, 278, 287 Mertens G, 300, 313 Messadi DV, 78, 98 Messent AJ, 352, 365 Messmore HL, 336, 346 Meuli M, 80, 100, 340, 348 Meurman LO, 136, 140 Meyer K, 23, 31, 74, 92, 136, 141 Meyer MF, 27, 33 Meyer TF, 357, 368 Meyers D, 243, 261 Meyrick B, 377, 392 Miao HQ, 246, 263 Midura RJ, 325, 331 Mijailovich SM, 57, 70 Mikecz K, 29, 34 Milani GF, 220, 236 Milani MR, 378, 395 Milewicz DM, 307, 321 Milici AJ, 64, 71 Millane RP, 336, 346 Miller EJ, 25, 26, 32 Miller LJ, 352, 365 Milner TA, 359, 369 Milstone LM, 15, 21 Min KU, 221, 237 Minami M, 377, 392 Mineau F, 246, 264 Minshall EM, 220, 237 Minshall RD, 357, 369 Mio T, 244, 259, 262, 267 Misenheimer TM, 203, 211

432 Miserocchi G, 147, 148, 150, 152, 153, 154, 155, 158, 160, 161, 162, 163, 165, 166, 167, 168, 342, 343, 349, 357, 368 Misevic GN, 50, 53 Mishra-Goruk K, 385, 397 Mitchell J, 195, 208 Mitchell L, 174, 185 Mitsui Y, 84, 102 Miura R, 200, 202, 209, 210 Miwa H, 300, 314 Miyamori I, 300, 313 Miyamoto S, 353, 365 Miyasaka M, 68, 72, 174, 186, 200, 201, 209, 363, 374 Miyauchi S, 2, 16, 24, 28, 32, 76, 95, 110, 126, 337, 346 Mizumoto N, 81, 88, 101 Mizutani A, 307, 321 Mjaatvedt CH, 28, 34, 201, 210 Moats-Staats BM, 223, 238 Modig J, 85, 103, 117, 130 Mohamadzadeh M, 81, 82, 88, 101, 120, 132 Mohapatra S, 116, 129 Mohri H, 357, 368 Moisander S, 299, 311 Molano I, 362, 373 Molet S, 361, 371 Molina C, 220, 236 Molist A, 243, 261 Moll J, 31, 35, 78, 79, 88, 97, 98, 99 Mongini T, 284, 289 Monick M, 244, 262 Monick MM, 343, 350 Monnens LA, 146, 165, 176, 188 Monnens LAH, 11, 20 Montandon D, 204, 211 Montano M, 243, 261, 359, 369 Montcourrier P, 114, 127 Montesano R, 83, 102 Monthouel MN, 361, 370 Monti G, 224, 239 Mooi WJ, 79, 98 Mooradian DL, 79, 88, 98

Author Index Moore D, 79, 99 Moore K, 120, 133 Morales TI, 75, 92 Moreglin M, 172, 184 Moreno R, 221, 237 Morgan JL, 214, 229 Morgelin M, 11, 20, 217, 232 Mori A, 174, 185 Mori Y, 75, 82, 93, 94 Morimoto K, 84, 103 Morin J, 10, 19, 56, 69, 147, 165, 170, 176, 183, 223, 238, 247, 265 Morisaki N, 377, 392 Morison HM, 325, 331 Morita H, 295, 309 Moriyama K, 361, 371 Morr H, 340, 349 Morrelli MA, 108, 124 Morris CP, 5, 17 Morris D, 86, 103 Morris DR, 385, 397 Morris HR, 387, 397 Morrison HM, 324, 325, 330, 331 Mort JS, 9, 10, 18, 19, 325, 331 Mortenson RL, 120, 132 Morton A, 221, 237 Morton LF, 352, 365 Moscatelli D, 377, 393 Moscatello DK, 10, 19, 174, 185, 304, 317 Moseley PL, 118, 132, 340, 348 Moses HL, 86, 104, 223, 238 Moses J, 214, 229, 337, 338, 347 Moses MA, 246, 263 Mosher DF, 203, 211, 356, 367 Moskal TL, 361, 370 Motomiya M, 244, 262, 280, 288 Mould AP, 357, 368 Mount LE, 57, 69 Mouta-Carreira C, 306, 320, 356, 367 Movat H, 39, 52 Mow VC, 55, 58, 63, 69, 71 Mowat M, 79, 99 Mueller SH, 377, 393 Mukherji B, 118, 131

Author Index

433

Muller E, 139, 142 Muller M, 88, 105, 113, 119, 120, 127, 132, 215, 230, 304, 318, 327, 333 Muller NL, 243, 261 Muller-Quernheim J, 362, 373 Mulligan MS, 363, 374 Mullins RD, 117, 129 Mullins S, 364, 376 Mulloy B, 337, 346, 379, 395 Mummert DI, 81, 88, 101 Mummert ME, 81, 88, 101 Munaim SI, 111, 126 Munger JS, 87, 105, 203, 211, 217, 233, 357, 369 Munn LL, 306, 320, 356, 367 Munoz A, 57, 70, 242, 260 Munro SB, 78, 97 Murakami H, 110, 126 Murakami T, 9, 18 Murdoch AD, 11, 20, 56, 69, 145, 146, 165, 294, 303, 308, 316, 378, 393 Murphy G, 218, 234 Murphy HS, 327, 332 Murphy LC, 175, 187 Murphy-Ullrich JE, 87, 105, 203, 211, 217, 233, 303, 316 Murray JF, 271, 287 Murray MJ, 362, 372 Murry CE, 355, 366 Musson RA, 117, 130 Mustoe TA, 356, 366 Muszynsky M, 159, 167

N Nackaerts K, 41, 50, 52 Nadal-Ginard B, 218, 233 Nagai H, 244, 262 Nagai S, 244, 262 Nagakubo D, 68, 72, 174, 186, 201, 209 Nagase H, 159, 167 Nagata M, 364, 375 Nagayasu Y, 357, 368 Nagura H, 228, 239 Nagy JI, 116, 117, 129

Naito M, 82, 101, 377, 392 Nakajima S, 364, 375 Nakamura T, 75, 82, 93, 148, 152, 166, 174, 186 Nakamura Y, 221, 237, 259, 267 Nambi P, 76, 96 Nance DM, 79, 99, 120, 132 Nannizzi L, 352, 365 Naot D, 76, 77, 96 Nara Y, 201, 209 Narayanan AS, 243, 261 Nardell EA, 61, 71 Narendra A, 340, 349 Narlid I, 10, 19, 173, 179, 184 Naso MF, 8, 18, 199, 208, 307, 321 Natali PG, 356, 366 Nathan CF, 120, 133, 363, 374 Nathan RM, 295, 311 Naughton MA, 352, 365 Naumann U, 181, 190 Navabi H, 136, 140 Nawroth P, 377, 392 Nayak S, 78, 97 Nayer A, 194, 207 Neame PJ, 8, 9, 10, 18, 19, 171, 172, 179, 183, 184, 213, 228, 292, 308 Needham SG, 323, 329 Negrini D, 147, 148, 151, 152, 153, 154, 155, 156, 158, 160, 161, 162, 163, 165, 166, 167, 168, 342, 343, 349, 357, 368 Nelimarkka L, 295, 299, 310, 311 Nelson FC, 220, 228, 236, 240 Nelson RM, 324, 330 Neri G, 14, 21 Nerlich A, 174, 186 Nettelbladt O, 26, 32, 87, 88, 105, 113, 118, 120, 126, 131, 132, 340, 349 Neufeld G, 304, 317 Newhouse MT, 244, 262, 344, 350 Newman RA, 361, 370 Newnham J, 85, 90, 103 Newsome AM, 82, 102 Ng-Eaton E, 299, 312 Nguyen HA, 24, 31, 85, 103

434 Nguyen Q, 9, 18 Nguyen T, 75, 95 Nguyen TK, 76, 96, 109, 125 Nicod lP, 253, 266 Nicodemus CF, 15, 21 Nicole S, 12, 20 Nicosia RF, 356, 366 Nielsen LS, 218, 234 Nietfeld JJ, 27, 32, 76, 96 Niggli V, 114, 127 Nijkamp FP, 364, 376 Nikkari ST, 41, 48, 52, 295, 309, 310 Nikkari T, 377, 392 Nikol S, 248, 265, 295, 309 Nilsson B, 214, 229 Ninomiya Y, 9, 18 Nishida T, 110, 126 Nishida Y, 8, 18, 27, 32, 76, 78, 96, 98 Nishimura S, 78, 98 Nishiyama A, 299, 304, 312, 317, 338, 347 Nistico P, 356, 366 Nitta Y, 225, 226, 228, 239 Nivens CM, 10, 19 Noble NA, 10, 19, 173, 185, 224, 238 Noble PW, 27, 30, 31, 33, 35, 75, 82, 88, 93, 102, 105, 108, 120, 122, 124, 125, 132 Nochi H, 75, 82, 93 Nochlin D, 307, 321 Nocks A, 78, 97 Noguchi N, 75, 94 Noite V, 247, 264, 306, 319 Noonan DM, 146, 165 Noordhoek JA, 66, 67, 72, 181, 189, 248, 251, 255, 265, 267, 326, 332 Norman M, 174, 185 North SL, 194, 208 Norton JD, 108, 113, 123, 124 Norton L, 138, 141 Norvel TM, 326, 332 Nowak G, 204, 212 Nowlen JK, 356, 367 Nugent HM, 301, 315 Nugent MA, 181, 189, 301, 315, 338, 347

Author Index Nunohiro T, 355, 362, 366, 372 Nurminen M, 137, 139, 141 Nylund L, 381, 395

O O’Blenes S, 47, 53 O’Brien JJ, 61, 70 O’Brien KD, 41, 48, 52, 295, 309 O’Byrne P, 225, 226, 239 O’Byrne PM, 325, 331 O’Connell BC, 301, 315 O’Connor CM, 147, 165, 245, 247, 263, 264, 324, 329 O’Connor GT, 181, 190 O’Connor K, 352, 365 O’Connor R, 87, 105 O’Connor RN, 203, 204, 211 O’Donnell MD, 245, 263 O’Driscoll LR, 147, 158, 165, 247, 264 O’Farrell S, 300, 314 O’Neill LA, 30, 31, 35 O’Neill SJ, 324, 330 O’Reilly MS, 306, 320 O’Sullivan DD, 270, 274, 281, 286, 287, 288 O’Toole E, 361, 372 Obata K, 246, 264 Ochiya T, 304, 317 Ockenhouse CF, 176, 187 Odajima R, 75, 94 Odland GF, 361, 371 Oegama TR, 79, 88, 98, 202, 210 Oertli B, 27, 31, 33, 109, 125 Ogata J, 307, 321 Ogawa Y, 75, 93 Oguri K, 357, 368 Oh BH, 223, 238 Oh ES, 12, 21, 303, 316 Ohashi PS, 29, 34, 79, 98 Ohashi Y, 75, 94 Ohazaki T, 359, 370 Ohe N, 361, 372 Ohkawa T, 75, 95 Ohkawara Y, 30, 31, 35 Ohlsson K, 324, 330

Author Index Ohmori K, 6, 17 Ohnishi K, 147, 166, 247, 264 Ohno I, 225, 226, 228, 239 Ohno S, 76, 95 Ohnuki Y, 2, 16, 76, 95, 110, 126, 337, 346 Ohnuku Y, 24, 28, 32 Ohtani H, 228, 239 Ohtsuka K, 51, 53 Oida K, 300, 313 Okada A, 27, 33 Okada E, 300, 313 Okada Y, 227, 239, 246, 258, 263, 267, 300, 313 Okamoto K, 108, 123 Okazawa M, 200, 208 Oksala O, 120, 132 Okubo T, 357, 368 Okuda S, 219, 235 Oldberg A, 10, 19, 56, 66, 69, 118, 131, 170, 171, 172, 173, 176, 179, 183, 184, 188, 214, 216, 217, 223, 229, 232, 238, 258, 267, 295, 300, 306, 309, 314, 319, 337, 338, 347 Oliferenko S, 30, 31, 35, 115, 128 Olin KL, 304, 318 Olivenstein R, 50, 53, 68, 72, 177, 188, 223, 238, 326, 331 Oliver BL, 30, 31, 35, 108, 125 Oliver RT, 118, 131 Olivieri D, 177, 188, 221, 228, 237, 240 Ollerenshaw SL, 220, 236 Olman MA, 25, 26, 32, 108, 124, 125 Olsen AS, 28, 33 Olsen BR, 306, 320 Olson JS, 24, 28, 31 Olson PJ, 243, 261 Olver RE, 162, 168 Olwin BB, 304, 317 Onley DJ, 352, 365 Onnerfjord P, 171, 183, 213, 228 Onnervik PO, 174, 186 Ono T, 272, 287 Onodera K, 361, 371 Onstan MW, 326, 332 Oohashi T, 9, 18

435 Ooi CCG, 326, 332 Oorni K, 304, 305, 318, 319 Opaskar-Hincman H, 270, 274, 286, 287 Orci L, 83, 102 Ordille S, 344, 350 Orellana A, 381, 395 Orford CR, 216, 231 Ornitz DM, 304, 317 Oronsky AL, 327, 333 Orsini LF, 339, 348 Ortega M, 323, 329 Ortonne JP, 361, 370 Osada R, 6, 17 Osame M, 364, 375 Osborn R, 362, 373 Osbourn JK, 295, 310 Osende JI, 307, 320 Osima B, 386, 397 Osman M, 26, 32, 87, 91, 93, 105, 106, 108, 123, 339, 348 Otterness IG, 64, 71 Overall CM, 200, 209, 246, 263, 359, 369 Overstreet JW, 27, 32 Overton JE, 61, 70 Owens GK, 203, 205, 211, 212 Owens R, 79, 99 Ozesmi M, 136, 140

P Paakko P, 324, 330 Pace E, 224, 226, 239 Pacific R, 363, 374 Page C, 181, 190 Page CP, 336, 346 Paige CJ, 29, 34, 79, 98 Paine D, 115, 118, 128 Pajak TF, 138, 141 Pajevic S, 269, 286 Pal S, 214, 229 Palecek SP, 201, 210 Palfrey AJ, 279, 288 Palmer GD, 6, 17 Palmer JW, 23, 31, 74, 92

436 Palmieri G, 361, 371 Palmucci L, 284, 289 Pan Q, 359, 370 Pan T, 113, 127 Panares R, 78, 98, 299, 312 Pang G, 244, 262, 323, 329 Panos RJ, 192, 207 Panoutsakopoulo V, 361, 370 Pansky A, 361, 371 Pantelidis P, 223, 238 Papakonstantinou E, 75, 94, 109, 125, 340, 341, 342, 343, 349, 350 Pappano WN, 9, 18 Parameswaran S, 113, 126 Pardo A, 147, 159, 166, 243, 247, 261, 264, 359, 369 Pare JA, 197, 201, 208 Pare PD, 113, 126, 197, 200, 201, 208, 209, 221, 237, 251, 265 Parekh T, 243, 261 Park JH, 223, 238 Park PW, 13, 20, 169, 171, 183 Park Y, 387, 398 Parker JC, 65, 72 Parker S, 307, 321 Parkos CA, 357, 368 Parks JS, 304, 318 Parks WC, 178, 188, 202, 210 Parmley RT, 284, 289 Parner J, 221, 237 Parshley MS, 119, 132 Partenheimer A, 295, 311 Parthasarathy N, 174, 185 Pasonen S, 76, 95, 109, 126 Pasqualini R, 299, 312 Passi A, 147, 151, 153, 155, 156, 158, 161, 162, 163, 165, 166, 167, 168, 305, 319, 342, 343, 349, 357, 368 Pasteur MC, 323, 329 Pastore C, 307, 320 Patel AS, 115, 128 Patel KD, 364, 375 Patterson E, 362, 372 Paul A, 270, 277, 286, 287 Paulsson M, 216, 232, 300, 315 Pavasant P, 75, 94

Author Index Payden DM, 363, 374 Payne BJ, 278, 288 Peach RJ, 77, 97 Peachey AR, 352, 365 Pearce RH, 10, 18 Pearson CA, 218, 234 Pearson CH, 214, 229 Pearson D, 218, 234 Pearson RSB, 220, 236 Peck D, 340, 348 Pellacani A, 78, 98, 299, 312 Pelletier JP, 64, 71, 243, 246, 262, 264 Pelliniemi L, 295, 310 Pelton RW, 223, 238 Peltonene L, 214, 230 Penc SF, 174, 186, 306, 320, 336, 346 Pendino KJ, 86, 104 Penettieiri RA, 78, 97 Penneys NS, 78, 98 Penno MB, 27, 31, 33, 41, 50, 52, 75, 82, 93, 120, 132 Pentikainen MO, 304, 305, 318, 319 Pentikainen O, 352, 365 Pentland AP, 362, 373 Penttinen CA, 218, 234 Pepinsky B, 364, 375 Pepinsky RB, 364, 376 Pepper C, 136, 140 Pepper MS, 83, 102 Perez J, 147, 159, 166 Perez RL, 363, 374 Perin JP, 299, 311 Peritt D, 78, 97 Perkett EA, 223, 238 Perlish JS, 216, 231 Perlmutter LS, 307, 321 Perrard J, 357, 368 Perrella MA, 78, 97, 98, 299, 312 Perruchoud AP, 109, 125, 340, 341, 343, 349, 350, 361, 371 Pertoft H, 83, 84, 102, 136, 140 Pesci A, 177, 188, 221, 228, 237, 240 Pesti D, 325, 331 Petaja J, 174, 185, 301, 316 Peten EP, 377, 393 Peters AM, 323, 329

Author Index Peters KW, 282, 288 Peters SP, 327, 332 Petersen F, 326, 332 Petersen OW, 361, 371 Peterson M, 117, 130 Peterson MW, 244, 262 Petitou JC, 378, 394 Petitou M, 378, 394 Petkov V, 180, 189 Petracek PJ, 387, 398 Petri JB, 361, 370 Pettersson I, 381, 395 Peuchmaur M, 224, 239 Pfeil U, 223, 238 Phan SH, 86, 104, 118, 120, 131, 195, 208, 362, 372 Philip A, 299, 312 Philips PG, 340, 349 Phillips DR, 352, 365 Phillips NC, 75, 93 Phipps RP, 243, 244, 261, 262 Piani S, 378, 395 Piantadosi CA, 280, 288 Picard J, 277, 287 Piccoli M, 361, 371 Picker LJ, 82, 101, 120, 132 Pidkiti D, 86, 104 Pienimaki JP, 76, 81, 95, 96, 109, 126 Pierce RA, 178, 188 Pierce RH, 280, 288 Pierschbacher M, 219, 235, 236, 295, 309 Pierschbacher MD, 87, 105, 173, 180, 185, 214, 227, 229, 239, 246, 263, 352, 365 Pietra GG, 42, 48, 52 Piez KA, 224, 239, 295, 311 Pignatelli M, 362, 373 Pilarski LM, 79, 80, 98, 99, 100, 115, 117, 118, 128, 129 Pilia G, 14, 21 Pineo GF, 344, 350 Pircher R, 217, 233 Pitossi F, 180, 189, 363, 374 Pittet JF, 86, 87, 103, 105, 118, 131, 203, 211, 217, 233, 357, 369

437 Pivirotto F, 220, 236 Plaas AH, 68, 72, 174, 186, 201, 209 Plaas AHK, 10, 19 Platt JL, 11, 20 Plenz G, 301, 315 Plug R, 79, 99, 116, 129 Pluschke G, 299, 312 Pociask D, 118, 131 Poelmann RE, 362, 373 Pogany G, 247, 264 Pohl W, 117, 130 Poiania GJ, 163, 168 Politano VA, 344, 350 Polizzi E, 363, 375 Polk DH, 85, 90, 103 Pollice MB, 327, 332 Pollman DJ, 25, 26, 32 Pomahac B, 174, 186, 306, 320, 336, 346 Ponce-Castaneda MV, 14, 21 Ponta H, 28, 31, 34, 35, 76, 77, 78, 79, 88, 96, 97, 98, 99, 116, 129, 340, 348 Ponting J, 108, 123, 306, 320 Poole AR, 214, 229, 325, 331 Pooler PM, 79, 84, 99, 114, 115, 117, 118, 127 Popescu O, 50, 53 Post M, 64, 71, 175, 177, 187 Postelthwaite AE, 86, 104 Postma DS, 66, 67, 72, 181, 189, 242, 248, 251, 255, 261, 265, 267, 325, 326, 331, 332 Potempa J, 324, 330 Potter-Perigo S, 175, 187, 202, 210, 304, 318 Pouliquen Y, 27, 33 Poulter LW, 117, 130, 323, 329 Povey HG, 205, 212 Powell A, 292, 308, 378, 394 Power C, 200, 209 Powers CJ, 336, 346 Pozzi A, 356, 367 Prakash S, 295, 310 Pralle H, 340, 349 Pratt BM, 75, 93 Pratt CW, 301, 316

438

Author Index

Pratt PC, 242, 261 Pratta M, 8, 17 Prehm P, 30, 34, 75, 79, 95, 99, 136, 140 Prestwich R, 115, 117, 128 Prevo R, 114, 127 Prince JT, 299, 312 Pringle GA, 214, 216, 229, 231 Prober DA, 80, 100, 116, 129 Prockop DJ, 204, 212 Proctor KG, 82, 102 Proud D, 91, 106 Pruski E, 115, 118, 128 Prydz K, 2, 4, 16, 336, 346 Ptela R, 355, 366 Pugin J, 362, 373 Pujol JP, 215, 218, 230, 234 Pukac LA, 385, 397 Pulkkinen L, 214, 230 Pullen M, 76, 96 Pure E, 29, 30, 31, 34, 35, 78, 97, 108, 114, 125, 127 Pye D, 84, 102

Q Qiu XL, 113, 126 Qu R, 299, 312 Quan C, 364, 376 Qui S, 361, 371 Quinn DA, 385, 397 Quinn MT, 357, 368 Quinones F, 26, 32 Qun L, 79, 99 Qwarnstrom EE, 303, 316

R Raab G, 246, 263 Raab M, 117, 129 Rabin CB, 135, 140 Rabinovitch M, 47, 49, 53, 75, 94, 300, 315, 360, 370 Rabinovitch PS, 244, 262 Racine-Samson L, 362, 373

Radhakrishnamurthy B, 304, 318, 336, 346 Rae F, 255, 267 Raghow R, 86, 104, 105, 118, 120, 131, 219, 235, 361, 370, 372 Raghu G, 194, 208, 243, 244, 248, 261, 262, 265 Rahemtulla F, 284, 289 Rahman A, 357, 369 Rahman I, 254, 267 Rahmanian M, 27, 33, 83, 84, 102, 145, 163, 164, 338, 347 Rahmoune H, 174, 186, 270, 286, 327, 333 Rain B, 224, 239 Raines EW, 201, 209, 295, 310, 355, 366 Raj JU, 163, 168 Raj NS, 11, 20 Rajagopal S, 361, 370 Rak J, 29, 34, 79, 98 Rall CJ, 78, 97 Ramamurthy P, 214, 230 Ramirez F, 86, 104 Ramirez R, 243, 261 Ramis I, 244, 262 Ramos C, 243, 261, 359, 369 Ramos MA, 356, 367 Ramos P, 305, 319 Ramos-Barbon D, 361, 371 Ramsden CA, 162, 168 Ranheim T, 299, 312 Rannels DE, 244, 262, 325, 331, 336, 345 Rapport CA, 377, 392 Rapraeger AC, 303, 304, 316, 317, 336, 346, 378, 394 Raskob GE, 344, 350 Rasmussen LM, 172, 173, 180, 184, 218, 235, 251, 265 Raspanti M, 40, 50, 52 Ratcliffe A, 6, 17, 63, 71 Rauch U, 214, 215, 230, 338, 347 Rauterberg J, 10, 19, 248, 265 Rawson T, 364, 376 Reardon IM, 254, 266

Author Index Reaves TA, 362, 373 Recklies AD, 9, 18, 176, 188, 218, 234 Redard M, 205, 212 Redding GJ, 90, 106 Redi L, 377, 392 Redington AE, 179, 188, 220, 221, 226, 236, 238, 239 Redini F, 215, 218, 230, 234 Reed RK, 136, 140 Reekmans G, 146, 165, 300, 313 Rees SG, 327, 333 Reeves G, 323, 329 Reggiani D, 378, 386, 395, 397 Rehkter MD, 195, 208 Reid L, 269, 270, 271, 279, 281, 284, 286, 287, 288, 289 Reid LM, 274, 278, 287, 288 Reijnen MM, 108, 124 Reilly CF, 301, 315, 336, 346, 384, 397 Reina M, 357, 368 Reiner A, 214, 229 Reinholt FP, 10, 19, 173, 184 Reisfeld RA, 300, 313 Reiss L, 10, 19 Reiter R, 214, 229 Reizes O, 13, 20, 169, 171, 183 Reizis B, 253, 266 Remington FL, 49, 53 Ren A, 323, 329 Ren XD, 353, 365 Renkin EM, 151, 166 Renkl AC, 79, 88, 99 Rennard SI, 259, 267, 362, 372 Rennke H, 357, 368 Renouf DV, 344, 350 Renshaw MW, 353, 365 Renz M, 364, 376 Repine AJ, 362, 373 Repine JE, 362, 373 Resifeld R, 299, 312 Retornaz F, 225, 226, 239 Reunanen H, 355, 366 Rey F, 135, 138, 140, 142 Reynolds CC, 284, 288 Reynolds JJ, 218, 234 Ribeiro SM, 217, 233

439 Ribeiro SP, 64, 72 Riccio A, 218, 234 Richards IM, 364, 375 Richardson PS, 278, 288 Riches DW, 31, 35, 75, 82, 93, 120, 132, 133 Richter H, 247, 264, 306, 319 Ricou B, 362, 373 Rider CC, 335, 345 Ridgway CC, 217, 233 Rieman DJ, 362, 373 Riese RJ, 258, 267 Riessen R, 248, 265, 295, 307, 309, 320 Rietveld FJR, 299, 300, 312, 313 Rifkin DB, 87, 105, 203, 211, 377, 393 Rigatti B, 4, 11, 16, 20 Riikonen T, 361, 371 Rijcken B, 242, 261 Riley DJ, 85, 103, 163, 168, 254, 267 Riley GP, 377, 393 Rilla K, 76, 81, 95, 96, 109, 126 Riolo RL, 284, 288 Risau W, 83, 102 Risher LW, 10, 18 Risso AM, 361, 371 Ritchie M, 172, 184, 216, 232 Rittling SR, 361, 370 Ritzenthaler J, 359, 363, 369, 374 Rivera KE, 25, 26, 32 Rivers PA, 6, 17 Rizzo A, 224, 226, 239 Robbins JR, 64, 71 Robenek H, 10, 19, 247, 248, 264, 265, 301, 306, 315, 319 Robert L, 27, 33 Roberts AB, 86, 104, 218, 224, 233, 238, 239 Roberts CJ, 243, 261 Roberts CR, 27, 32, 41, 52, 55, 56, 61, 68, 68, 69, 70, 72, 108, 113, 123, 126, 144, 145, 146, 163, 164, 178, 188, 196, 197, 200, 201, 202, 206, 208, 209, 212, 222, 223, 238, 295, 310, 326, 331 Roberts DD, 203, 211 Roberts R, 377, 393

440 Robertson GW, 57, 69 Robey PG, 171, 172, 183, 184, 221, 238, 258, 267, 295, 309 Robin E, 84, 103 Robinson J, 336, 345 Robinson MK, 364, 375 Robinson SD, 356, 367 Roboz J, 137, 139, 141 Roby JD, 178, 188, 202, 210 Rocamora N, 357, 368 Roche NS, 86, 104, 218, 233 Roche WR, 179, 188, 204, 212, 220, 221, 226, 236, 238, 239 Rochester CL, 340, 348 Rochon Y, 357, 368 Rockey DC, 359, 362, 369, 373 Rockey RC, 108, 125 Rodbard D, 272, 287 Roden L, 284, 289 Rodriguez I, 29, 34 Rodriguez-Mazzaneque JC, 202, 211, 327, 332 Rodriquez I, 81, 101 Rogers AV, 220, 228, 237, 240 Roghley PJ, 326, 332 Rohrback KA, 49, 53 Rokaw SN, 242, 260 Roller ML, 79, 99 Rom WN, 202, 203, 211, 340, 349, 359, 370 Roman J, 117, 130, 359, 363, 369, 374 Romanic AM, 362, 373 Romaris M, 172, 173, 180, 184, 218, 235, 243, 251, 261, 265 Romberger DJ, 259, 267, 362, 372 Romer LH, 356, 366 Ron D, 304, 317 Ronnov-Jessen L, 361, 371 Rooney P, 84, 102, 108, 113, 123, 124, 306, 320 Roongta U, 117, 130, 201, 209 Roos D, 79, 98, 357, 369 Roos E, 79, 98 Ropraz P, 361, 371 Rosada C, 41, 50, 52 Rose JW, 352, 365

Author Index Rose MC, 278, 282, 287, 288 Rosen DM, 224, 239, 295, 311 Rosenberg L, 357, 368 Rosenberg LC, 9, 18, 214, 217, 229, 233, 301, 303, 306, 315, 316, 319 Rosenberg RD, 146, 165, 300, 314, 336, 346, 377, 378, 392, 395 Rosenfeld L, 91, 106 Rosengren B, 304, 305, 317, 318 Rosenhall L, 85, 103, 117, 130 Rosenquist TH, 362, 372 Roser S, 363, 374 Rosner B, 242, 260 Ross OH, 8, 17 Ross R, 201, 202, 209, 211, 295, 307, 310, 320, 355, 366 Rosseau S, 340, 349 Rossi F, 363, 374 Rossi GA, 117, 130 Rossing TH, 270, 286 Roswit WT, 362, 373 Roth M, 340, 342, 343, 349, 350, 361, 371 Roth MR, 75, 94, 109, 125, 175, 187, 295, 311 Roth SI, 356, 366 Rothman VL, 305, 318 Roughley PJ, 9, 10, 18, 19, 55, 56, 58, 59, 60, 64, 66, 67, 69, 71, 72, 113, 127, 147, 165, 170, 172, 175, 176, 178, 183, 184, 187, 188, 218, 223, 234, 238, 247, 265, 325, 331, 340, 348 Rouillard D, 361, 370 Roussel P, 270, 286, 327, 333 Roy C, 114, 127 Rubin K, 204, 211, 362, 372 Rubinchik E, 244, 262 Ruch J, 338, 347 Rudd CE, 117, 129 Ruegg MA, 146, 165 Ruggieri A, 40, 50, 52 Ruggiero M, 378, 393 Ruggiero SL, 81, 100 Ruiter DJ, 299, 300, 312, 313 Ruiz V, 147, 159, 166

Author Index

441

Rulop C, 76, 95 Ruoslahti E, 8, 10, 17, 19, 67, 72, 171, 172, 173, 180, 183, 184, 199, 200, 202, 208, 209, 210, 214, 218, 219, 229, 234, 235, 251, 252, 265, 266, 278, 287, 292, 294, 295, 299, 300, 304, 308, 309, 312, 314, 317, 335, 336, 345, 346, 352, 365 Rusch V, 244, 262 Russel LR, 147, 158, 165 Russell KJ, 247, 264, 324, 329 Rustgi AK, 78, 97 Ryan GB, 204, 211 Ryan ST, 87, 105, 219, 235 Rymaszewski Z, 307, 321

S Saamanen AM, 75, 93 Saari H, 27, 33 Sable CL, 120, 132 Sacchi A, 356, 366 Sache E, 378, 394 Saed-Nejad F, 63, 71 Saetta M, 220, 236, 242, 261 Saetta MP, 242, 261 Saez AO, 251, 265 Safran M, 306, 320 Sagan S, 27, 32 Sage EH, 306, 319, 320 Sahin AA, 136, 140 Sahu S, 270, 280, 284, 286, 288, 289, 328, 333 Saijan SU, 270, 286 Saika S, 11, 19 Saiki I, 357, 368 Saint MS, 244, 262 Saito N, 364, 375 Saito Y, 377, 392 Saitoh D, 78, 97 Saitoh H, 324, 330 Saksela O, 218, 234, 377, 393 Salathe M, 79, 91, 99, 106 Salminen H, 295, 310 Salmivirta M, 304, 306, 317, 320, 378, 394

Salmon M, 364, 375 Salo T, 120, 132 Salustri A, 75, 94 Sampson PM, 26, 32, 87, 105, 108, 119, 123, 132, 340, 348 Samson D, 12, 20 Samuel SK, 75, 79, 80, 88, 94, 108, 115, 116, 124, 129 Samuels P, 80, 100 Samuelsson T, 85, 103, 117, 130 San Antonio JD, 172, 174, 179, 184, 185, 216, 231, 300, 314 Sanborn TA, 299, 312 Sanchack K, 301, 315 Sanchez E, 363, 374 Sanchez-Madrid F, 82, 101 Sanchirico ME, 361, 370 Sandell LJ, 202, 211, 214, 229, 295, 306, 310, 319 Sanderson RD, 56, 59, 69, 303, 316, 337, 347 Sandig M, 324, 329 Sando K, 27, 33 Sandy JD, 202, 210, 327, 332 Sanes JR, 356, 367 Sanmugalingham D, 364, 376 Sannes PL, 338, 347 Sannohe S, 364, 375 Santana A, 224, 238 Santoni A, 361, 371 Santoro SA, 172, 179, 184, 216, 231, 355, 362, 366, 372 Santra M, 173, 174, 175, 185, 187, 304, 317 Saoncella S, 306, 320, 356, 367 Sapsford RJ, 326, 332 Sarembock IJ, 205, 212 Sarnoff RB, 91, 106 Sarnstrand B, 56, 66, 69, 75, 93, 118, 131, 170, 174, 176, 180, 183, 186, 188, 189, 203, 211, 223, 238, 243, 244, 261, 262 Sarris G, 216, 231 Sartipy P, 304, 305, 317, 319 Sasaki M, 6, 17 Sasaki T, 115, 128, 304, 317, 356, 367

442 Sasisekharan R, 335, 345 Sasntra M, 215, 230 Sasrris G, 172, 179, 184 Sata M, 60, 70 Satake S, 356, 367 Sato A, 244, 262 Sato H, 244, 262, 280, 288 Sato S, 60, 70 Sato T, 201, 209 Satoh K, 244, 262 Satomi S, 147, 166, 247, 264 Satomura K, 10, 19, 172, 184, 258, 267 Sattar A, 84, 102 Sauer U, 174, 186 Saumon G, 64, 72, 162, 167 Saunders GF, 300, 314 Saunders S, 300, 314 Savage CO, 340, 348 Savani RC, 75, 76, 79, 80, 81, 82, 87, 88, 91, 93, 97, 99, 100, 108, 110, 113, 114, 115, 116, 117, 118, 119, 120, 122, 124, 126, 127, 129, 132, 326, 331 Saverymuttu SJ, 323, 329 Sawa Y, 307, 321 Sawai T, 2, 16, 24, 27, 28, 32, 33, 76, 95, 110, 126, 337, 346 Sawamura SJ, 75, 93 Sawhney RS, 214, 229 Saxena B, 224, 238 Sayre JW, 242, 260 Scanu AM, 304, 318 Scarborough RM, 352, 365 Scatena M, 356, 366 Sceid CR, 385, 397 Schaaf B, 324, 330 Schachner M, 200, 209 Schaefer L, 301, 315 Schechter GL, 115, 128 Scheid A, 340, 348 Scheidegger JJ, 284, 288 Schellenberg RR, 200, 208 Schenholm M, 26, 32, 87, 105, 118, 120, 131 Schenker M, 242, 260 Scherrer M, 66, 72 Scheynius A, 29, 34, 118, 120, 131

Author Index Schick BP, 300, 314 Schiedt MJ, 86, 104 Schiller V, 15, 21 Schipper CA, 79, 98 Schiro JA, 362, 373 Schittny JC, 56, 69, 146, 165, 301, 315, 336, 346 Schlaak M, 362, 373 Schleicher ED, 174, 186 Schlessinger D, 14, 21 Schlingemann RO, 299, 300, 312, 313 Schmekel B, 85, 103 Schmid P, 299, 312 Schmidt A, 214, 230 Schmidt G, 173, 185, 215, 217, 230, 233, 247, 264, 306, 319, 356, 367 Schmidt K, 273, 287 Schmidt M, 219, 236 Schmidt MB, 55, 58, 69, 173, 180, 185 Schmidtchen A, 174, 186, 214, 229 Schmits R, 29, 34, 79, 98 Schnapp LM, 359, 369 Schnarr RL, 202, 210 Schneider T, 364, 376 Schnohr P, 221, 237 Schoen FJ, 242, 261 Scholtz JM, 355, 366 Scholzen T, 214, 229 Schon P, 215, 231 Schonherr E, 10, 15, 18, 19, 21, 202, 211, 213, 215, 216, 218, 219, 228, 230, 232, 235, 248, 265, 299, 301, 304, 311, 315, 318, 327, 333, 338, 347 Schopf E, 79, 88, 99 Schor AM, 75, 94 Schor K, 204, 212 Schor SL, 75, 94 Schrager JA, 79, 98 Schrappe M, 300, 313 Schreiber SS, 301, 315 Schrum S, 362, 373 Schubert M, 341, 349 Schuessler TF, 66, 72 Schuh D, 88, 105, 113, 119, 120, 127, 132 Schulman J, 244, 262

Author Index Schulman LL, 247, 264 Schulz-Cherry S, 203, 211 Schuppan D, 172, 184, 356, 357, 367, 368 Schutte H, 340, 349 Schuyler W, 363, 374 Schwarting R, 56, 69 Schwartz JJ, 146, 165, 300, 314 Schwartz MA, 353, 365 Schwartz N, 336, 345 Schwartz NB, 90, 106, 339, 348 Schwartz SM, 355, 366 Schwartz WM, 295, 309 Schwarz K, 295, 311 Schwarz MI, 117, 130 Schworak NW, 146, 165 Sciore P, 64, 71 Scofield L, 61, 71, 90, 106 Scott I, 84, 102 Scott IC, 9, 18 Scott JE, 39, 52, 56, 58, 69, 70, 172, 184, 216, 231, 232, 247, 264, 300, 315, 325, 331, 336, 339, 341, 345, 346, 348, 349 Scott LJ, 75, 94, 295, 309 Scott PG, 214, 229, 339, 347 Screaton GR, 77, 97 Scurer B, 49, 53 Scwartz NB, 61, 71 Seddon M, 218, 234 Sedgwick JB, 364, 375, 376 Sedin G, 75, 85, 95, 110, 126 Seeberger K, 115, 118, 128 Seed B, 77, 97 Seed MP, 108, 123 Seeger W, 340, 349 Segarini PR, 295, 311 Seghatchian J, 336, 346 Segura Valdez L, 147, 159, 166, 247, 264 Sekhon HS, 181, 189, 244, 262 Sekiguchi K, 361, 372 Sekiguchi R, 307, 321 Seldin MF, 28, 33 Selman M, 147, 159, 166, 243, 247, 261, 264, 359, 369 Seltzer J, 270, 286

443 Selzer MG, 108, 115, 117, 118, 119, 124, 344, 350 Sempowski GD, 243, 244, 261, 262 Senaldi G, 29, 34, 79, 98 Senior RM, 247, 264 Seo JW, 221, 237 Seo JY, 221, 237 Sepa HEJ, 377, 392 Serini G, 361, 371 Seuwen K, 337, 346 Seyedin SM, 224, 239, 295, 311 Seyer JM, 86, 104, 118, 120, 131, 361, 370 Shahinian A, 29, 34, 79, 98 Shahzeidi S, 25, 26, 32, 118, 120, 130, 340, 349 Shanahan CM, 295, 310 Shanley TP, 327, 332, 363, 374 Shannon JM, 113, 127 Shao L, 114, 115, 128 Shapiro SD, 26, 32, 108, 125, 202, 210, 247, 258, 264, 267 Shappell SB, 357, 368 Shaver JR, 327, 332 Shaw AR, 79, 98 Shaw DM, 27, 33 Shaw HA, 181, 190 Shaw J, 86, 104 Shaw LM, 355, 365 Shaw NJ, 90, 106 Shaw RJ, 224, 239 Shay AK, 64, 71 Shazeidi S, 223, 238 Sheehan JK, 269, 278, 286, 288 Sheibani K, 41, 50, 52, 137, 141 Shen YJ, 339, 347 Shepherd K, 357, 368 Sheppard D, 86, 87, 103, 105, 118, 131, 203, 211, 220, 236, 355, 357, 359, 366, 369 Sherman L, 28, 34, 76, 77, 96 Sherrill DL, 242, 260 Shete SS, 307, 321 Sheykhnazari M, 344, 350 Shi C, 78, 97, 299, 312 Shi W, 300, 313 Shi X, 359, 370

444 Shibahara S, 218, 234 Shields J, 78, 97 Shimada S, 75, 82, 93 Shimada Y, 78, 97 Shimokawa H, 361, 371 Shimonaka M, 200, 209 Shimura S, 61, 70, 324, 330 Shin KY, 387, 398 Shing Y, 306, 320 Shinkai H, 76, 95, 109, 110, 126 Shinomiya K, 246, 263 Shinomura T, 2, 8, 9, 16, 18, 24, 28, 32, 76, 95, 110, 126, 200, 202, 209, 210, 213, 228, 337, 346 Shiraishi A, 11, 19 Shiraishi M, 110, 126 Shirakura R, 307, 321 Shirato K, 30, 31, 35, 225, 226, 228, 239, 324, 330 Shirk RA, 174, 185 Shishiba Y, 75, 94 Shizari M, 24, 31, 85, 103, 119, 132 Shizari T, 75, 94 Shock A, 364, 375 Shore SH, 284, 289 Showalter LJ, 300, 314 Shrive NG, 64, 71 Shteyngart B, 91, 92, 106, 108, 123, 145, 164 Shuhei Y, 381, 395 Shukla A, 86, 104 Shum D, 324, 325, 330, 331 Shute JK, 259, 267 Shworak NW, 300, 314 Sibalic A, 109, 125 Sibinga NE, 78, 97 Sibinga NES, 299, 312 Sibley L, 151, 166 Sicks S, 353, 365 Siebert C, 340, 349 Siebert JW, 81, 100 Siegal GP, 78, 98 Siegelman MH, 29, 34, 82, 101, 120, 132 Sielczak MW, 364, 375 Sigurdson SL, 359, 369 Silbert JE, 377, 392

Author Index Silcock D, 361, 372 Silides D, 137, 139, 141 Silver MM, 299, 312 Silvera MR, 243, 261 Silverman N, 12, 20 Silverstein R, 87, 105 Sim JJ, 221, 237 Simard JJ, 29, 34, 79, 98 Sime PJ, 47, 49, 53, 118, 131, 147, 165, 173, 180, 181, 185, 189, 219, 227, 236, 239, 244, 253, 262, 266, 340, 349 Simmons WL, 25, 26, 32 Simon JC, 30, 34, 79, 88, 99, 117, 129 Simon-Morrisey E, 159, 167 Simonneau G, 224, 239 Simons E, 79, 80, 82, 87, 88, 99, 110, 117, 119, 122, 126, 326, 331 Sinay P, 378, 394 Singer SJ, 82, 101 Singh RK, 78, 98 Singleton D, 253, 254, 266 Sionov RV, 76, 77, 96 Siracusa LD, 214, 229 Sires UI, 202, 210 Sironen RK, 76, 95, 109, 126 Sisson JH, 259, 267 Skeffington BS, 30, 31, 35 Skinner MP, 355, 366 Skold CM, 145, 164, 362, 372 Skorski T, 173, 174, 185 Skutelsky E, 223, 238 Sleeman J, 28, 34, 76, 77, 79, 96, 99, 116, 129 Sleeman JP, 78, 79, 97, 98 Sleijfer S, 86, 104 Slevin M, 27, 31, 33, 108, 114, 124, 127, 306, 320 Slutsky AS, 64, 71, 72 Sly P, 85, 90, 103 Smadja-Joffe F, 84, 103 Small JV, 30, 31, 35, 115, 128 Smedsrod B, 136, 140 Smethurst PA, 352, 365 Smit R, 175, 187 Smith A, 176, 177, 187 Smith AE, 381, 395

Author Index Smith CA, 255, 267 Smith CW, 357, 368 Smith E, 10, 19, 172, 184, 258, 267 Smith JM, 86, 104, 108, 113, 123, 124, 218, 224, 233, 239 Smith JR, 118, 131 Smith JW, 352, 365 Smith LL, 355, 366 Smith P, 82, 101 Smith RS, 244, 262 Smith TJ, 244, 262 Smolen JE, 324, 330 Smorenburg SM, 378, 394 Smyth RL, 90, 106 Snell L, 175, 187 Snider GL, 61, 70, 158, 167, 181, 190, 219, 235, 241, 251, 260, 265 Snow AD, 47, 53, 307, 321 So WY, 325, 331 Sobonya RE, 191, 207 Sodek J, 359, 369 Soderberg M, 281, 288 Solakivi T, 377, 392 Solheim OP, 139, 142 Solursh M, 202, 210, 214, 229 Solway J, 57, 70 Somer A, 377, 393 Somerhoff C, 270, 286 Somerman MJ, 359, 370 Somers KD, 115, 128 Sommarin Y, 153, 167 Sommer B, 10, 19, 172, 184, 258, 267 Sommerhoff C, 270, 286 Song YL, 220, 237 Sont JK, 228, 240 Soombo AA, 205, 212 Soria C, 84, 103 Soriani A, 361, 371 Sorio C, 363, 374 Sottile J, 361, 372 Spearman MA, 75, 79, 80, 88, 94, 115, 129 Speer CP, 90, 106 Speizer FE, 242, 260 Spence CR, 343, 344, 350, 378, 383, 393, 396 Spengler DM, 246, 263

445 Spicer AP, 2, 16, 24, 28, 30, 31, 33, 34, 75, 76, 79, 93, 95, 96, 99, 108, 110, 113, 119, 125, 126, 337, 346 Spillmann D, 304, 317, 378, 394 Spiro RC, 300, 313 Spisani S, 108, 124 Spiteller G, 108, 124 Spokojny AM, 299, 312 Spooner E, 377, 393 Sporn MB, 86, 104, 105, 218, 219, 224, 233, 235, 238, 239 Sprague AG, 364, 376 Spring F, 78, 97 Spring J, 294, 309, 378, 394 Springer TA, 82, 101, 352, 357, 365, 368 Spurzem JR, 259, 267, 362, 372 Sraer JD, 307, 321 Srimal S, 363, 374 Srinivasan SR, 304, 318, 336, 346 St John PL, 324, 329 St John T, 299, 312 Stabellini G, 339, 348 Staffordt AR, 379, 395 Stahl RC, 300, 314 Stallcup WB, 299, 304, 312, 317, 356, 367 Stamenkovic I, 28, 29, 34, 77, 78, 81, 86, 97, 98, 101, 104, 246, 263 Stamenovic D, 57, 70 Standaert TA, 90, 106 Stander M, 181, 190 Stanley HE, 269, 285 Stanley MJ, 56, 59, 69, 303, 316 Stanton K, 253, 254, 266 Stanway G, 352, 365 Starcher BC, 61, 70 Starkie CM, 325, 331 Starosta R, 304, 318 Statkov PR, 204, 211 Stauffer UG, 340, 348 Stazzone EJ, 63, 71 Steadman R, 324, 329 Steigman D, 383, 396 Steigman DM, 344, 350 Stein PD, 344, 350 Steinberg KP, 362, 373

446 Steinfeld R, 146, 165, 300, 313 Stellmach V, 217, 233 Stellrecht CM, 300, 314 Stelter-Stevenson WG, 246, 264 Stenmark K, 159, 167 Sterk PJ, 228, 240 Stern D, 377, 392 Stern M, 81, 100 Stern R, 79, 80, 82, 87, 88, 99, 110, 117, 119, 120, 122, 126, 132, 326, 331 Sterner-Kock A, 359, 369 Stetler-Stevenson WG, 158, 167 Stevens RL, 15, 21 Stewart JA, 181, 189, 245, 263 Sthanam M, 11, 20 Stiles AD, 223, 238 Stipp CS, 294, 300, 309, 313 Stockley RA, 246, 263, 324, 325, 330, 331 Stone OL, 206, 212 Stone PJ, 158, 167, 181, 190 Strachan RK, 82, 101 Strain AJ, 86, 104 Strang LB, 162, 168 Streit M, 306, 320 Stricklin G, 243, 261 Strieter RM, 27, 30, 31, 33, 35, 75, 82, 93, 108, 120, 123, 125, 132 Striker GE, 194, 208 Striker JE, 377, 393 Striker LJ, 194, 208, 377, 393 Stringer SE, 3, 16 Strobl B, 27, 33 Struble M, 364, 376 Strydom DJ, 378, 394 Stubbs PJ, 301, 316 Stulz P, 343, 350 Sturgess JM, 279, 288 Su WD, 9, 18 Suchard SJ, 363, 374 Sud S, 357, 369 Sudarshan C, 361, 372 Sugahara K, 335, 336, 345, 381, 387, 395, 397 Suganuma H, 244, 262 Sugihara T, 61, 70

Author Index Sugiura M, 387, 397 Sukhova GH, 159, 167 Suki B, 57, 70 Sulavik SB, 118, 120, 131 Sun FF, 364, 375 Sun L, 109, 125 Sundberg C, 204, 211 Suter PM, 362, 373 Suzuki J, 228, 239 Suzuki M, 75, 95, 246, 263, 361, 371 Suzuki S, 200, 201, 209, 210, 214, 229, 295, 303, 309, 316 Suzuki Y, 75, 82, 93, 101, 138, 142 Svahn CM, 377, 393 Svee K, 117, 130, 201, 209 Svensson L, 10, 19, 172, 173, 184, 216, 232, 304, 306, 317, 319 Swawamura M, 201, 209 Swenson TL, 305, 318 Swiderski RE, 26, 32 Swiedler SJ, 381, 395 Sylvester KG, 81, 100 Symon FA, 364, 375, 376 Syrokou A, 339, 347 Szefler SJ, 220, 237 Sznaider JI, 147, 159, 166 Sztrolovics R, 325, 331

T Tabas I, 304, 318 Tada M, 75, 82, 93 Tager IB, 242, 260 Taggart C, 324, 330 Taha R, 50, 53, 68, 72, 177, 188, 223, 238, 326, 331, 361, 371 Tai-Ping F, 84, 102 Taipale J, 252, 266 Takagi M, 147, 166, 247, 264 Takahashi F, 359, 370 Takahashi H, 324, 330 Takahashi K, 60, 70, 359, 370 Takahashi M, 75, 95 Takahashi S, 300, 313 Takahashi T, 217, 232 Takai Y, 115, 128 Takami H, 11, 20

Author Index Takashima A, 81, 88, 101 Takehara H, 75, 94 Takeuchi J, 202, 210 Takeuchi T, 295, 309 Takeuchi Y, 219, 235 Tam EM, 246, 263 Tamburro AM, 108, 124 Tammi M, 2, 16, 28, 33, 75, 76, 81, 93, 96, 101, 109, 125, 340, 341, 343, 349, 350, 361, 371 Tammi MI, 76, 95, 109, 126 Tammi R, 75, 76, 81, 93, 96, 101, 109, 120, 126, 132 Tammi RH, 76, 95 Tamoto K, 75, 82, 93 Tamura G, 30, 31, 35, 225, 226, 228, 239 Tamura R, 244, 262 Tan AK, 80, 100 Tan CT, 118, 131 Tanaka A, 30, 31, 35 Tanaka R, 61, 62, 66, 70, 71, 178, 188, 326, 332 Tang L, 357, 368 Tang LH, 214, 229 Taniguchi N, 27, 33 Tanimoto K, 76, 95 Tanne K, 76, 95 Tanno Y, 228, 239 Taooka Y, 118, 131 Tapanadechopone P, 11, 20 Tarone G, 24, 31, 42, 52, 77, 97, 284, 289 Tasheva ES, 175, 187, 295, 311 Tashiro K, 200, 209 Tashkin DP, 242, 260 Tate L, 259, 267 Taugchi T, 75, 95 Taylor AE, 152, 166 Taylor BM, 364, 375 Taylor GM, 224, 239 Taylor JC, 254, 266 Taylor S, 305, 318 Teder P, 79, 87, 88, 99, 105, 113, 117, 118, 119, 127, 130, 132, 137, 139, 141, 340, 349 Teeling P, 50, 53

447 Teichman LJ, 300, 314 Teixeira MM, 364, 375 Ten Dijke P, 226, 239 Tengblad A, 26, 32, 82, 87, 102, 105, 113, 118, 120, 126, 131, 136, 137, 138, 140, 141 Terada LS, 362, 373 Terao T, 75, 93 Termeer CC, 30, 34, 117, 129 Termine JD, 9, 18, 171, 183, 214, 216, 221, 229, 231, 238, 278, 287, 295, 309 Terracio L, 362, 373 Terry DE, 342, 349 Testi R, 228, 240 Tetley TD, 254, 267 Tewarei D, 175, 187, 215, 230 Tezuka N, 174, 185 Thaanawiroon C, 391, 398 Thakker-Varia S, 163, 168 Thelin S, 75, 95 Theocharis AD, 279, 288, 307, 321 Thesleff I, 306, 320 Thet LA, 218, 234 Thieringer R, 363, 375 Thieszen SL, 362, 372 Thomas A, 120, 133 Thomas GJ, 108, 124, 324, 329 Thomas K, 76, 96 Thomas L, 78, 98 Thomlinson AM, 58, 70, 172, 184, 216, 232 Thompson AY, 295, 311 Thompson BT, 343, 344, 350, 378, 382, 383, 385, 386, 387, 389, 390, 393, 394, 396, 397, 398 Thompson JF, 175, 187 Thompson NL, 224, 238, 239 Thompson S, 283, 288 Thornton DJ, 278, 288 Thornton SC, 377, 393 Thrall RS, 118, 120, 131, 219, 235 Thunell M, 117, 130 Thung LY, 357, 369 Thurlbeck WM, 181, 189, 241, 243, 244, 260, 261, 262 Thylen A, 137, 139, 141, 142

448 Tiedermann K, 75, 94, 171, 175, 183, 186 Tierney DF, 64, 72 Till KJ, 115, 118, 128 Tillet E, 356, 367 Tillie I, 228, 240 Timans J, 78, 97 Timens W, 66, 67, 72, 181, 189, 248, 251, 255, 265, 267, 326, 332 Timpl R, 11, 20, 172, 184, 217, 232, 304, 317, 356, 367, 378, 394 Tiozzo R, 378, 386, 395, 397 Tiruppathi C, 357, 369 Tobetto K, 75, 82, 93 Tocci MJ, 246, 264 Tohda G, 300, 313 Toida T, 381, 387, 395, 398 Tolg C, 78, 97 Tolker E, 254, 266 Tollefsen DM, 174, 185 Tomasek JJ, 361, 371 Tomita K, 63, 71 Tomlinson AM, 325, 331 Tomoike H, 60, 70 Tonnel AB, 225, 226, 228, 239, 240 Toole BP, 28, 30, 34, 74, 75, 76, 92, 93, 96, 108, 111, 113, 125, 126, 127, 163, 168, 299, 312 Tootell E, 181, 189 Topaloglu H, 12, 20 Toribatake Y, 63, 71 Torri G, 378, 394 Tortorella MD, 8, 17 Tournier JM, 278, 287 Tozzi CA, 163, 168 Tran HC, 65, 72 Tran ND, 301, 315 Tran PK, 303, 316 Tranniello S, 108, 124 Tranquillo RT, 79, 88, 98 Trask BC, 85, 173 Trask TM, 173, 185, 215, 231 Trautman MS, 163, 168 Trautwein C, 204, 211 Travis J, 324, 330 Travis LB, 136, 140 Travis WD, 136, 140, 158, 167

Author Index Treanor RE, 301, 316 Tredget EE, 339, 347 Tremblay GM, 181, 189 Tremblay L, 64, 72 Tremble P, 202, 210, 301, 315, 357, 368 Trigg CJ, 228, 240 Trochon V, 84, 103 Troedel JM, 9, 18 Trotter JA, 216, 232 Trounce JR, 220, 236 Troup S, 175, 187 Trudeau JB, 226, 239 Truog WE, 90, 106 Tsai SY, 118, 131 Tsang KWT, 326, 332 Tschernig T, 113, 127 Tsegenidis T, 339, 347 Tsicopoulos A, 225, 226, 239 Tsifrina E, 114, 127 Tsoi CK, 303, 316, 357, 368 Tsolakis I, 307, 321 Tsuji T, 202, 210 Tuan RS, 11, 20, 56, 69 Tuan TL, 355, 366 Tuckwell DS, 352, 365 Tuder RM, 340, 348 Tufveson G, 82, 102, 109, 125 Tufvesson E, 175, 180, 186 Tumova S, 13, 21, 300, 313, 338, 347, 356, 367 Turato G, 242, 261 Turcotte H, 177, 188, 221, 237 Turi TG, 175, 187 Turino GM, 26, 32, 56, 60, 69, 80, 81, 82, 91, 92, 93, 100, 106, 108, 117, 119, 123, 132, 145, 164, 339, 348 Turley EA, 75, 76, 79, 80, 81, 88, 91, 93, 94, 96, 97, 98, 99, 100, 106, 108, 110, 113, 115, 116, 117, 118, 120, 124, 126, 128, 129, 132 Turley M, 108, 116, 124 Turley S, 116, 129 Turnbull J, 292, 308, 378, 394 Turnbull JE, 304, 317 Turner BS, 269, 270, 285, 286 Turner CE, 356, 366

Author Index

449

Turner D, 28, 34, 201, 210 Tuszynski GP, 305, 318 Twardzik DR, 172, 173, 180, 184, 218, 235, 251, 265 Tzanakakis G, 339, 347 Tzanakakis GN, 75, 95

U Ueda M, 377, 392 Uede T, 359, 370 Ueki N, 75, 95 Uemura Y, 108, 123 Uhal BD, 247, 264 Uitto J, 175, 187, 215, 218, 230, 234 Ujita M, 200, 209 Ulisse S, 75, 94 Ulloa L, 180, 189 Ulmsten U, 174, 176, 185, 187 Ulrik CS, 221, 237, 238 Umino T, 362, 372 Underhill CB, 24, 30, 31, 34, 41, 42, 50, 52, 75, 76, 77, 85, 94, 97, 103, 119, 132, 137, 141, 299, 312 Underwood DC, 362, 373 Underwood PA, 113, 127, 303, 316 Unemori EN, 159, 167 Ungefroren H, 175, 179, 186 Unger E, 381, 395 Unruh HW, 203, 204, 211 Urayama O, 364, 375 Urena JM, 357, 368 Urman B, 82, 102 Urtizberea JA, 12, 20 Usui N, 27, 33 Utani A, 76, 95, 109, 110, 126

V Vacherot F, 174, 186 Vaidya S, 363, 375 Vaillant P, 117, 130, 201, 209 Vainio S, 306, 320 Valentin E, 356, 367 Valentine KA, 344, 350 Valenza F, 64, 72 Valhmu WB, 6, 17, 63, 71

Valiyaveettil M, 176, 187 Valli M, 172, 184, 216, 232 Van AI, 364, 376 Van Boeckel CAA, 378, 394 van de Heuvel LPWJ, 11, 20 Van de Kerkhof PC, 300, 313 Van de Lest CH, 177, 181, 188, 252, 266 Van de Leur E, 75, 94 Van de Velden TJ, 146, 165 Van de Woestijme KP, 66, 67, 72 Van den Berghe H, 243, 261, 300, 313 Van den Born J, 146, 165 Van den BP, 174, 186 Van den Heuvel LP, 176, 188 Van der Berghe H, 300, 314 Van der Heuvel LP, 146, 165 Van der Linde HJ, 364, 376 Van der Schueren B, 300, 314 Van der Woude FJ, 338, 347 Van Goor H, 108, 124 Van Iperen L, 362, 373 Van Krieken JHJM, 228, 240 Van Kuppevelt TH, 11, 20, 176, 177, 181, 188, 252, 266 Van Lier RA, 357, 369 Van Noorden CJ, 378, 394 Van Obberghen-Schilling E, 224, 238 Van Oosterhout AJ, 364, 376 Van Pee D, 225, 226, 239 Van Straaten JFM, 66, 67, 72, 181, 189, 248, 251, 255, 259, 265, 267, 326, 332 Van Tussenbroek F, 174, 186 van Weering DHJ, 306, 320 Van Weyenberg J, 361, 370 Vandeligt K, 75, 76, 93 Vandenbroucke JP, 228, 240 Vanek M, 299, 312 Vannucchi S, 378, 393 Varani J, 327, 332 Varga J, 218, 234 Vartion T, 194, 208 Vaschieri C, 361, 370 Vasios G, 306, 320 Vassalli P, 29, 34, 81, 101 Vaughan CJ, 307, 321

450 Vaughan MB, 361, 371 Vazquez JJ, 220, 236 Veerkamp JH, 11, 20, 146, 165, 176, 177, 181, 188, 252, 266 Veissiere D, 277, 287 Velluti G, 117, 130 Vender RL, 159, 167 Veness-Meehan KA, 223, 238 Venkataraman G, 335, 345 Venkatesan N, 61, 62, 66, 70, 72, 113, 127, 178, 188, 326, 332 Venturoli D, 153, 154, 155, 166 Verbanac KM, 324, 330 Verbeken E, 41, 50, 52 Verbeken EK, 66, 67, 72 Verbloet D, 225, 226, 239 Vergnes JP, 10, 19, 171, 183, 214, 229 Verhoeven A, 357, 369 Vermeesch J, 146, 165, 300, 313 Vermylen J, 300, 313 Vernon RB, 306, 320 Verscharen C, 202, 210, 327, 332 Versnel MA, 137, 139, 141 Versteeg EM, 177, 181, 188, 252, 266 Vesely I, 325, 331 Vestbo J, 221, 237 Vetter U, 9, 18, 171, 183, 214, 215, 230 Veugelers M, 146, 165, 300, 313 Vignaud JM, 117, 130 Vignola AM, 224, 226, 239 Vihinen P, 361, 371 Vijayagopal P, 304, 318, 336, 346 Vilar RE, 91, 106 Vilchis-Landeros MM, 14, 21 Vink J, 78, 98 Virolle T, 361, 370 Virtanen I, 220, 237 Vischer P, 215, 231, 301, 315 Viswanathan HL, 359, 370 Vlahakis NE, 68, 72 Vlahova PI, 381, 395 Vlodavsky I, 246, 263, 357, 368, 377, 393 Voelkel NF, 340, 348 Vogel KG, 64, 67, 71, 72, 176, 177, 187, 216, 232, 247, 264, 278, 287, 300, 315

Author Index Vogel W, 9, 18, 171, 183, 214, 215, 230 Vogelzang NJ, 138, 142 Voith U, 30, 34 Von Kemp K, 174, 186 Von Stein O, 79, 99, 116, 129 Vooys W, 306, 320 Voss B, 221, 238 Vracko R, 205, 212 Vuori K, 353, 365

W Wagenvoort CA, 42, 48, 52 Wagner E, 135, 140 Wagner JC, 135, 140 Wagner P, 50, 53 Wagner W, 295, 310 Wagner WD, 174, 185, 304, 306, 318, 319 Wahl LM, 86, 104 Wahl SM, 86, 104 Wainwright D, 77, 97, 340, 348 Wakefield D, 246, 263 Wakefield LM, 86, 104, 218, 233 Wakeham A, 29, 34, 79, 98 Waldenstrom A, 82, 102 Walenga JM, 387, 398 Walker A, 381, 395 Walker J, 352, 365 Wallace D, 253, 266 Wallaert B, 228, 240 Wallin J, 139, 142 Wallis GA, 6, 17 Walsh GM, 364, 375 Walters DV, 162, 168 Waltz TA, 300, 313 Wang C, 79, 80, 82, 87, 88, 99, 100, 108, 110, 113, 115, 116, 117, 118, 119, 120, 122, 124, 126, 132, 326, 331 Wang DY, 84, 102 Wang H, 305, 319, 361, 362, 370, 372 Wang J, 228, 240, 338, 347 Wang L, 301, 315 Wang MH, 78, 98, 108, 123, 306, 320 Wang Q, 87, 105, 219, 235

Author Index Wang T, 385, 397 Wang XF, 299, 312 Wang Y, 87, 105, 219, 235, 385, 397 Wang Z, 258, 267 Wangoo A, 224, 239 Warburton D, 147, 165, 219, 236 Ward PA, 118, 120, 131, 219, 235, 363, 374 Wardell JR, 278, 288 Wardlaw AJ, 220, 236, 364, 375, 376 Ware LB, 362, 373 Warkentin TE, 303, 316 Warner A, 284, 289 Warner RL, 327, 332 Warren PW, 203, 204, 211 Warshamana GS, 118, 131 Wary KK, 355, 356, 365, 367 Wasilenko WJ, 115, 128 Wasmuth JJ, 79, 99, 307, 321 Watanabe H, 8, 9, 11, 17, 18, 20 Watanabe K, 300, 313 Watkins RH, 223, 238 Watson CE, 76, 96 Watt SM, 377, 393 Waxler B, 136, 140 Wayner E, 299, 312 Webb EF, 362, 373 Webb HH, 64, 72 Webb SC, 323, 329 Webber C, 214, 229 Weber GF, 342, 349, 361, 370 Weber IT, 216, 232 Weening RS, 357, 369 Wei DC, 344, 350 Wei J, 355, 365 Wei Z, 381, 395 Weiber ER, 144, 164 Weigel PH, 2, 16, 28, 33, 75, 76, 93, 96, 109, 125 Weigert C, 174, 186 Weinacker A, 220, 236 Weinberg RA, 299, 312 Weis JH, 15, 21 Weiss JJ, 176, 177, 187 Weiss JM, 30, 34, 79, 88, 99 Weiss SJ, 327, 332 Weiss ST, 242, 260

451 Weissberg PL, 295, 310 Weissenbach J, 12, 20 Weissman B, 23, 31, 74, 92 Weissman G, 324, 330 Weissman SM, 15, 21 Welch MP, 361, 371 Weldner N, 136, 140 Welgus HG, 202, 210, 325, 331 Weller M, 181, 190 Welles S, 111, 126 Wells AF, 78, 82, 98, 102 23, 24, 31 Wells DE, 28, 33 Wellstein A, 336, 346 Welsh MJ, 283, 288 Wen FQ, 362, 372 Wenzel KW, 223, 238 Wenzel SE, 220, 226, 237, 239 Werb Z, 202, 210, 301, 315, 357, 368 Wert SE, 227, 239 Werth VP, 76, 96 West DC, 27, 30, 33, 83, 84, 102, 103, 306, 320 West JB, 65, 72 Westergren-Thorsson G, 56, 66, 69, 75, 93, 94, 109, 118, 125, 131, 170, 174, 175, 176, 180, 181, 183, 186, 187, 188, 189, 190, 203, 211, 223, 238, 243, 258, 259, 261, 267 Westermarck J, 355, 366 Westin U, 324, 330 Westling J, 202, 210, 327, 332 Whinna HC, 301, 316 Whitby DJ, 206, 212 White DA, 86, 103 White J, 117, 130, 201, 209 White KE, 108, 124 White PS, 12, 20 White RJ, 9, 10, 18, 19, 325, 331 Whitelock J, 295, 310 Whitelock JM, 303, 316 Whitham SE, 218, 234 Whitman C, 118, 132 Whitsett JA, 227, 239 Whitson SW, 214, 229 Whittemore AS, 242, 260 Whittle N, 78, 97 Whyte A, 88, 105, 119, 120, 132

452 Wiberg C, 172, 184, 217, 232 Wick M, 117, 130 Wickline SA, 202, 210 Widdicombe JG, 57, 69 Widstrom C, 27, 33, 145, 163, 164, 338, 347 Wieghorst A, 324, 330 Wiesel P, 78, 98, 299, 312 Wietzerbin J, 361, 370 Wiggs BR, 200, 208, 221, 237, 251, 265 Wight TN, 41, 47, 48, 52, 53, 55, 56, 61, 68, 71, 76, 79, 85, 90, 96, 99, 103, 111, 115, 120, 126, 128, 132, 144, 145, 146, 163, 164, 165, 175, 187, 201, 202, 209, 210, 211, 248, 265, 295, 300, 303, 304, 306, 307, 309, 310, 315, 316, 317, 318, 319, 320, 321, 327, 332, 336, 346, 356, 357, 367, 368 Wihl JA, 324, 330 Wiig H, 151, 153, 166 Wilcox WR, 12, 20 Wilcox-Adelman S, 306, 320 Willems LNA, 228, 240 Willet KE, 85, 90, 103 Williams JH, 220, 236 Williams KJ, 76, 96, 304, 305, 318 Williams MV, 118, 131 Willis RA, 243, 261 Willougby DA, 108, 123 Wilpoth M, 204, 212 Wilson JE, 204, 212, 295, 310 Wilson JW, 220, 236 Winkler T, 174, 186, 306, 320, 336, 346 Winnemoller M, 173, 185, 215, 217, 231, 233, 356, 367 Winters GL, 295, 310 Witsch-Prehm P, 10, 19, 248, 265 Witschi H, 194, 208 Witt DP, 305, 319 Witte L, 304, 317 Wittkowsky AK, 378, 394 Wittmann K, 180, 189 Wizniak J, 115, 118, 128

Author Index Wlad H, 381, 395 Woessner JF, 147, 165 Wolfe JT, 136, 140 Wolheim FA, 181, 189 Wong AJ, 174, 185, 304, 317 Wong C, 79, 99 Wong MJ, 300, 313 Wong SH, 299, 312 Wong WW, 377, 393 Wong ZM, 300, 313 Wood JG, 6, 17 Woods A, 12, 13, 21, 248, 265, 300, 303, 313, 316, 338, 347, 356, 367, 377, 393 Woolcock AJ, 220, 236 Woolley K, 225, 226, 239 Wooten RM, 176, 177, 187 Worthley SG, 307, 320 Wrana JL, 14, 21, 359, 369 Wright CD, 324, 330 Wright CJ, 50, 53 Wright JA, 75, 79, 80, 88, 94, 100, 115, 116, 129 Wright JL, 251, 258, 265, 267 Wright JR, 280, 288 Wright MJ, 306, 319 Wright SD, 363, 374 Wright TC, 378, 385, 394, 395, 397 Wright TM, 30, 31, 35, 108, 125 Wrocklage C, 295, 311 Wu C, 361, 372 Wu H, 76, 96 Wu J, 27, 31, 33, 75, 82, 87, 93, 105, 108, 122, 124, 203, 211, 217, 233, 357, 369 Wu JF, 357, 369 Wu KK, 300, 313 Wu M, 242, 260 Wu R, 146, 165 Wu RR, 11, 20 Wu SJ, 387, 398 Wu T, 118, 131 Wu X, 324, 330 Wuthrich RP, 27, 31, 33, 109, 125 Wynford-Thomas D, 136, 140 Wysoka M, 108, 125

Author Index

453 X

Xing Z, 173, 180, 181, 185, 189, 219, 227, 236, 239, 340, 349 Xu H, 304, 306, 318, 319 Xu J, 64, 71, 175, 177, 187, 355, 362, 366, 372, 373 Xu JH, 304, 318 Xu L, 174, 185 Xu N, 357, 369 Xu Q, 295, 310 Xu T, 10, 19, 172, 184, 258, 267

Y Yager D, 81, 100 Yagoda A, 118, 131 Yamada H, 300, 313 Yamada K, 295, 309 Yamada KM, 200, 201, 209, 210, 243, 253, 261, 266, 303, 316, 353, 365 Yamada S, 387, 397 Yamada SS, 243, 261 Yamada Y, 2, 6, 8, 9, 11, 12, 16, 17, 18, 20, 24, 28, 32, 75, 76, 95, 110, 126, 337, 346, 356, 367 Yamagata M, 200, 201, 202, 209, 210, 303, 316 Yamaguchi T, 82, 101 Yamaguchi Y, 6, 10, 17, 19, 200, 202, 209, 210, 218, 219, 234, 235, 251, 252, 265, 266, 300, 304, 313, 317, 335, 336, 345, 346 Yamakawa N, 202, 210 Yamakawa T, 307, 321 Yamakido M, 118, 131 Yamamoto C, 175, 187 Yamamoto Hokayama M, 357, 364, 368, 376 Yamamoto T, 175, 187 Yamamoto Y, 75, 82, 93 Yamamura H, 28, 34, 201, 210 Yamanaka M, 147, 166 Yamanaka N, 201, 209 Yamashita H, 75, 95

Yamauchi K, 225, 226, 228, 239 Yamori Y, 201, 209 Yan SR, 363, 374 Yanagishita M, 4, 16, 75, 94, 214, 229, 247, 265, 272, 287, 335, 345 Yang B, 76, 79, 80, 97, 99, 100, 108, 116, 120, 124, 129, 132 Yang BB, 201, 210 Yang BL, 76, 80, 97, 116, 129, 201, 210 Yang EY, 81, 100 Yang J, 362, 373 Yang VC, 84, 102 Yang VWC, 214, 229 Yang XL, 41, 50, 52, 108, 116, 124 Yang Z, 381, 395 Yankaskas JR, 283, 288 Yannas IV, 344, 350 Yano K, 362, 372 Yashiro M, 78, 98 Yasui K, 75, 82, 93 Yasunari K, 377, 392 Yaswen L, 361, 372 Yauch RL, 355, 365 Yayon A, 246, 263, 304, 306, 317, 320 Ybot-Gonzales P, 303, 316 Yednock TA, 202, 210 Yeo TK, 202, 210 Yohida T, 280, 288 Yokokawa K, 377, 392 Yokozeki M, 361, 371 Yoneda M, 200, 209 Yonekura H, 357, 368 Yoon HJ, 221, 237 Yoshida K, 214, 229, 387, 397 Yoshida M, 2, 16, 24, 28, 32, 75, 76, 95, 110, 126, 337, 346 Yoshida S, 377, 392 Yoshida Y, 2, 16, 24, 28, 32, 76, 95, 110, 126, 337, 346 Yoshie O, 200, 209 Yoshikawa J, 377, 392 Yoshimura K, 324, 330 Yoshioka H, 9, 18 You XM, 47, 53 Young C, 118, 131

454

Author Index

Young MF, 9, 10, 18, 19, 171, 172, 183, 184, 214, 215, 221, 230, 238, 258, 267, 295, 301, 309, 315 Young MR, 214, 229, 278, 287 Young SG, 357, 369 Younker WR, 86, 104 Younkin EV, 327, 332 Ytterberg D, 75, 95 Yu G, 387, 398 Yu MC, 359, 370 Yu Q, 28, 29, 34, 86, 104, 113, 127, 246, 263 Yu RJ, 117, 118, 119, 122, 130 Yudin AI, 27, 32 Yuen KY, 326, 332 Yung S, 108, 124 Yurchenco PD, 56, 69, 336, 346 Yurchenko PD, 146, 165

Z Zafarullah M, 243, 262 Zagris N, 339, 348 Zaidi SHE, 47, 53 Zakarian A, 29, 34, 79, 98 Zako M, 13, 20, 169, 171, 183 Zaman A, 79, 84, 99, 114, 115, 117, 118, 127 Zambruno G, 361, 370 Zantrilli J, 327, 332 Zardi L, 217, 233, 306, 319, 361, 371 Zawaideh S, 361, 370 Zettergren L, 136, 137, 140, 141 Zhan X, 115, 128 Zhang HY, 362, 372 Zhang J, 300, 314 Zhang K, 195, 208, 362, 372 Zhang L, 30, 34, 146, 165, 206, 212, 300, 314 Zhang LJ, 4, 16 Zhang M, 78, 98 Zhang Q, 359, 369 Zhang S, 79, 99, 108, 116, 120, 124, 129, 132

Zhang SF, 175, 187 Zhang W, 76, 96 Zhang Y, 181, 189, 201, 210, 359, 370 Zhao B, 304, 318 Zhao C, 10, 19, 172, 184, 258, 267 Zhao HW, 117, 118, 119, 122, 130 Zhao J, 147, 165, 219, 236 Zhao LL, 356, 366 Zheng T, 258, 267 Zhou D, 363, 375 Zhou MY, 357, 369 Zhou Z, 79, 84, 99, 114, 115, 117, 118, 127 Zhous I, 227, 239 Zhu D, 78, 97, 114, 128 Zhu H, 78, 97, 114, 128 Zhu L, 304, 318 Zhu W, 55, 58, 69, 305, 318 Zhu X, 355, 366 Zhu YJ, 382, 396 Zhu YK, 362, 372 Zhu Z, 258, 267 Ziebell M, 115, 117, 128 Ziegenhagen MW, 362, 373 Ziegler S, 340, 349 Ziesche R, 180, 189 Zimmermann CN, 364, 376 Zimmermann DR, 8, 17, 18, 67, 72, 199, 202, 208, 210, 211, 295, 309, 327, 332, 341, 349 Zimmermann TS, 301, 315 Zipfel PF, 362, 373 Zissel G, 362, 373 Zlokovic BV, 301, 315 Zola H, 79, 98 Zuin R, 242, 261 Zumwalt RE, 191, 207 Zuo F, 86, 103 Zuo L, 118, 132 Zuraw BI, 91, 106 Zutter M, 355, 362, 366, 372 Zuzel M, 115, 118, 128 Zylka D, 80, 100, 116, 129

SUBJECT INDEX

A Acharan sulfate sulfonation, 390 Aggrecan function, 7 response to mechanical stimulation, 64 structure, 6 Agrin in blood vessels, 299 expression, 146 structure, 11 Asthma airway fibrosis, 220 integrins, 363

B Bamacan in blood vessels, 299 structure, 11, 146 Betaglycan in blood vessels, 299 function, 14, 146 interaction with TGFβ, 253 structure, 14

Biglycan asthma, 68, 177, 326 bleomycin-induced injury, 66, 179, 326 in blood vessels, 295 chronic obstructive pulmonary disease (COPD), 181 emphysema, 67, 248, 326 expression, 171 fibrotic lung disease, 66, 196 function, 10 interaction with TGFβ, 10, 173 knockout, 10, 172 lipoprotein interaction, 305 in lung, 56 lung biomechanics, 61 regulation by TGFβ, 174 response to mechanical stimulation, 64 structure, 9 systemic sclerosis, 181 ventilation-induced lung injury, 66 Bronchial mucus in asthma, 280 cellular origin, 270 in chronic bronchitis, 279 in cystic fibrosis, 282

455

456

Subject Index

[Bronchial mucus] mucin, 269 proteoglycans, 271 in pulmonary alveolar proeinosis, 280 in quadroplegic patients, 281 Bronchiectasis description, 323 proteoglycans, 325 role of neutrophil, 323

C CD44 expression, 78 function, 15, 29, 114 gene, 77, 114 knockout, 30 structure, 15, 28, 77, 114 Chondroitin sulfate structure, 3, 338 sulfonation, 390

D Decorin angiogenesis, 306 asthma, 68, 177, 221 bleomycin-induced injury, 66, 179, 219 in blood vessels, 295 chronic obstructive pulmonary disease (COPD), 181 emphysema, 67, 248, 326 endocytosis, 175, 215 expression, 171 fibrotic lung disease, 196 function, 10 gene, 214 interaction with bacteria, 176 interaction with EGF receptor, 10, 173 interaction with fibronectin, 217 interaction with lipoprotein, 305 interaction with TGFβ, 10, 173, 217 interaction with thrombospondin I, 217

[Decorin] interaction with type I collagen, 216 interaction with type VI collagen, 217 knockout, 10, 172 in lung, 56 lung biomechanics, 58 regulation by TNFα, 175 response to mechanical stimulation, 64 structure, 9, 214 systemic sclerosis, 181 Dermatan sulfate interaction with heparin cofactor II, 174 interaction with basic FGF, 174 regulation by TGFβ, 175 structure, 3, 338 sulfonation, 390

E Edema hydraulic, 153 induced by hypoxia, 159 lesional, 153 newborn lung, 162 Emphysema description, 241 extracellular matrix, 245 fibroblasts, 243 pathogenesis, 254 proteinases, 246 proteoglycans, 248 types, 242

F Fibromodulin bleomycin-induced injury, 66, 179, 326 expression, 171 function, 10 interaction with TGFβ, 173 knockout, 10, 173 structure, 10 Fibrosis description, 191

Subject Index

457

[Fibrosis] myofibroblasts, 194, 361 integrins, 359

G Glypican in blood vessels, 299 function, 14 in lung, 146 structure, 14 Glycosaminoglycans functions in lung, 339 hypoxia, 340 pulmonary hypertension, 342 structure, 2, 335 synthesis, 2

H Heparan sulfate structure, 3, 337, 381 sulfonation, 389 Heparin anticoagulant activity, 378, 390 antiproliferative activity, 386 cardiac output, 383 function, 377 helicity, 380 inhibition of SMC growth, 384 intrinsic factor X inhibition, 381 low molecular weight, 380 protein kinase C activation, 385 prothrombic activity, 381 pulmonary hypertension, 382 structure, 337, 379 structure/activity relationship, 385 sulfonation, 387 Hyaluronan analysis, 136 angiogenesis, 83 asthma, 25, 68 bleomycin-induced injury, 25, 87, 118 bronchial mucus, 279 bronchoalveolar lavage fluid, 24, 117

[Hyaluronan] bronchopulmonary dysplasia, 90 degradation, 27, 113 emphysema, 91 function, 30, 75, 110, 145 hypersensitivity pneumonia, 25 idiopathic pulmonary fibrosis, 25, 117 inflammation, 81 intracellular, 76 leukocyte trafficking, 119 in lung, 56, 145 lung biomechanics, 63 lung development, 24, 85 localization by biotinylated probe, 41 macrophage activation, 119 malignant mesothelioma, 136 mucosal host defenses, 91 Na⫹/H⫹ exchange, 385 pleural fluid, 139 pulmonary hypertension, 48 respiratory distress syndrome (ARDS), 24, 89, 117 response to growth factors, 27 sarcoidosis, 25, 117 serum, 137 structure, 2, 23, 74, 108, 136, 336 synthesis, 2, 75 sulfonation, 389 vasculogenesis, 83 wound healing, 80 Hyaluronan receptor CD44, 28, 77 RHAMM, 79 Hyaluronan synthase (HAS) expression, 24, 28, 109 knockout, 28, 76 structure, 75 Hyaluronidase function, 27

I Integrin acute lung injury, 362 asthma, 363 fibrosis, 359

458

Subject Index

[Integrin] signal transduction, 352 structure, 352

K Keratan sulfate structure, 4, 339

L Link protein function, 8 knockout, 9 Lung bronchial mucus, 269 development, 85 interstitial pressure, 147 interstitium, 143 microvascular fluid exchange, 149 proteoglycans, 144, 247 Lumican asthma, 68, 177, 326 expression, 171 function, 10 interaction with TGFβ, 173 knockout, 11, 173 in lung, 56 regulation by basic FGF, 175 response to mechanical stimulation, 64 structure, 10

M Mesothelioma description, 135

P Perlecan asthma, 177 bleomycin-induced injury, 66 in blood vessels, 299 chronic obstructive pulmonary disease (COPD), 181 emphysema, 67, 251 function, 11

[Perlecan] knockout, 11 in lung, 56, 146 mutation, 12 structure, 11, 146 ventilation-induced lung injury, 66 Platelet-derived growth factor (PDGF), role in lung, 27, 75, 202, 342 Proteoglycans amyloid, 308 angiogenesis, 306 atherosclerosis, 307 atomic force microscopy, 50 basement membrane, 11 blood vessels, 295 cell adhesion, migration and proliferation, 303 cell surface, 12 changes in hypoxia, 159 classification, 1, 292 confocal microscopy, 50 contribution to edema, 153, 155 contribution to microvascular fluid exchange, 150 contribution to pulmonary interstitial pressure, 148 degradation, 5, 147, 327 diabetes, 308 electron microscopy, 49 extracellular matrix, 5 hemostasis and thrombosis, 301 histochemistry, 39 immunohistochemistry, 40 inflammation, 305 in situ hybridization, 49 interaction with growth factors, 252 intracellular, 15 lipid accumulation, 304 lung biomechanics, 56 lung development, 162 morphometry, 50 magnetic resonance imaging, 51 pulmonary hypertension, 48 restenosis, 307 synthesis, 4

Subject Index

459

Pulmonary vascular hypertension congenital cardiac shunts, 47 description, 42 emphysema, 47 pathology, 42 thromboembolism, 48 venous obstruction, 48

R RHAMM expression, 80 function, 116 gene, 79 structure, 79

S Serglycin in blood vessels, 300 function, 15 structure, 15 Syndecan in blood vessels, 299 function, 13 lipoprotein accumulation, 305 in lung, 146 structure, 12

T Transforming growth factor β (TGFβ), role in lung, 27, 75, 86, 202, 224, 341

V Versican asthma, 68, 177, 326 bleomycin-induced injury, 66, 179, 326 in blood vessels, 295 bronchiolitis obliterans organizing pneumonia (BOOP), 178 cell adhesion, migration and proliferation, 200 chronic obstructive pulmonary disease (COPD), 181 fibrotic lung disease, 66, 178, 196, 325 function, 8, 199 knockout, 28 lipoprotein interaction, 304 in lung, 56, 145 lung biomechanics, 59 respiratory distress syndrome (ARDS), 178 response to mechanical stimulation, 64 sarcoidosis, 178 structure, 8, 145, 199 systemic sclerosis, 181 ventilation-induced lung injury, 66 wound healing, 203

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Lung Development and Regeneration (Lung Biology in Health and Disease)

Pleural Disease, Second Edition (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 186 Pleural Disease

Lung Biology in Health & Disease Volume 151 Fetal Origins of Cardiovascular and Lung Disease

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

Lung Biology in Health & Disease Volume 211 Tropical Lung Disease 2nd Edition

Monitoring Asthma (Lung Biology in Health and Disease, Volume 207)

Severe Pneumonia (Lung Biology in Health and Disease, Volume 206)

Regulation of Breathing (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 176 Non-Neoplastic Advanced Lung Disease

Pneumocystis Pneumonia, 3rd Edition (Lung Biology in Health and Disease)

Lung Biology in Health and Disease Volume 153 Environmental Asthma

Bronchopulmonary Dysplasia, Volume 240 (Lung Biology in Health and Disease)

Cystic Fibrosis (Lung Biology in Health and Disease)

Lung Biology in Health & Disease Volume 195 Asthma Prevention

Lung Biology in Health & Disease Volume 135 Control of Breathing in Health and Disease

Lung Biology in Health & Disease Volume 180 Venous Thromboembolism

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Lung Biology in Health & Disease Volume 157 Respiratory-Circulatory Interactions in Health & Disease

Lung Biology in Health & Disease Volume 210 Sarcoidosis

Lung Biology in Health & Disease Volume 201 Lung Surfactant Function and Disorder

Lung Biology in Health & Disease Volume 160 Gravity and the Lung: Lessons from Microgravity

Genetics of Asthma and Chronic Obstructive Pulmonary Disease (Lung Biology in Health and Disease 218)

Lung Biology in Health and Disease Volume 214 Practical Pulmonary and Critical Care Medicine: Disease Management

Lung Biology in Health & Disease Volume 191 Long-Term Intervention in Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 182 Pharmacotherapy in Chronic Obstructive Pulmonary Disease

Lung Biology in Health & Disease Volume 184 Lung Volume Reduction Surgery for Emphysema

Lung Biology in Health & Disease Volume 200 Functional Lung Imaging

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