Osteoarthritis, inflammation, and degradation: a continuum

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OSTEOARTHRITIS, INFLAMMATION AND DEGRADATION: A CONTINUUM

Biomedical and Health Research Volume 70 Recently published in this series: Vol. 69. Vol. 68. Vol. 67. Vol. 66. Vol. 65. Vol. 64.

Vol. 63. Vol. 62.

Vol. 61. Vol. 60. Vol. 59. Vol. 58. Vol. 57. Vol. 56. Vol. 55. Vol. 54. Vol. 53. Vol. 52. Vol. 51. Vol. 50. Vol. 49. Vol. 48. Vol. 47.

O.K. Baskurt, M.R. Hardeman, M.W. Rampling and H.J. Meiselman (Eds.), Handbook of Hemorheology and Hemodynamics J.-F. Stoltz (Ed.), Mechanobiology: Cartilage and Chondrocyte – Volume 4 R.J. Schwartzman, Differential Diagnosis in Neurology H. Strasser (Ed.), Traditional Rating of Noise Versus Physiological Costs of Sound Exposures to the Hearing T. Silverstone, Eating Disorders and Obesity: How Drugs Can Help S. Eberhardt, C. Stoklossa and J.-M. Graf von der Schulenberg (Eds.), EUROMET 2004: The Influence of Economic Evaluation Studies on Health Care Decision-Making – A European Survey M. Parveen and S. Kumar (Eds.), Recent Trends in the Acetylcholinesterase System I.G. Farreras, C. Hannaway and V.A. Harden (Eds.), Mind, Brain, Body, and Behavior – Foundations of Neuroscience and Behavioral Research at the National Institutes of Health J.-F. Stoltz (Ed.), Mechanobiology: Cartilage and Chondrocyte – Volume 3 J.-M. Graf von der Schulenburg and M. Blanke (Eds.), Rationing of Medical Services in Europe: An Empirical Study – A European Survey M. Wolman and R. Manor, Doctors’ Errors and Mistakes of Medicine: Must Health Care Deteriorate? S. Holm and M. Jonas (Eds.), Engaging the World: The Use of Empirical Research in Bioethics and the Regulation of Biotechnology A. Nosikov and C. Gudex (Eds.), EUROHIS: Developing Common Instruments for Health Surveys P. Chauvin and the Europromed Working Group (Eds.), Prevention and Health Promotion for the Excluded and the Destitute in Europe J. Matsoukas and T. Mavromoustakos (Eds.), Drug Discovery and Design: Medical Aspects I.M. Shapiro, B.D. Boyan and H.C. Anderson (Eds.), The Growth Plate C. Huttin (Ed.), Patient Charges and Decision Making Behaviours of Consumers and Physicians J.-F. Stoltz (Ed.), Mechanobiology: Cartilage and Chondrocyte, Vol. 2 G. Lebeer (Ed.), Ethical Function in Hospital Ethics Committees R. Busse, M. Wismar and P.C. Berman (Eds.), The European Union and Health Services T. Reilly (Ed.), Musculoskeletal Disorders in Health-Related Occupations H. ten Have and R. Janssens (Eds.), Palliative Care in Europe – Concepts and Policies H. Aldskogius and J. Fraher (Eds.), Glial Interfaces in the Nervous System – Role in Repair and Plasticity ISSN 0929-6743

Osteoarthritis, Inflammation and Degradation: A Continuum

Edited by

Joseph A. Buckwalter University of Iowa, Department of Orthopaedics and Rehabilitation, Iowa City, USA

Martin Lotz Division of Arthritis Research, The Scripps Research Institute, La Jolla, USA

and

Jean-François Stoltz Groupe Ingénierie et Thérapie Cellulaire UMR CNRS – UHP 7563 – Faculté de Médecine Vandoeuvre Lès Nancy – France and Unité de Thérapie Cellulaire et Tissus CHU NANCY – Vandoeuvre lès Nancy – France

Amsterdam • Berlin • Oxford • Tokyo • Washington, DC

© 2007 The authors and IOS Press. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without prior written permission from the publisher. ISBN 978-1-58603-773-4 Library of Congress Control Number: 2007938066 Publisher IOS Press Nieuwe Hemweg 6B 1013 BG Amsterdam Netherlands fax: +31 20 687 0019 e-mail: [email protected] Distributor in the UK and Ireland Gazelle Books Services Ltd. White Cross Mills Hightown Lancaster LA1 4XS United Kingdom fax: +44 1524 63232 e-mail: [email protected]

Distributor in the USA and Canada IOS Press, Inc. 4502 Rachael Manor Drive Fairfax, VA 22032 USA fax: +1 703 323 3668 e-mail: [email protected]

LEGAL NOTICE The publisher is not responsible for the use which might be made of the following information. PRINTED IN THE NETHERLANDS

Osteoarthritis, Inflammation and Degradation: A Continuum J. Buckwalter et al. (Eds.) IOS Press, 2007 © 2007 The authors and IOS Press. All rights reserved.

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Preface Osteoarthritis is a major public health issue due to its impact in term of handicap. Moreover, Ageing of the world population and outbreak of obesity in industrialized and non-industrialized countries will dramatically increase its incidence in the next years. Regarded as a multi-factorial disease, today mechanistic and inflammatory theories are no more opposed but, on the contrary, are framed within the same continuum: osteoarthritis, inflammation and degeneration. In order to collect major information in a benchmark book on the fundamental aspects of this disease, internationally well-known authors, from multiple specialties, gathered to analyse, dissect and finally try to understand the secrets of a disease which should no more be regarded as the common and relentless result of ageing or of passive wear but much more as an active disease able to benefit from the best targeted pharmacological (anti-cytokines, inhibitors of signalling pathways, inhibitors of proteases, etc.) and non-pharmacological (cellular therapy, gene therapy, cartilage engineering etc.) therapies, current and future. Update of these therapies goes through a sharp knowledge of the different pathophysiological mechanisms of osteoarthritis. Major new paradigms have emerged in this field in the very last years. For example, it is noteworthy that cartilage used to be the unique tissue involved in the OA process. But in this book, many chapters refer to novel findings on the role of other tissues like bone or synovial tissue which should be of critical importance for the degradative process of the OA joint. Another challenge refers to the possibility in the future to evaluate the potential severity of the disease in a single patient from the very early stage. The recent new advances in imaging and biomarkers detailed in this volume suggest that we are not so far from this capacity. These recent advances have been compiled in this monograph, that should captivate a large audience, scientists and clinicians. F. Berenbaum Elected President of OARSI

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Acknowledgments Writing a monograph is a difficult and collective task. That is the reason why, on behalf of Joseph Buckwalter, and Martin Lotz, I would like to thank all the authors who have contributed to this work. A special acknowledgements to Drs Martine Burger and Jean Gavaudan (MD, FCP) for their friendly and efficient help. I would not forget the secretaries of my department, Isabelle and Estelle, who have followed up the administrative work and maintained contacts between the authors and the publisher. J.F. Stoltz

Contributors Aigner Thomas Osteoarticular and Molecular Pathology Institute of Pathology University of Leipzig Germany [email protected] Berenbaum Francis 1 – Unité Mixte de Recherche CNRS 7079 Physiology and Physiopathology Laboratory University Paris 6, 7 quai St. Bernard, Bât A France 2 – Department of Rheumatology UFR Pierre et Marie Curie Saint-Antoine hospital 75012 Paris France [email protected] Bianchi Arnaud Laboratoire de Physiopathologie et Pharmacologie Articulaires (LPPA) UMR 7561 CNRS-Nancy Université Avenue de la forêt de Haye, BP 184 54505 VANDŒUVRE-lès-NANCY France [email protected]

Blanco Franscisco J. Osteoarticular and Aging Research Lab Biomedical Research Center CH Universitario Juan Canalejo A Coruña Spain Phone: 34-981-178272 Fax: 34-981-178273 [email protected] [email protected] Buckwalter Joseph Professor, Head and Steindler Chair Department of Orthopaedics and Rehabilitation University of Lowa 01008-A JPP, 200 Hawkins Drive Iowa City, IA 52242 USA Phone: 319-356-3595 Fax: 319-356-8999 [email protected] De Isla Natalia Groupe d’Ingénierie Cellulaire et Tissulaire, LEMTA-UMR CNRS 7563 Faculté de Médecine Université Henri Poincaré, 9 av. de Haye 54505 Vandoeuvre les Nancy France [email protected]

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Ding Lei William E. Cornatzer Chair in Biochemistry Department of Biochemistry and Molecular Biology University of North Dakota School of Medicine and Health Sciences Box 9037 501 N. Columbia Road Grand Forks, ND 58202 USA [email protected] Dumas Dominique Groupe d’Ingénierie Cellulaire et Tissulaire, Faculté de Médecine Université Henri Poincéré UMR CNRS 7563 LEMTA 54505 Vandoeuvre lès Nancy France [email protected] Gabay Odile Unité Mixte de Recherche CNRS 7079 Physiology and Physiopathology Laboratory, University Paris 6, 7 quai St. Bernard, Bât A France [email protected] Galteau Marie-Madeleine Nancy Université UHP, Laboratoire de Physiopathologie et Pharmacologie Articulaires UMR 7561 CNRS Avenue de la forêt de Haye, BP 184 54505 Vandoeuvre lès nancy France [email protected] Goldring Mary B. Laboratory for Cartolage Biology Hospita for Special Surgery Weill College of Medicine of Cornell University Caspary Research Building, Room 528 535 E. 70th Street

New York, NY 10021 USA Tel.: 212-774-7564 Fax: 212-249-2373 [email protected] http://www.hss.edu/researchstaff_goldring-mary.asp Gomez Rodolfo Santiago University Clinical Hospital NEIRID Lab, NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases Research Area, Laboratory nº4 Trav. Choupana sn 15706 Santiago de Compostela Spain Ph. & Fax: 34+981+950905 [email protected] Gomez-Reino Juan J. Santiago University Clinical Hospital Division of Rheumatology Door 36. Trav. Choupana sn 15706 Santiago de Compostela Spain Phone: 34+981+951036 [email protected] Gosset Marjolaine UMR CNRS 7079 Physiology and Physiopathology Laboratory University Paris 6, 7 quai St. Bernard Bât A, 75 000 Paris France [email protected] Gualillo Oreste Santiago University Clinical Hospital NEIRID Lab, NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases Research Area, Laboratory nº4 Trav. Choupana sn 15706 Santiago de Compostela Spain Ph. & Fax: 34+981+950905 [email protected] [email protected]

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Guo Danping William E. Cornatzer Chair in Biochemistry Department of Biochemistry and Molecular Biology University of North Dakota School of Medicine and Health Sciences Box 9037 501 N. Columbia Road Grand Forks, ND 58202 USA [email protected] Homandberg Gene A. Professor and Chair William E. Cornatzer Chair in Biochemistry Department of Biochemistry and Molecular Biology University of North Dakota School of Medicine and Health Sciences Box 9037 501 N. Columbia Road Grand Forks, ND 58202 USA Phone: 701-777-6422 Fax: 701-777-2382 [email protected] Jimenez Segio Jefferson Institute of Molecular Medicine Thomas Jefferson University Suite 509 Bluemle Life Sciences Building 233 South 10th Street Philadelphia, PA, 19107 USA [email protected] Jouzeau Jean-Yves Nancy université UHP, Laboratoire de Physiopathologie et Pharmacologie Articulaires UMR 7561 CNRS Avenue de la forêt de Haye, BP 184 54505 Vandoeuvre Lès Nancy France [email protected]

Kirchmeyer Melanie Nancy université UHP, Laboratoire de Physiopathologie et Pharmacologie Articulaires UMR 7561 CNRS Avenue de la forêt de Haye, BP 184 54505 Vandoeuvre Lès Nancy France [email protected] Lago Francisca Laboratory of Molecular and Cellular Cardiology Santiago University Clinical Hospital Research Laboratory 1 Trav. Choupana sn 15706 Santiago de Compostela Spain Phone: 34+981+950902 Fax: 34+981+950905 [email protected] Lago Rocio Santiago University Clinical Hospital NEIRID Lab NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases Research Area, Laboratory nº4 Trav. Choupana sn 15706 Santiago de Compostela Spain Ph. & Fax: 34+981+950905 [email protected] Lajeunesse Daniel Osteoarthritis Research Unit University of Montreal Hospital Centre Notre-Dame Hospital 1560 Sherbrooke Street East Montreal, Quebec, H2L 4M1 Canada Tel.: 514-890-8000, ext. 28914 Fax: 514-412-7583 [email protected]

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Lopez-Armada Maria J. Osteoarticular and Aging Research Lab Biomedical Research Center CH Universitario Juan Canalejo A Coruña Spain [email protected] Lotz Martin Division of Arthritis Research The Scripps Research Institute Div MEM 161 10550 North Torrey Pines Road La Jolla, CA 92037 USA [email protected] Malemud Charles J. Charles J. Malemud, Ph.D. Case Western Reserve University and University Hospitals Case Medical Center Department of Medicine Division of Rheumatic Diseases 2061 Cornell Road, Rm. 207 Cleveland, Ohio 44106-5076 USA [email protected] Martel-Pelletier Johanne Director, Osteoarthritis Research Unit and Osteoarthritis Chair University of Montreal Hospital CHUM, Notre-Dame Hospital 1560 Sherbrooke Street East Montreal, Quebec, H2L 4M1 Canada Tel.: 514-890-8000, ext. 26658 Fax: 514-412-7582 [email protected] Martin James A. University of Iowa Department of Orthopaedics and Rehabilitation 200 Hawkins Drive Iowa City, IA 52242 USA

Oliviero Francesca Rheumatology Unit Department of clinical and Experimental Medicine University of Padova Via Giustiniani 2 35128 Padova Italy Phone: +39 049 8212190 Fax: +39 049 8212191 [email protected] Otero Miguel Santiago University Clinical Hospital NEIRID Lab, NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases Research Area, Laboratory nº4 Trav. Choupana sn 15706 Santiago de Compostela Spain Ph. & Fax: 34+981+950905 Present address is: Hospital for Special Surgery Caspary Research Building 5th Floor, 535 E. 70th Street New York, NY, 10021 USA Phone: 212-774-755 Pedersen Douglas R. University of Iowa Department of Orthopaedics and Rehabilitation 200 Hawkins Drive Iowa City, IA 52242 USA Pelletier Jean-Pierre Head, Arthritis Centre University of Montreal Director, Osteoarthritis Researche Unit and Osteoarthritis Chair University of Montreal CHUM, Notre-Dame hospital 1560 Sherbrooke Street East Montreal, Quebec, H2L 4M1 Canada [email protected]

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Piera-Velazquez Sonsoles Jefferson Institute of Molecular Medicine Thomas Jefferson University Suite 509 Bluemle Life Sciences Building 233 South 10th Street Philadelphia, PA, 19107 USA Punzi Leonardo Rheumatology Unit Department of clinical and Experimental Medicine University of Padova Via Giustiniani 2, 35128 Padova Italy Phone: +39 049 8212190 Fax: +39 049 8212191 [email protected] Rego Ignacio Osteoarticular and Aging Research Lab Biomedical Research Center CH Universitario Juan Canalejo A Coruña Spain [email protected] Riquelme Bibiana Areas Física e Inmunología Facultad de Ciencias Bioquímicas y Farmacéuticas Universidad Nacional de Rosario Suipacha 531, 2000 Rosario Argentina [email protected] Sandell Linda Department of Orthopaedic Surgery and Cell Biology Washington University School of Medicine, MS 8233 660 S. Euclid Ave. St. Louis, MO 63110 USA Tel.: 314-454-7800 Fax: 314-454-5900 [email protected]

Sfriso Paolo Rheumatology Unit Department of clinical and Experimental Medicine University of Padova Via Giustiniani 2, 35128 Padova Italy Phone: +39 049 8212190 Fax: +39 049 8212191 [email protected] Smith Robert Lane Rehabilitation Research and Development Center VA Palo Alto Health Care System Palo Alto, CA Department of Orthopaedic Surgery Stanford University School of Medicine Stanford, CA USA [email protected] Stoltz J.F. Directeur du groupe d’Ingénierie Cellulaire et Tissulaire LEMTA-UMR CNRS 7563 Faculté de Médecine Université Henri Poincaré 9 av. de Haye (Nancy Université-UHP) 54505 Vandoeuvre Lès Nancy France and Chef de service Unité de Thérapie Cellulaire et Tissulaire Brabois – 54 500 Vandoeuvre Lès Nancy France Tel.: +33 3 83 15 37 79 [email protected] Terkeltaub Robert, MD Chief, VA Rheumatology Section Professor of Medicine, UCSD 111K, VAMC 3350 La Jolla Village Drive San Diego, CA 92161 USA Tel.: 858-642-3519 Fax: 858-552-7425 [email protected]

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Thedens Daniel R. Department of Radiology The University of Iowa City IA 52242 USA [email protected] Van Den Berg Wim Experimental Rheumatology & Advanced Therapeutics Nijmegen Centre For Molecular Life Sciences Radboud University Medical Centre

Nijmegen The Netherlands [email protected] Van Der Kraan Peter Experimental Rheumatology & Advanced Therapeutics Nijmegen Centre For Molecular Life Sciences Radboud University Medical Centre Nijmegen The Netherlands [email protected]

This monograph was published in appreciation of OARSI and of European Society on Cell and Tissue Engineering and Therapy and under the patronage of Université Henri Poincaré (Nancy université) with an unrestricted educational grant of Negma-Lerads (Wockhardt international).

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Contents Preface F. Berenbaum Acknowledgments and Contributors J.F. Stoltz

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Part I. Extra Cellular Stimuli I. Inflammatory Factors Involved in Osteoarthritis Johanne Martel-Pelletier and Jean-Pierre Pelletier

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II. Mechanical Loading Effects on Articular Cartilage Matrix Metabolism and Osteoarthritis Robert Lane Smith

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III. Aging, Inflammation, and Altered Chondrocyte Differentation in Articular Cartilage Calcification and Osteoarthritis Robert A. Terkeltaub

31

IV. Leptin, the Prototype of Adipokines: Molecules at the Crossroads of Inflammation and Metabolism Rodolfo Gómez, Rocío Lago, Francisca Lago, Juan J. Gómez-Reino, Miguel Otero and Oreste Gualillo V. The Role of Extracellular Matrix Fragments in the Autocrine Regulation of Cartilage Metabolism Gene A. Homandberg, Lei Ding and Danping Guo VI. Pathophysiological Relevance of PPAR to Osteoarthritis: From the Control of Inflammation to Cartilage Protection? Arnaud Bianchi, Mélanie Kirchmeyer, Marie-Madeleine Galteau and Jean-Yves Jouzeau

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56

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Part II. Signalling Mechanisms VII. MAP Kinases Charles J. Malemud VIII. Transcriptional Control of Chondrocyte Gene Expression Mary B. Goldring and Linda J. Sandell IX. Gene Expression Profiling of Human Articular Chondrocytes and Osteoarthritis Sergio A. Jimenez and Sonsoles Piera-Velazquez

99 118

143

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Part III. Effectors and Different Pathways X. Prostaglandin E2 and Osteoarthritis: The Role of Cyclooxygenases, Prostaglandin E Synthases and 15-Prostaglandin Dehydrogenases Odile Gabay, Marjolaine Gosset and Francis Berenbaum

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XI. NO and Other Radicals in the Pathogenesis of Osteoarthritis Martin Lotz

182

XII. Mitochondria and Chondrocytes: Role in Osteoarthritis Francisco J. Blanco, María J. López-Armada and Ignacio Rego

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XIII. Subchondral Bone and Osteoarthritis Progression: A Very Significant Role Johanne Martel-Pelletier, Daniel Lajeunesse and Jean-Pierre Pelletier

206

XIV. Osteoarthritis and Inflammation – Inflammatory Changes in Osteoarthritic Synoviopathy Thomas Aigner, Peter van der Kraan and Wim van den Berg

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Part IV. Imaging and Clinical Applications XV. Magnetic Resonance Imaging of Cartilage: New Imaging and Clinical Approaches Daniel R. Thedens, James A. Martin and Douglas R. Pedersen

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XVI. Multimodality of Microscopy Imaging Applied to Cartilage Tissue Engineering D. Dumas, B. Riquelme, E. Werkmeister, N.D. Isla and J.F. Stoltz

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XVII. Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis Leonardo Punzi, Francesca Oliviero and Paolo Sfriso

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XVIII. Cartilage Engineering J.F. Stoltz, M. Lotz and J. Buckwalter

280

XIX. Therapeutics and Osteoarthritis J. Buckwalter, M. Lotz and J.F. Stoltz

287

Author Index

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Part I Extra Cellular Stimuli



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I Inflammatory Factors Involved in Osteoarthritis Johanne MARTEL-PELLETIER ∗, PhD and Jean-Pierre PELLETIER, MD Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada, H2L 4M1 Abstract. Osteoarthritis (OA) is a disease that predominantly, but not solely, affects the diarthrodial joints and results from an interaction between a number of complex mechanical and biological processes. Knowledge of the etiopathogenesis of OA has progressed significantly in the past few decades. A major characteristic of OA is articular cartilage destruction, yet it has become obvious that synovial inflammation, although not a primary cause of the disease, is among the significant structural changes that take place during its development. There is compelling evidence suggesting that secreted inflammatory mediators impact on the matrix homeostasis of articular tissue cells by altering their metabolism. Among these mediators that are responsible for the progression of the disease, evidence points to the proinflammatory cytokine interleukin-1 beta (IL-1ß) as the most important factor responsible for the catabolic process in OA. New members of the IL-1 superfamily have recently been identified (ILF5-ILF10), some of which are suggested to be of interest for the arthritic diseases. Other proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-17, IL-18, and IL-8, are also considered potential contributing factors in the pathogenesis of OA. However, the exact role and importance of each in the OA process is not yet clearly established. In addition to cytokines, other inflammatory mediators also play a major role in the OA pathological process. These include nitric oxide (NO), eicosanoids (prostaglandins and leukotriene), and a newly identified cell membrane receptor family, the protease-activated receptors (PARs), in which an important role for PAR-2 in chronic arthritis has been suggested. All these topics will be discussed in this review and should help the reader to better understand the most recent advances concerning the inflammatory factors involved in the pathophysiology of OA.

Introduction Osteoarthritis (OA) is a disease closely associated with the aging process and therefore represents a growing public health cost, not only for the Western countries but worldwide. Interestingly, arthritis is the second most expansive disease category in North ∗ Corresponding Author: Johanne Martel-Pelletier, PhD, Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada, H2L 4M1, Phone: 514-890-8000, ext. 26658, Fax: 514-412-7582, E-mail: [email protected].

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J. Martel-Pelletier and J.-P. Pelletier / Inflammatory Factors Involved in Osteoarthritis

America, second only to cardiovascular disease and followed by cancer. OA is the most prevalent arthritic disease and the one most seen and treated by rheumatologists and general practitioners. Since there is, as yet, no cure for this disease, the economical impact of OA on our health economy is an important concern in the context of an aging population. Indeed, this disease affects 10 to 15 percent of the world’s population, and its frequency increases with aging; its incidence is higher than 60 percent in the population over 65 years of age. The aetiology of OA is multifactorial, yet this disease is characterized by a number of articular structural changes, including cartilage destruction and alterations in synovial membrane and subchondral bone, which impair joint movement and cause pain. Cartilage destruction is associated with, and it is believed that it may even be preceded by, subchondral bone alterations. During the course of OA, intermittent flares, which reflect the presence of an inflammatory process, appear at the synovial membrane. There is a general consensus that synovial inflammation in OA, although not a primary phenomenon in this disease, contributes to its progression. This review focuses on bringing to the reader an understanding of which of the inflammatory factors participate in the complex interaction in OA tissues, and lead to the progression of structural changes observed in this disease. This knowledge is essential not only to a further understanding of the pathophysiology of the disease per se, but also to the development of new therapeutic strategies that can modify the progression of the disease.

Pathophysiology It is now well established that in OA, the earliest histopathological alteration that occurs in cartilage is a depletion of major matrix macromolecules including collagen and aggrecan. Collagen is of particular importance as its breakdown results in the loss of the structural integrity of the tissue. It appears that alterations of the collagen network, as well as the aggrecan, result from an increased level of proteolytic enzymes synthesized by chondrocytes. A great deal of attention has been given to identifying the protease most likely responsible for the occurrence of matrix degradation. It is widely accepted that the metalloprotease (MMP) family comprises a major involvement in the disease process [1]. Of this family, collagenases (enzymes responsible for collagen degradation) and aggrecanases (enzymes responsible for the aggrecan cleavage found in OA synovial fluid) have been suggested to play major roles in the degradation of the extracellular matrix observed in OA. The increase in the level of collagenases (collagenase-1 [MMP-1], collagenase-2 [MMP-8], and collagenase-3 [MMP-13]) found in human OA cartilage provide strong evidence to this effect. Although all three collagenases are active on collagen fibrils, differences in their in situ roles and in mechanisms regulating their expressions have been reported. Taking into account the specificity of each of these collagenases, neutralizing the synthesis/activity of collagenase-3 (MMP-13) seems to be the most promising strategy to halt cartilage breakdown [2]. As for the aggrecanases, the importance of two such enzymes has been reported: aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5). However, recent studies have demonstrated that ADAMTS-5 appears to be the predominant enzyme involved in the OA degradative process [3,4].


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Other enzymes from the serine-dependent protease family, such as the plasminogen activator/plasmin system, are also likely to play a role, but, in cartilage, primarily as activators of MMPs. Enzymes from the cysteine protease family, including cathepsin members, also appear to be involved in articular tissue degradation. The enzymatic alterations in cartilage matrix may explain the exhaustive degradation of this tissue but do not provide an explanation for the factors involved in the upregulation of the expression and synthesis of enzymes in this disease. The following hypothesis has been put forward to explain the pathological development of OA at the clinical stage of the disease. The cartilage matrix breakdown, produced by proteolytic enzymes, releases increased amounts of matrix macromolecule fragments and neoantigens into the synovial fluid which, upon phagocytosis, promote inflammation in the synovial membrane. In turn, the inflamed synovial membrane releases several mediators capable of creating a vicious cycle by inducing increased cartilage degradation and subsequently triggering further inflammation.

Inflammatory Mediators Proinflammatory Cytokines Considerable evidence has accumulated to indicate that the proinflammatory cytokines are crucial in mediating inflammation and tissue destruction in OA. It is claimed, and substantiated by studies on animal models, that interleukin-1-ß (IL-1ß) is of pivotal importance in OA cartilage destruction and considered to be the principal mover of the enzyme system [5–7]. The catabolic effects of IL-1ß are multiple. This cytokine is able to stimulate its own production, to increase the synthesis of enzymes (MMPs, plasminogen activator/plasmin), to inhibit the synthesis of the major physiological inhibitors of these enzymes (TIMPs, PAI-1), to inhibit the synthesis of matrix constituents such as collagen and proteoglycans, and to stimulate the synthesis and release of some eicosanoids including prostaglandins and leukotrienes. The action of this proinflammatory cytokine on the enzyme process combined with the suppression of matrix synthesis results in severe degradation of articular tissues and the appearance of conditions that we know to be characteristic of OA. This cytokine also plays important roles in normal physiology, including stimulation of the turnover of extracellular matrix. Hence, control mechanisms exist to limit the extent of cytokine activation and to avoid potential tissue injury. One of these control mechanisms, unique to the IL-1 system, is a physiological inhibitor of its receptor known as the IL-1 receptor antagonist (IL-1Ra). IL-1Ra is a structural derivative of IL-1 that binds to IL-1 receptors but does not activate target cells [8]. IL-1Ra blocks the effects of IL-1 in the immediate cell environment by competing for binding to cell-surface receptors. Although IL-1Ra was originally described as a secreted form (sIL-1Ra), it is now known that IL-1Ra consists of a family of molecules. Three additional intracellular structural variants of IL-1Ra (icIL-1Ra1, 2, 3) have been identified and are formed by alternate transcriptional splice mechanisms. These isoforms of IL-1Ra do not possess leader sequences and are therefore synthesized in the cytoplasm but are not usually secreted from cells. The icIL-1Ra1 also binds to IL-1 receptors with equal avidity as the secreted form (sIL-1Ra), and can be secreted from cells under certain conditions. Its role is suggested to be the inhibition of IL-1 binding to extracellular receptors in specific situations. icIL-1Ra2 has been described in cells only at the

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mRNA level and may not normally exist as a protein. icIL-1Ra3 is a lower molecular weight protein that is produced in large amounts by neutrophils and hepatocytes, and binds poorly to IL-1 receptors. In order to verify the effects of blocking IL-1β, in vivo studies were first conducted in animal models of OA, in which IL-1Ra was administered by intra-articular injection or by gene therapy. Data showed such in vivo treatment was therapeutically beneficial [9–11]. IL-1Ra injection in patients with symptomatic knee OA also showed significant improvement on symptoms [12]. However, the results of this pilot study could not be confirmed in another recent Phase II double-blind study [13]. Other proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), IL-17 and IL-18, as well as some chemokines such as IL-8, are also considered potential contributing factors in the pathogenesis of OA. It has been shown that all of these cytokines are expressed in OA tissues; however, the exact role and importance of each in the OA process is not yet clearly established, and it is not known whether a functional hierarchy exists between them. TNF-α is a potent cytokine that exerts diverse effects by stimulating a variety of cells. The best-studied aspect of TNF-α is its ability to promote inflammation. This proinflammatory cytokine’s dominant role in rheumatoid arthritis is well illustrated by the blocking of its activity in vivo in studies on animal models and, more recently, on humans. This cytokine is also present in OA but at a severe stage of the disease. Recent studies provide evidence that OSM, a member of the IL-6 family, plays a role in the inflammatory response. However, because this cytokine is also involved in physiological as well as pathological functions, its exact role is not known. Indeed, although most of the in vitro findings point to the catabolic effects of OSM (14–17], some in vivo studies suggest anabolic effects, whereby OSM promotes wound healing and bone formation in addition to having anti-inflammatory effects (14–21]. Interestingly, OSM is the only member of this cytokine family to cause proteolytic release of proteoglycan and collagen from human articular cartilage. Although OSM upregulates a spectrum of protease inhibitors, including TIMP and serine protease inhibitors (α1-protease inhibitor, antichymotrypsin and PAI-1), its proinflammatory role in arthritis may rely on the upregulation of the synthesis of some MMPs and PGE2. Also of interest is the capacity of OSM to synergize the action of other inflammatory mediators [17,22–24], including IL-1, TNF-α, IL-17 and lipopolysaccharide. More particularly, in chondrocytes this striking synergistic effect appears to occur through the induction of the expression of the collagenases, stromelysin-1, MT1-MMP and aggrecanases. Among actions relevant to joint inflammation, OSM also induces IL-6. However, the role of IL-6 in inflammation remains unclear, since IL-6 can induce the production of TIMP-1, IL-1Ra and the soluble TNF receptor 55. Among the other cytokines, it has been suggested that IL-17 and IL-18 play a role in OA pathophysiology as they both share many properties with IL-1. However, on articular tissue cells, these cytokines also demonstrate effects independent of IL-1ß. Moreover, both seem to be involved in the early phase of the inflammatory process. Local in vivo overexpression of IL-17 has been shown to promote destructive arthritis [25]. In collagen-induced arthritis in mice, the blocking of endogenous IL-17 resulted in suppression of arthritis, including a clear suppression of joint damage [26]. It is interesting to note that this study also showed that neutralization of IL-1 had no effect on IL-17-induced inflammation and joint damage, identifying the IL-1-


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independent role of IL-17. Furthermore, IL-17 induced the expression of the receptor activator of NF-κB ligand, the RANKL, in osteoblasts, a crucial factor in bone resorption [27]. In addition, in vivo studies using adenoviral overexpression of IL-17 in the joint showed that this cytokine induced focal erosions in the bone. Consequently, this factor could be of great relevance in OA, as subchondral bone remodelling appears to be intimately involved in the early phase of the disease. IL-18 is a pleiotropic cytokine belonging to the IL-1 family. Although this cytokine is a critical factor in developing immune responses, in vivo data in experimental animals indicate that the net effect of IL-18 in the development of arthritis is proinflammatory. In addition, IL-18 can induce other pro-inflammatory cytokines such as IL-1, creating an amplifying loop. Its potential involvement in OA has been suggested based on its enhanced presence in OA cartilage and synovial membrane [28,29]. Six new members of the IL-1 family were identified primarily through the use of DNA database searches for IL-1 homologues, and named IL-1F5 to IL-1F10 [30]. Although expression patterns and the biological functions of these new IL-1 family members have not yet been well characterized, some of them could be of interest for the arthritic diseases. IL-1F7 forms a complex with IL-18 binding protein, which might bind to and sequester IL-18R [31]. IL-1F10 is a low nonagonistic ligand for IL-1R1 [32]. IL-1F9 as well as IL-1F6 and IL-1F8 activate NF-κB (one of the most important inducers of inflammation), and IL-1F5 might be an IL-1F9 antagonist [33,34]. Finally, IL-1F8 has been shown to exert proinflammatory effects in human joint cells [35]. Nitric Oxide and Eicosanoids In addition to proinflammatory cytokines, other inflammatory mediators could also play major roles in the OA process, the principals being nitric oxide (NO) and the eicosanoids, including prostaglandins and leukotrienes. NO acts as a mediator in various physiological and pathophysiological processes in the human body. NO generated by the inducible NO synthase (iNOS) has regulatory, proinflammatory and destructive effects. OA cartilage produces a large amount of NO (and reactive oxygen species), and high levels of nitrites/nitrates have been found in the synovial fluid and serum of arthritis patients. NO has been shown to be involved in the promotion of cartilage catabolism in OA through a number of mechanisms, including the induction of synovial inflammation. It can inhibit the synthesis of cartilage matrix macromolecules, such as aggrecans, enhance MMP activity, and reduce the synthesis of IL-1Ra by chondrocytes. NO also plays a role in chondrocyte apoptosis and induces cyclooxygenase (COX)-2/prostaglandin E2 (PGE2) synthesis. Also demonstrated was that exogenous PGE2 could sensitize human OA chondrocytes to cell death induced by NO [36,37]. In vivo findings in studies done using OA experimental animal models in which oral administration of therapeutic dosages of a specific inhibitor of iNOS demonstrated positive therapeutic benefits on the progression of lesions [37–39]. Collectively, inducible NO acts by reducing the major anabolic processes and increasing the catabolic processes, making it a complete factor favouring joint destruction. Moreover, the present knowledge points to a possible therapeutic value for iNOS inhibitors in the treatment of OA as chondroprotective, anti-inflammatory and analgesic compounds. This molecule is therefore believed to be an attractive target in OA, because reducing its excess production may not only slow the disease progression, but is also likely to reduce the symptoms, making it able to reach two targets simultaneously.

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Other major inflammatory factors involved in OA pathophysiology are the prostaglandins and leukotrienes. Prostaglandins are synthesized from arachidonic acid via the actions of the COX enzymes, either constitutively or in response to cell-specific trauma, stimuli, or signalling molecules. The most abundant prostanoid in the human body is PGE2. Dependent upon context, PGE2 exerts a homeostatic or inflammatory effect. Inhibition of PGE2 synthesis by non-steroidal anti-inflammatory drugs (NSAIDs) has been an important anti-inflammatory strategy for more than a century. In addition to exacerbating joint inflammation, PGE2 can also potentiate the effects of other mediators of inflammation. It can affect cartilage remodelling directly or function indirectly as an autocrine regulatory factor. PGE2 may also contribute to joint damage by promoting MMP production, osteoclastic bone resorption, and angiogenesis. COX (COX-1 and -2) activity had been considered the key step in prostaglandin synthesis. Recently, other splice variants of COX-1 have been identified and named COX-3 and PCOX-1 [40,41]. They were first identified in canine tissues. Recently the presence of a COX-3 mRNA transcript was confirmed in human cells. The regulation of COX-3 appears to be identical to that of COX-1, and one of the PCOX, the PCOX1a, was shown to lack the COX activity. Moreover, splice variants of COX-2 have also been reported, but have failed to show enzymatic activity [42]. Metabolism of arachidonic acid by COX-1 or COX-2 yields to the unstable intermediary PGH2, which can be further metabolized into PGE2, PGD2, PGF2α, PGI2 (prostacyclin) or tromboxane A2. The enzyme responsible for the isomerization of PGH2 was not known until recent identification of PG synthase (PGS) as the terminal enzyme responsible for prostanoid synthesis. In the case of PGE2, studies suggest the presence of at least three distinct PGES, named cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1), and mPGES-2 [43–46]. cPGES is constitutively and ubiquitously expressed and is preferentially coupled with COX-1, promoting immediate production of PGE2 [45–47]. COX-2 and mPGES-1 protein expression are concordantly induced by IL-1ß, consistent with the hypothesis that mPGES-1 and COX-2 are co-regulated and that stimulated PGE2 synthesis may depend on upregulation of both of these enzymes. mPGES-2, the most recently identified PGES, is constitutively expressed in diverse tissues. While its role remains elusive, it has been found to be functionally linked to both COX-1 and COX-2. However, and although mPGES-2 can couple with COX-1 to produce PGE2 in response to acute inflammation and to COX-2 in response to chronic inflammation, data have demonstrated a modest preference for coordination with COX-2. Studies from mPGES-1-deficient mice and animal models of inflammatory arthritis strongly suggest the role of mPGES-1 in inducible PGE2 production and arthritis [48,49]; and accumulating evidence implicates mPGES-1 in the pathogenesis of OA. Hence, mPGES-1 is localized in the superficial layers of human OA cartilage, areas where IL-1ß is also found, which is consistent with a role for IL-1ß in stimulating chondrocyte mPGES-1 [50,51]. Moreover, PGE2 secretion from OA chondrocytes correlates well with mPGES-1 concentrations following stimulation [52]. Since the controversy surrounding selective COX-2 inhibitors owing to an apparent increase in the risk of cardiovascular disease and stroke, researchers have been looking at extensively the potential utility of clinically targeting mPGES-1, yet no specific pharmacologic inhibitors are currently available. The use of NSAIDs or COX-2 selective inhibitors has shown that PGE2 inhibition alone does not seem to delay the natural history of progressive OA. In recent years, it has been shown that PGE2 synthesis is only one part of the arachidonic acid pathway. The precursor, arachidonic acid, is a substrate that gives origin to many other lipid me-


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diators, including leukotrienes. Leukotrienes themselves play a major role in the development and persistence of the inflammatory process, and it is now clear that prostaglandins and leukotrienes have complementary effects. Leukotrienes are produced by the enzyme 5-lipoxygenase (5-LOX). Leukotriene A4 (LTA4) is the first to be synthesized and is then processed into LTB4 or LTC4, then LTD4 and LTE4, which are potent chemotactic and inflammatory factors. Levels of LTB4 and LTC4 are increased in OA synovial tissue [53,54]. Studies have also revealed that, on human OA synovial membrane, LTB4 potently stimulates the release of proinflammatory cytokines such as IL-1ß and TNF-α [54–58]. Thus, the failure of NSAIDs to impact OA progression could be due to the fact that inhibiting only the COX pathways leads to a shunt to leukotriene production in these tissues [59–63]. From this concept, it is hypothesized that blocking production of both leukotrienes and prostaglandins could have a synergistic effect in achieving optimal or a wider-spectrum of anti-inflammatory activity. Data on a disease modifying OA drug (DMOAD) Phase III clinical trial of such a dual inhibitor of COX and 5-LOX revealed that such a drug can significantly reduce the progression of knee OA structural changes using quantitative magnetic resonance imaging (qMRI) [64]. More particularly, this was found for the cartilage volume. This drug was also equally effective as a known NSAID, naproxen, at reducing OA symptoms. Protease-Activated Receptors Some members of the protease-activated receptors (PARs) have been recently shown to be involved in inflammatory pathways, and, more specifically, an important role for PAR-2 in chronic arthritis has been demonstrated. These receptors belong to a novel family of seven-transmembrane G-protein-coupled receptors that are activated through a unique process. Cleavage of their N-terminal domains by proteases unmasks a new N-terminal sequence that acts as a tethered ligand, binding and activating the receptor itself [65–67]. Once activated, this process is irreversible. To date, four members of this family have been identified and designated PAR-1 to -4. They exhibit differential tissue expression as well as selectivity in activation. The enzymes activating PARs belong to the serine protease family; thrombin activates PAR-1, -3, and -4, trypsin PAR-1, -2 and -4, tryptase and membrane-type serine protease-1 PAR-2, and cathepsin G as well as plasmin triggers PAR-4 activation [68,70]. PARs, particularly PAR-2, have been reported to be involved in multiple cellular responses related to tissue injury and repair, angiogenesis, nociception and neurogenic inflammation, and recently inflammatory conditions including those in rheumatoid arthritis and, more recently, in OA. In that regard, an important role for PAR-2 in chronic arthritis has been shown by using a PAR-2 gene knockout mouse in which inflammation was significantly delayed with the adjuvant-induced arthritis model [71,72]. PAR-2 expression has recently been found in human chondrocytes and synovial fibroblasts [73–75], and was modulated by the proinflammatory cytokines IL-1ß and TNF-α as well as the growth factors bFGF and TGF-ß. TGF-ß, however, differentially regulates normal and OA human chondrocytes [74]. The inflammatory function of this receptor has been found to be associated, depending on the cell systems, to NO, COX-2, PGE2, and MMPs. It is also suggested that activation of PAR-1 and -2 induce the production of the proinflammatory cytokines IL-1ß, IL-6, IL-8, IL-18 and TNF-α. Considering the pro-algesic and -inflammatory effects of PAR-2, this receptor might constitute a novel alternative therapeutic target for OA.

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Conclusion OA is mediated by a multitude of complex autocrine and paracrine anabolic and catabolic factors that act upon diverse cells from articular tissues. Various pathways result in alterations in transcription factors that transduce signals intracellularly. The end result of these pathways is the production of proinflammatory cytokines in which multiple lines of research have established the central role of IL-1ß, as well as other inflammatory mediators such as other cytokines and the factors NO, prostaglandins, leukotrienes and PARs. Therapies targeted at any of these steps, both upstream and downstream may prove to be beneficial.

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[61] Paredes Y, Massicotte F, Pelletier JP, Martel-Pelletier J, Laufer S, Lajeunesse D: Study of the role of leukotriene B4 in abnormal function of human subchondral osteoarthritis osteoblasts: effects of cyclooxygenase and/or 5-lipoxygenase inhibition. Arthritis Rheum 2002;46: 1804-1812. [62] Celotti F, Durand T: The metabolic effects of inhibitors of 5-lipoxygenase and of cyclooxygenase 1 and 2 are an advancement in the efficacy and safety of anti-inflammatory therapy. Prostaglandins Other Lipid Mediat 2003;71: 147-162. [63] Marcouiller P, Pelletier JP, Guévremont M, Martel-Pelletier J, Ranger P, Laufer S, et al: Leukotriene and prostaglandin synthesis pathways in osteoarthritic synovial membranes: regulating factors for IL-1beta synthesis. J Rheumatol 2005;32: 704-712. [64] Pelletier JP, Raynauld JP, Bias P, Laufer S, Haraoui B, Choquette D, Abram F, Vignon E, MartelPelletier J: Licofelone, a 5-lipoxygenase and cyclooxygenase inhibitor, reduces the progression of knee osteoarthritis (OA): A double blind, multicentre two-year study using quantitative MRI. Late-breaking abstract – Podium presentation. Annual American College of Rheumatology (ACR) Scientific meeting. 2006 (online). [65] Dery O, Corvera CU, Steinhoff M, Bunnett NW: Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol 1998;274: C1429-1452. [66] Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R: Proteinase-activated receptors. Pharmacol Rev 2001;53: 245-282. [67] Hollenberg MD: Proteinase-mediated signaling: proteinase-activated receptors (PARs) and much more. Life Sci 2003;74: 237-246. [68] Nystedt S, Emilsson K, Wahlestedt C, Sundelin J: Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci U S A 1994;91: 9208-9212. [69] Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, et al: Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 1997;272: 4043-4049. [70] Ossovskaya VS, Bunnett NW: Protease-activated receptors: contribution to physiology and disease. Physiol Rev 2004;84: 579-621. [71] Lindner JR, Kahn ML, Coughlin SR, Sambrano GR, Schauble E, Bernstein D, et al: Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 2000;165: 6504-6510. [72] Ferrell WR, Lockhart JC, Kelso EB, Dunning L, Plevin R, Meek SE, et al: Essential role for proteinaseactivated receptor-2 in arthritis. J Clin Invest 2003;111: 35-41. [73] Abe K, Aslam A, Walls AF, Sato T, Inoue H: Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts. Life Sci 2006;79: 898-904. [74] Boileau C, Martel-Pelletier J, Amiable N, Fahmi H, Mineau F, Geng C, et al: Protein-activated receptor (PAR)-2 in human osteoarthritic tissues: Regulation of the receptor for the synthesis of catabolic factors. Ann Rheum Dis 2006;65: THU0004 (Abstract). [75] Xiang Y, Masuko-Hongo K, Sekine T, Nakamura H, Yudoh K, Nishioka K, et al: Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1beta, TNF-alpha and TGF-beta. Osteoarthritis Cartilage (online).

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II Mechanical Loading Effects on Articular Cartilage Matrix Metabolism and Osteoarthritis Robert Lane SMITH, PhD Rehabilitation Research and Development Center VA Palo Alto Health Care System, Palo Alto, CA Department of Orthopaedic Surgery Stanford University School of Medicine Stanford, CA Abstract. This chapter examines the effects of mechanical loading on cartilage metabolism in explant culture and as isolated chondrocytes in high density monolayer and agarose/scaffold cultures. The mechanical loading effects are defined in terms of the two stress states that arise within cartilage, shear stress and hydrostatic pressure. The paper examines possible signaling pathways that could contribute to transduction of changes in the physical environment of articular chondrocyte to modulation of chondrocyte gene and protein expression.

Introduction Osteoarthritis (OA) is a disabling disease that is estimated to impact 1 in 3 individuals over the age of sixty [1]. The precise etiology of the disease remains unclear in part because the onset of OA and progression of the disease rest with a multiplicity of physical and biological factors [2]. This chapter will explore the overlap between mechanical load-mediated events and chondrocyte-based metabolic responses that may contribute to the progressive joint destruction characteristic of OA. Early manifestations of OA are generally silent and have only recently become amenable to discovery through improvements in imaging methods, arthroscopic visualization and analysis of gait abnormalities [3–5]. New imaging techniques can now reveal localized thinning of the cartilage extracellular matrix. Arthroscopic examination can pinpoint localized defects and detect surface irregularities [6]. Analysis of gait can establish how weight transfer with activity may generate harmful joint loading patterns [7]. As OA continues, patient disability ultimately results from destruction of the articular cartilage causing severely painful joint motion as bone meets bone [8,9]. There are two classifications of OA. In primary disease, although the exact cause remains unclear, onset of cartilage destruction appears to be associated with some element of abnormal joint biomechanics. In secondary disease, injury, infection, hereditary factors, developmental processes and metabolic or neurological disorders influence

R.L. Smith / Mechanical Loading Effects on Articular Cartilage Matrix Metabolism and OA

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joint tissue metabolism and can initiate cartilage breakdown [10]. While the onset of OA and joint loading appear linked, the search for molecular signaling pathways regulating articular chondrocyte cartilage matrix metabolism continues to be a subject of intense investigation [11]. Understanding the basis of mechanical signaling in cartilage is central for discovery of therapeutic agents to prevent progression of OA and development of methods for cartilage repair and/or regeneration.

1. Mechanical Loading and Cartilage Homeostasis In normal joints, the combined effects of mechanical loading and multiple interacting physiological mediators sustain chondrocyte viability and promote cartilage matrix homeostasis [12,13]. In normal adult cartilage, chondrocyte proliferation and anabolic metabolism remain relatively quiescent so that extracellular matrix longevity and functionality is preserved [14]. Stabilization of the balance between matrix synthesis and degradation by the chondrocytes is critical for prevention of joint degeneration [15]. Defining the cartilage catabolic response to injurious stimuli confirmed that matrix metabolism must remain under stringent control by genetic and epigenetic mechanisms for joints to remain healthy [16]. From a mechanical perspective, activities of daily living generate compressive loads across the diarthroidal joints due to surface sliding, rolling, spinning and fluid exudation [17,18]. The motion at the joint surfaces creates two types of fundamental stresses, shear stress and hydrostatic stress (pressure) within the cartilage extracellular matrix [19]. The specific type of motion and degree of loading will generate varying levels of shear and hydrostatic stress within the cartilage. Our in vitro studies demonstrate that both types of stress differentially influence chondrocyte metabolism [20–28]. Articular chondrocytes exposed to shear stress increase expression of the proinflammatory mediators, nitric oxide, prostaglandin E2 (PGE2) and interleukin-6, and decrease expression of the cartilage matrix macromolecules [20–24]. In contrast, applying hydrostatic pressure increases cartilage matrix gene expression and enhance matrix production [25–28]. In other experimental models where shear stress predominates, an increase in proinflammatory cytokines and other factors is associated with inhibition of hormonal and growth factor effects on cartilage metabolism [29–31]. In spite of clinical and experimental data implicating mechanical effects in OA, a precise mechanism is lacking regarding ways in which day to day diarthroidal joint loading contributes to cartilage degeneration. This is in spite of human clinical observations implicating ligamentous injury, limb alignment, obesity, trauma and genetics as risk factors for OA [32–36]. Each of these clinical conditions alters the profile of joint loads and each is recognized as a potential cause of progressive loss of cartilage. While normal joint loads are essential for articular cartilage matrix homeostasis, the strong association of OA with dense connective tissue injuries that disrupt joint mechanics points to a prominent role for inappropriate stress states to induce cartilage degeneration.

2. Articular Cartilage: Biochemistry and Biomechanics In diarthroidal joints, articular cartilage distributes loads that reach up to 7 times body weight as a result of specialized matrix macromolecules that provide compressive resil-

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ience and tensile strength. In an intact cartilage matrix, joint motion remains essentially pain free. This aspect of tissue functionality is due primarily to three matrix constituents, water, aggrecans, and type II collagen [37,38]. Aggrecans provide resistance to compression due to the hydrophilic and electronegative properties of covalently attached glycosaminoglycans [39]. Type II collagen provides tensile strength through an organized network of cross-linked fibrils. Hyaluronan together with a limited complement of glycoproteins and low molecular weight proteoglycans ensure matrix organization [40]. The cartilage matrix constituents originate from chondrocytes distributed in zones extending from the flattened cells of the superficial layer to the rounded cells within deeper layers adjacent to the subchondral bone interface [41]. Mathematical models of joint loading resolve two principal stresses, shear and hydrostatic, that arise within cartilage when compressive forces are applied [19,42,43]. Major shifts in the relative proportion and/or distribution of these stresses in cartilage might be a major factor regulation of chondrocyte metabolism. The mechanical changes associated with OA risk factors, such as laxity or excessive loads, may selectively alter the level and distribution of shear stress in the cartilage while not influencing pressure. At present, the precise levels of shear stress that can incite cartilage degradation in vivo are unknown. There is, however, evidence linking the incidence of localized inflammation in OA joints to changes in load distribution [44]. It is likely that continuous exposure to inappropriate stresses contributes to a catabolic phenotype in chondrocytes. Cartilage catabolism is accompanied by release of proinflammatory cytokines and degradative enzymes into the joint space and into the serum [45]. The range of chondrocyte-derived inflammatory factors will be examined below with respect to vitro studies of cartilage degradation induced by mechanical loading.

3. Cartilage Explants: Mechanical Loading and Metabolism Effects of mechanical loading on articular cartilage metabolism have been investigated in vitro with cartilage explants and with isolated chondrocytes. Explant cultures provide a quasi-tissue systems approach to examine how mechanical loads alter cartilage metabolism since the morphology and zonal distribution of the cells remain organized in a three-dimensional matrix. Under defined culture conditions, chondrocyte viability persists in all zones of full-thickness explants and aggrecan and collagen content remain stable throughout a three-week culture period [46]. In addition, dynamic and equilibrium mechanical properties also remain stable. With significant variation in the loading conditions, cartilage explant cultures can exhibit distinct metabolic behavior depending on the type, duration and magnitude of the physical stimulus. For instance, injurious compression alters the physical properties of cartilage due to onset of chondrocyte-mediated turnover and release of proteoglycans from the matrix [47,48]. Similarly, disruption of the normal joint architecture results in degradation of the flowindependent viscoelastic and equilibrium properties of articular cartilage [49,50]. The response of cartilage explants to mechanical loading induces production of multiple chemical and biological molecules that coincide with early and late metabolic changes in chondrocytes. When dynamic compression is applied to cartilage explants, an initial up-regulation of aggrecan and type II collagen expression is subsequently followed by decreased synthesis patterns for both matrix macromolecules [51,52]. The chondrocyte response remains subject in part to the distribution of stress states within the matrix. In full-thickness explants retaining a thin layer of bone, cyclic loading in


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unconfined compression produces a weakening of the collagen network and an increase in hydraulic permeability [53]. In explant cultures not exposed to loading, less denaturation of type II collagen occurs, a finding consistent with low turnover and consistent with less tissue remodeling. The exposure to cyclic compression also increased release of fragments of the major matrix proteins into the culture medium when compared to unloaded cultures. The observations imply that, on some level, cyclic loads destabilize matrix homeostasis depending on loading conditions. In a study where static compression is applied to the explant cultures, aggrecan and type II collagen synthesis rates were generally decreased from the outset of loading [54]. The mRNA signal levels for aggrecan and type II collagen exhibited a transient increase at 0.5 hours but then decreased between 4 and 24 hours after compression was applied. The protein levels decreased ahead of the mRNA level suggesting that matrix biosynthesis involves pathways other than mRNA expression. In studies where the compressive loading of cartilage explants consisted of injurious levels of peak stresses (>20 MPa), chondrocyte apoptosis was maximally induced by 24 hours after ending the loading protocol [55]. At high peak stresses, the equilibrium and dynamic stiffness of the explants decreased with the severity of the load in uniaxial confined compression. When cartilage mechanical properties were assessed in radially unconfined compression, the equilibrium and dynamic stiffness values were decreased a peak stresses in the range of 7 to 12 MPa [55]. The change in mechanical properties was accompanied by degradation of the collagen fibrillar network, loss of glycosaminoglycans and increased tissue swelling. One hypothesis for the effects of compressive loading on the cartilage explants is that increased shear stresses act directly on the cells as they undergo a distortional change in shape. Examination of chondrocytes within a compressed cartilage matrix show that both the cell body and the nucleus undergo changes in shape that are dependent on the stress levels being applied [56,57]. The assumption has to be advanced that chondrocytes in explants become subject to distortion because of the cut edges of the tissue samples where some fluid loss can occur, particularly in unconfined compressive loading. The extent to which the same degree of deformation might occur in the intact joint surface cartilage remains unclear.

4. Isolated Chondrocytes: Mechanical Loading and Metabolism Under in vitro loading conditions, the metabolic responses observed for cartilage explants under compression appear to represent patterns where injury leads to decreased matrix biosynthesis and loss of matrix macromolecules. The results obtained from studies of compressive loading of cartilage explants is mirrored to a significant degree by studies examining the response of isolated articular chondrocytes to physical stimulation. In these types of in vitro experiments, the chondrocytes are typically subjected to mechanical loading in the absence of an assembled extracellular matrix. A number of model systems have been used to assess strain dependent effects on chondrocyte metabolism at the level of gene and protein expression. One system used to study mechanical effects relies on preservation of the articular chondrocyte phenotype by placing the cells in primary high density monolayer culture [58]. The high density cultures are defined by having the cells present in concentrations of 1×10 6/cm2 without the cells ever having been passaged following isolated by collagenase digestion. In our use of this model, low levels of shear stress applied to articular chondro-

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cytes through the application of fluid motion by a cone viscometer activated release of proinflammatory mediators and inhibited matrix protein synthesis [20–24]. Exposure of human OA chondrocytes to a continuously applied shear stress (1.64 Pa) increased nitric oxide (NO) release by 1.8-, 2.4-, and 3.5-fold at 2, 6, and 24 h, respectively, into the culture medium. NO is a short-lived but highly soluble and diffusible signaling factor that is implicated in the regulation of cartilage matrix protein gene expression. Shear stress also significantly increased gene expression of the inducible form of nitric oxide synthase. Exposure of chondrocytes to shear stress for 2, 6, and 24 h inhibited type II collagen mRNA signal levels by 27%, 18% and 20% after a constant post-shear incubation period of 24 h. Aggrecan mRNA signal levels were inhibited by 30%, 32% and 41% under identical conditions. Addition of an NO antagonist increased type II collagen mRNA signal levels by an average of 1.8-fold (137% of the un-sheared control) and reestablished the aggrecan mRNA signal levels by an average of 1.4-fold after shear stress (92% of the un-sheared control) (ANOVA p < 0.05). Applying shear stress to human OA chondrocytes also results in an increase in apoptosis as evidenced by presentation of membrane phosphatidylserine and increased nucleosomal degradation. As might be expected, expression of the anti-apoptotic factor, bcl-2, was decreased by shear stress and addition of the nitric oxide antagonists, L-N(5)-(1-iminoethyl) ornithine and N-omega-nitro-L-arginine methyl ester (L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%, respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coincided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to shear stress in the absence of L-NAME. These results support the fact that shifts in mechanical stresses in cartilage, even at low levels, may induce factors such as nitric oxide that can then lead to joint degeneration through effects on chondrocyte viability. A number of other model systems in which chondrocytes placed in agarose have been used to determine the effects of varying levels of compression on matrix metabolism [59–66]. Chondrocytes isolated from fetal, young and aged bovine cartilage and cultured in agarose gels showed that fetal and young chondrocytes were similar with respect to cell proliferation and proteoglycan accumulation whereas aged chondrocytes exhibited diminished capacity with respect to both activities [59]. Collagen accumulation was also reduced in the aged chondrocytes by approximately 55% when compared to younger cells. The differences in metabolism of the chondrocytes resulted in the production of an extracellular matrix that exhibited a significant reduction in stiffness when compared to the matrix produced by younger chondrocytes [59]. Other studies of chondrocytes placed in agarose demonstrate that the cells maintain fidelity with respect to the extracellular matrix synthesis representative of the zone of articular cartilage from which the cells originated. Superficial zone cells from the upper 15 to 20% region of the cartilage responded differently when compared to the deeper tissue chondrocytes [60]. In agarose culture, superficial cell proliferation was stimulated by dynamic strain whereas the deeper cells remained unchanged. The deeper cells did exhibit an increase of glycosaminoglycan synthesis under dynamic strain at a frequency of 1 Hz with amplitude of 15%. These data imply cell specific responsiveness to compressive strain. The reactivity of articular chondrocytes to dynamic compression is also apparent through studies on matrix biosynthesis in an agarose disk model [61]. Although dynamic compression stimulates extracellular synthesis, the deposition of the matrix macromolecules and the physical properties of the matrix varied depending on whether the cells were in the center or in the periphery of the disk. These data imply that cell-matrix interactions may have equal importance to matrix-Independent cell deformation and


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transport limitations for maintenance of an organized load-bearing matrix. Studies on cell seeding density together with comparison studies of different types of support scaffolds suggest that direct mechanical effects occur through complex and overlapping signaling mechanisms [62,63]. Dynamic compression also has functional effects on chondrocyte metabolism and has been shown to counteract effects of interleukin-1beta induced release of nitric oxide and prostaglandin E2 [64–66]. The question remains as to the mechanism by which dynamic loading modulates cell metabolism. Recent studies show that ion channel inhibitors can differentially influence the chondrocyte responses to mechanical stimuli in such a way that either glycosaminoglycan synthesis or protein synthesis may show altered rates depending on the type of channel being blocked [67]. The fact that the differences occur while the type and level of mechanical loading is held constant reinforces the hypothesis that redundant multi-level regulatory mechanisms must be active in regulation of cartilage homeostasis. Efforts to quantify the relationship of intracellular cytoskeletal components with cell-surface integrin-mediated extracellular matrix attachments are just beginning. However, initial studies provide evidence that the cell-matrix transition points are dynamic and under continual time-dependent reorganization in response to the application of mechanical loading [68]. The cytoskeletal data are compatible with early cell culture studies and finite element modeling that attempted to discern the behavior of the articular chondrocyte under uniaxial compressive loading. One early study showed that the cells decreased in cross-sectional area depending on the level of compressive strain [69]. The authors suggested that the chondrocyte may alter its intracellular composition by cellular processes to compensate for the compressive load. Given our increasing understanding of intracellular signaling pathways, these observations suggest that changes in cell morphology may provide both signaling mechanisms as well as protection under excessively high compressive loads. Compression induced changes in chondrocyte morphology vary with local tissue strains [70] and have been shown to coincide with differential expression of collagen types [71] and release of latent degradative enzymes, such as procollagenase [72,73]. Thus, cellular deformation may be a primary determinant in the regulation of cartilage metabolism by mechanical loading. The question remains to what extent that sensitivity to shear stress in normal and OA chondrocytes underlies proinflammatory mediator expression that subsequently leads to matrix degeneration through the induction of catabolic processes in cartilage and synovial tissue. The second major stress type that occurs within diarthroidal joints is hydrostatic pressure. Within the matrix the levels of hydrostatic pressure are proportional to the contact stresses that are generated with joint surface loads. A number of studies show that contact stresses in the major joints can reach levels up to 20 to 25 MPa depending on the activity [74,75]. These contact stress are accompanied by the generation of hydrostatic pressure in cartilage in the range of 2 to 15 MPa [74,75]. In vitro, levels of hydrostatic pressure at 5 to 15 MPa increase matrix synthesis as quantified by incorporation of radiolabeled sulfate and proline in adult bovine articular cartilage explants [76]. Organ culture experiments demonstrate that sites of proteoglycan production coincide with regions of pure hydrostatic pressure [77]. Our in vitro studies show that physiological levels of hydrostatic pressure results in anabolic patterns of chondrocyte gene expression with a counteracting effect on catabolic factors [25–28]. Normal bovine articular chondrocytes exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and

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24 hrs showed that type II collagen mRNA signal levels exhibited a biphasic pattern in response to continuously applied loading. There was an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. In separate experiments, chondrocytes were exposed to intermittent hydrostatic pressure for a period of 4 hrs per day for 4 days. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data confirmed that mechanical loading in vitro with time-specific protocols produced changes in chondrocyte metabolism consistent with predictions from mathematical models. In separate studies, aggrecan mRNA signal levels increased 1.3- and 1.5-fold at 5 and 10 MPa, respectively, relative to beta-actin mRNA, when exposed to intermittent hydrostatic pressure for 4 hrs/day for 1 day. Extending the loading period to 4 hrs/day for 4 days (4×4) increased aggrecan mRNA signal levels by 1.4-, 1.8- and 1.9-fold at loads of 1, 5 and 10 MPa, respectively. Type II collagen mRNA signal levels were increased at loads of 5 and 10 MPa with the 4×4 loading regimen. Western blotting confirmed that IHP increased aggrecan and type II collagen in the chondrocyte extracts. Other effects of intermittent hydrostatic pressure (IHP) include reversal of inhibitory effects of bacterial antigen (LPS) on chondrocyte metabolism. Intermittent hydrostatic pressure applied to LPS-activated chondrocytes decreased both nitric oxide synthase mRNA signal levels and nitric oxide released into the culture medium. Exposure of LPS-activated chondrocytes to IHP also increased type II collagen and aggrecan mRNA signal levels by 1.7-fold, when compared to chondrocytes activated by LPS and maintained without loading. Applying IHP decreased the signal levels for monocyte chemotactic factor-1 and matrix metalloproteinase-2 following LPS activation by 45% and 15%, respectively. These data confirmed that IHP counteracts effects of inflammatory agents, such as bacterial LPS, on chondrocytes. Application of intermittent hydrostatic pressure (IHP) reduced the levels of NO induced by shear but did not alter release NO from chondrocytes not exposed to shear stress. NO induced by shear stress or by addition of an NO donor (sodium nitroprusside) decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and type II collagen. IHP blocked the inhibitory effects of the NO donor on matrix gene expression but failed to alter the inhibitory effects of shear stress on matrix mRNA levels. This data suggest that shear stress inhibits matrix synthesis through multiple mediators and/or overlapping pathways with hydrostatic pressure possibly acting through different pathways. Interestingly, in alginate cultures of bovine chondrocytes hydrostatic pressure decreased expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 [78]. Cyclic tension in this system downregulated TIMP-1 while increasing expression of expression of hypertrophic chondrocyte markers, consistent with differential responsiveness of chondrocytes to stress states. The precise mechanism by which hydrostatic pressure modulates chondrocyte metabolism remains unclear. A recent study implicates changes in calcium ion distribution, both intracellular and extracellular, as a signaling mechanism for hydrostatic loading [79]. A five minute exposure of chondrocytes on cultured on cover slips to constant hydrostatic pressure at a level of 0.5 MPa increased free calcium ion levels by two-fold for cells isolated from middle zone of cartilage whereas cells of the superficial and deep zone exhibited a 1.5-fold increase in free calcium ion. Effects of inhibitors of calcium ion flux showed that a calcium channel blocker (verapamil) did not block the effect of hydrostatic pressure on the chondrocytes whereas a stretch-activated channel


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blocker and an intracellular storage blocker partially reduced the effects of hydrostatic pressure on calcium ion distribution. These data were interpreted as suggesting the involvement of inositol 1,4,5-triphosphate (IP3)-mediated calcium increase, similar to the processes active in bone cells.

5. Cartilage Metabolism and Matrix Degradation During joint development and through adult life, cartilage metabolism is continually influenced by biological and mechanical factors [80,81]. In early cartilage development, the growth hormone/IGF-1 dependent pathway is a primary stimulus for chondrocyte proliferation and matrix synthesis [82]. With skeletal maturation, the IGF-1 effect on chondrocyte metabolism through specific receptors is modulated in part through fluctuating levels of IGF-1 binding proteins that are present in the serum [83,84]. In OA, articular chondrocytes exhibit a refractory response to IGF-I, that appears independent of functional plasma membrane receptors [85]. OA joints also exhibit increased levels of proinflammatory mediators that alter chondrocyte metabolism [86]. These inflammatory mediators include nitric oxide, interleukin-1, interleukin-6 and tumor necrosis factor-alpha. Interleukin-1alpha and interleukin-1beta (IL-1) inhibit matrix synthesis [87,88] and together with tumor necrosis factor alpha [89,90] induce matrix degrading enzyme expression by chondrocytes [91–93]. Other inflammatory mediators including nitric oxide and IL-6 are associated with induction of degradative enzymes [94] and inhibition of aggrecan synthesis [95], respectively. Experimental animal models that produce symptoms consistent with OA in include immobilization [96], increased instability (excision of anterior cruciate ligament, 97), excessive impact or blunt traumatic injury [98,99]. Other causes OA have been advanced such as a stiffening of subchondral surfaces so that load distribution to bone becomes limited [100]. The chondrocyte responses to varying levels of the two types of cartilage stress states may provide the metabolic link to all of these risk factors. For example, increased shear stress may act as an inducer of the matrix metalloproteinases (MMPs) associated with arthritis [101]. Cartilage damaged by trauma exhibit increased release of collagenase (MMP-1) and stromelysin (MMP-3), either of which may degrade proteoglycans [102–105]. Collagenases may then initiate degradation of type II collagen after activation [106,107]. Stromelysin is activated by plasmin and subsequently activates other MMPs [108]. Collagenase and stromelysin together may also destabilize the matrix by degrading minor components, such as link protein and other glycoproteins [109,110]. Three unique collagenases, the fibroblast collagenase (MMP-1), the neutrophil collagenase (MMP-8) and the collagenase 3 (MMP-13) are proposal as integral elements in matrix degradation [111]. In naturally occurring OA in dogs, MMP-9 was the matrix metalloproteinase that correlated with onset and progression of disease [112]. Under conditions of a shear stress of 16 dyn/cm2 (1.6 Pa), MMP-9 expression was increased in rabbit chondrocytes. In this model, transfection of the chondrocytes with a dominant negative mutant of cJun NH2-terminal kinase [113] attenuated the MMP-9 expression. Our studies of OA cartilage demonstrated that expression of the MMP-9 was highest in areas of the cartilage that exhibited the greatest amount of fibrillation [114,115]. Of interest, even the most normal appearing cartilage in the OA joint showed significant elevation of MMP-9 expression. This data fits with the proposed role of active neutral matrix metalloproteinases in the pathogenesis of arthritis [116–118]. Degradation products from

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metalloproteinases are elevated during periods of active joint destruction [119–121]. The enzyme activities include aggrecanases that degrade the cartilage proteoglycans [122,123]. Core protein peptides corresponding to an aggrecanase cleavage site, between Glu373 and Ala374 are elevated in human joint fluid [124,125]. Multiple enzymes must be active since neoepitopes of aggrecan globular domain G1 generated by MMP and aggrecanase activity are present in OA cartilage [126].

6. Chondrocyte Mechanotransduction Transmittal of the mechanical forces to chondrocytes localized within an extracellular matrix is assumed to involve interactions between proteins of cell plasma membrane and matrix components [127–129]. Modes for transfer of the mechanical stimulus for regulation of transcriptional and translational processes depend on signaling pathways linked to ion channels, membrane receptors, and cytoskeletal proteins [130]. Selective integrin subunits present in the plasma membrane are involved in when chondrocytes respond to fluid shear, compressive loading and stretch [131,132]. The interactions between the chondrocytes and matrix then activate intracellular second messenger pathways that orchestrate multiple metabolic reactions to changes in mechanical environment [133]. In setting a hierarchical pattern by which extracellular stimuli are converted into cellular responses, the mitogen-activated protein kinases (MAPKs) emerge as a primary effectors for positive and negative changes in cellular metabolism [134–136]. Downstream effects of the MAPKs follow receptor-mediated phosphorylation of the MAPK-kinase kinase (MEK kinases) [137]. Activation of MEK kinases leads to phosphorylation of the MEKS or MKKs that then phosphorylate the MAPKs. The MAPKs are divided into three families, the extracellular regulated kinases (ERK-1, -2), the stress-activated protein kinases (SAPK) or c-jun NH2-terminal kinases (JNK) and the p38 MAPKs [138]. The p38 MAPKs originate from four genes and exhibit specificity for upstream activators and downstream substrates. Mechanical loads alter chondrocyte metabolism through activation of specific intracellular kinase pathways depending on the type of load applied [139–142]. Compressive loading induces a phased phosphorylation of the extracellular regulated kinases 1 and 2 (ERK 1/2) where ERK2 exhibits a persistent phosphorylated state for up to 24 hours [143]. In embryonic limb bud, compressive loading increases collagen and aggrecan expression while decreasing IL-1beta expression [124]. Other kinases, p38 mitogen-activated kinase (p38) and a member of c-Jun N-terminal kinase (JNK) pathway (SEK1), exhibited more transient responses (10 minutes to 1 hour) [144]. In endothelial cells, kinase activation induces transcription genes encoding monocyte chemotactic protein-1 (MCP-1) and c-Fos [145]. Mechanical signaling events that activate or suppress gene expression involve translocation of signaling molecules into the nucleus for activation of the transcription factor proteins that bind to the promoter sequences of gene as enhancers or suppressors of mRNA synthesis [146]. The p38 MAPKs play crucial roles in the regulation of gene expression either by activating transcription factors or by stabilizing RNA and protein turnover [147]. Transcription factors act through interactions with binding motifs located in the upstream 5’-flanking regulatory DNA sequences [148] and in combination with co-activators and initiation factors that bind to RNA polymerase II [149]. In growing cartilage, type II collagen expression depends on the transcription factor, SOX-9,


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but in adult tissue type II collagen expression may be less reliant on this factor [150]. Hydrostatic pressure increases transcription factor activation [151] and shear stress is implicated in the translocation of the STAT proteins [152]. Stimulation of chondrocytes by proinflammatory mediators, such as IL-1, involves activation of nuclear factor kappa B (NF-kappaB), NF-IL6 (CREB), AP-1 [153]. Similarly, MMP expression is controlled by the transcription factors, AP-1, NF-kappaB, Sp1, and STAT [154]. The extent to which specific levels of shear stress also act through nuclear protein activation remains of interest and will be focus of one aspect of this proposal.

7. Summary The available data from in vitro studies of cartilage explants and isolated chondrocytes together with clinical experience leads one to a conclusion that at some critical level shear stress is deleterious to decades-long preservation of articular cartilage. A range of studies show that the cause is linked to activation of proinflammatory mediator release from the articular chondrocytes. However, a full understanding of how the threshold for this mechanical stimulus to incite articular cartilage degeneration is set remains lacking. A challenge for the future is establish at a molecular level how the balance between the two different types of mechanical stresses that arise in the cartilage can preserve matrix stability over 8 to 9 decades in some individuals. Such knowledge will contribute to three important long term goals. First, understanding the role of mechanical loading on cartilage matrix production will contribute to fundamental information to improve the success of cartilage repair by tissue engineering. Second, understanding the precise role of mechanical loading on cartilage repair will advance surgical procedures that could forestall the need for total joint arthroplasty. Third, the information may contribute to the development of new classes of drugs to prevent progression of early OA.

Acknowledgements This work was supported by a Department of Veterans Affairs, RR&D Merit Review Proposal A2128-RC and a Research Career Scientist Award (RLS).

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III Aging, Inflammation, and Altered Chondrocyte Differentation in Articular Cartilage Calcification and Osteoarthritis Robert A. TERKELTAUB, MD Section Chief of Rheumatology Allergy-Immunology, VA Medical Center Professor of Medicine, University of California San Diego Division of Rheumatology, 3350 La Jolla Village Drive, San Diego, CA 92161 Phone: 858-642-3519, Fax: 858-552-7425, E-mail: [email protected] Abstract. Extracellular matrix calcification with calcium pyrophosphate dihydrate (CPPD) and/or hydroxyapatite crystals commonly develops in osteoarthritic (OA) cartilage. Primary forms of articular cartilage CPPD crystal deposition and less commonly hydroxyapatite deposition can present as degenerative joint disease. Moreover, CPPD and hydroxyapatite crystals can traffic from cartilage to synovium and induce cytotoxic, catabolic, and inflammatory responses of chondrocytes and synovial lining cells, and promote synovial proliferation. Such changes have the potential to not only contribute to low-grade synovitis and inflammatory symptoms in OA but also to accelerate the progression of OA. In addition, CPPD and hydroxyapatite crystals are commonly found in joints with advanced OA. However, it is not clear that CPPD and hydroxyapatite crystal deposition actually worsen the course of primary OA. Instead, it appears that CPPD and hydroxyapatite crystal deposition in OA articular cartilage reflect aging, inflammation, altered IGF-I and TGFβ responsiveness, chondrocyte hypertrophic differentiation, changes in the closely linked metabolism of ATP, PPi, and Pi, and possibly local changes in PTHrP expression and systemic changes in PTH. As such, OA pathogenesis richly informs us on mechanisms that drive articular cartilage calcification. Conversely, the presence of cartilage calcification informs us about pathogenesis and progression factors in subsets of affected subjects with OA. Keywords. PTH, PTHrP, NPP1, ANKH, PPi, transglutaminase, chondrocyte hypertrophy, chondrocalcinosis, TGFβ, IGF-I, Cartilage Intermediate Layer Protein

Introduction Scope of the Problem of Joint Cartilage Calcification Extracellular matrix calcification commonly develops in osteoarthritic (OA) cartilage. CPPD and basic calcium phosphate (BCP) crystals (principally hydroxyapatite) are by far the commonest forms of calcium-containing crystals in articular cartilages [1–4]. CPPD and hydroxyapatite crystals can traffic from cartilage to synovium and directly turn on cells and activate complement to induce cytotoxic, catabolic, and inflammatory

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R.A. Terkeltaub / Articular Cartilage Calcification and Osteoarthritis

responses of chondrocytes and synovial lining cells [5–7]. In addition, both CPPD and HA crystal ingestion by synovial fibroblasts promotes proliferation, mediated partly by calcium release from crystal dissolution as well as by mitogen activated protein kinase (MAPK) signaling [5,6]. As such, CPPD and hydroxyapatite crystal deposition have the potential to not only contribute to low-grade synovitis and inflammatory symptoms such as pain and stiffness in OA but also to accelerate the progression of OA. There is a particularly high frequency of positive knee joint synovial fluids for CPPD and/or BCP crystals in aged patients with advanced degenerative arthritis at the time of joint arthroplasty [1]. The presence of BCP crystals in affected joints correlates directly with OA severity [1,2]. However, the apparent sources of joint fluid BCP crystals in advanced OA include not only perichondrocytic crystal deposits (especially in the superficial zone) but also bone shards embedded in cartilage and bony debris exposed by cartilage erosion in established OA. Primary degenerative arthropathy due to intra-articular BCP crystal deposition occurs [6]. However, secondary forms of BCP crystal deposition arthropathy appears to be much more common, particularly in association with periarticular disease such as mechanical instability of the shoulder joint due to chronic rotator cuff tear [8]. CPPD crystals are commonly found in meniscal fibrocartilage and less commonly in hyaline articular cartilage of the knee in association with OA and aging [9]. Decreased hydration and unknown distinctions in extracellular matrix composition may account for preferential localization of CPPD crystals to meniscal fibrocartilage [9]. Monoclinic CPPD crystals have a greater inflammatory potential than triclinic CPPD crystals [10], one of the likely reasons that clinical manifestations of CPPD deposition are so variable. Cartilage CPPD crystal deposition can be asymptomatic or can mimic OA, gout, rheumatoid arthritis, or neuropathic joint disease [11,12]. Cartilage degenerative changes in CPPD deposition disease can be observed in not only typical joints affected by primary OA such as the knee but also in atypical sites for primary OA joints such as the glenohumeral, wrist, and metacarpophalangeal joints [12]. Local disturbances in PPi metabolism occur in OA cartilage, whereas heterogeneous but systemic disturbance in PPi metabolism clinically manifested primarily in the joint may underlie the distinct distribution of arthropathy in idiopathic/sporadic, familial, and secondary metabolic disease-associated CPPD deposition [13,14].

CPPD Crystal Deposition and Prognosis in OA The presence of CPPD crystals in primary knee OA was initially suggested to be a predictive factor for more frequent knee replacement surgery [15]. In addition, higher mean radiographic scores correlated with the presence of calcium-containing crystals in OA in a recent study of patients at the time of total joint arthroplasty [1]. However, degenerative cartilage disease associated with sporadic CPPD crystal deposition disease may be less destructive than that observed in primary OA. For example, prospective analysis of CPPD deposition disease of the knee suggested that radiographic worsening of degenerative arthritis was slow [16]. Typically, changes in radiographic extent of chondrocalcinosis are observed over time [16]. There is no clear correlation between the extent of calcification and progression of CPPD deposition arthropathy. In the recent Boston OA Knee Study (BOKS) and in the Health, Aging and Body Composition (Health ABC) Study [17], Neogi et al. prospectively evaluated the relationship between chondrocalcinosis and the progression of knee OA longitudinally


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Figure 1. Mechanistic convergence of OA and cartilage matrix calcification. The figure schematizes fundamental disease processes within cartilage that ultimately promote both OA and matrix calcification, as reviewed in the text.

using MRI. In BOKS, knees with chondrocalcinosis had a decreased risk of cartilage loss compared with knees without chondrocalcinosis and there was no difference in risk in Health ABC. Stratification by the presence of intact or damaged knee menisci produced comparable results within each cohort. In the setting of OA, the processes leading to matrix calcification had been regarded to reflect passive secondary consequences of advanced cartilage pathology. Moreover, joint inflammation induced by deposited calcific crystals was thought to be determine the primary significance of chondrocalcinosis for the progression of OA. The findings of Neogi et al. [17], and recent advances in understanding the pathogeneses of OA and chondrocalcinosis suggest quite different ways of looking at the significance of chondrocalcinosis in OA. For example, the dysregulated cartilage matrix repair that generates cartilage calcification may be more effective at slowing cartilage tissue failure than other forms of cartilage repair in OA. Below, I discuss the argument that cartilage matrix calcification and OA reflect the convergence of heretogeneous processes that actively drive stereotypical patterns of cartilage injury and repair (Fig. 1). Processes given particular attention in this review are (i) certain low grade inflammatory alterations in cartilage that promote chondrocal-

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cinosis by modulating chondrocyte maturation to hypertrophy, (ii) chondrocyte responses to PTH and PTHrP, (iii) dysregulated ATP and PPi metabolism in cartilage aging, and (iv) imbalance in chondrocyte responses to the growth factors TGFβ and IGF-I.

Altered Chondrocyte Differentiation and Matrix Calcification in OA: Chondrocyte Hypertrophy Unlike growth plate cartilage, articular cartilage is specialized to resist matrix calcification. However, the physiologic regulated changes in chondrocyte differentiation and viability characteristic of growth plate chondrocytes, including proliferation, hypertrophy, and apoptosis, as well as changes in mitochondrial function and Pi transport, can be partially recapitulated in articular cartilage chondrocytes as a feature of the pathology of OA [18–25]. The development of grossly enlarged chodrocytes that express hypertrophic differentiation markers such as type X collagen, vascular endothelial growth factor (VEGF), and the transglutaminases (TGs) TG2 and FXIIIA is wellrecognized in OA cartilage [18–21,26–28]. The orderly and sequential transitions of growth plate chondrocytes from resting to proliferating, proliferating to prehypertrophic, and prehypertrophic to terminal hypertrophic differentiation are tightly regulated by multiple mediators with opposing and, in some cases, direct mutually antagonistic effects [29–32]. Figure 2 though far from a complete listing, illustrates major aligned forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth plate. Some of these factors are already implicated in OA pathogenesis, and inflammatory mediators such as S100A11, CXCL8, TNFα, and the role of RAGE and TG2, in chondrocyte hypertrophy in OA cartilage receive intensive discussion below. Chondrocyte hypertrophy is a differentiation state specialized for calcification, in part by alteration of extracellular matrix composition, the enhanced release of matrix vesicles to promote mineral seeding, increased generation of PPi and Pi, and increased TG2 and FXIIIA expression (Fig. 3) [14,21,26,27,33]. VEGF release by hypertrophic chondrocytes has the potential to promote synovial angiogenesis [19] and thereby contribute to the synovitis that is observed to a variable degree in the OA joint. The increased susceptibility of hypertrophic chondrocytes to apoptotic death also is partly significant because of the pro-mineralizing effects of chondrocyte apoptosis [23,35,36].

Inflammatory Mediators in Extracellular Matrix Modification for Calcification Alteration of the articular cartilage extracellular matrix by hypertrophic chondrocytes that is a fundamental factor in preparing the matrix for calcification also has ramifications for the biomechanical and signaling properties of the matrix in OA and aging. For example, stabilization of pericellular matrix proteins via transamidation-catalyzed cross-linking by TG2 and FXIIIA may not only promote calcification [37] but also stabilize the matrix. Collagen II is down-regulated, but collagen X, MMP-13 and ADAMts5 are up-regulated in hypertrophic chondrocytes. Taken together, it is conceivable that the dysregulated matrix repair response of hypertrophic chondrocytes in OA may at least be superior as a tissue repair mechanism to that exerted by non-viable chondrocytes.


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Figure 2. Aligned forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth plate, sone of which are also active in OA cartilage. The Figure, though far from a complete listing, depicts major aligned forces promoting and suppressing chondrocyte maturation to hypertrophy in the growth plate. Some of these factors are already implicated in OA pathogenesis, as discussed in the text. Inflammatory mediators such as S100A11, CXCL1, CXCL8, TNFα, and the role of RAGE and TG2, in chondrocyte hypertrophy in OA cartilage receive intensive discussion in the text.

Two distinct TGs, TG2 and Factor XIIIA are expressed in temporal and spatial association with chondrocyte hypertrophy and matrix calcification in the growth plate and are up-regulated in hypertrophic cells in the superficial and deep zones of knee OA articular cartilage and the central (chondrocytic) zone of OA menisci [26,28,38]. IL-1β, TNFα, the chemokines CXCL1 and CXCL8, nitric oxide (NO) and the potent oxidant peroxynitrite induce increased chondrocyte TG catalytic activity [26,28,38]. OA severity-related, donor age-dependent, and particularly marked age-dependent IL-1-induced increases in TG activity occur in chondrocytes from human knee menisci [28]. Increased Factor XIIIA and TG2 activities directly induce calcification by chondrocytes [28]. TG2 also promotes activation from latency of TGFβ [39]. We recently discovered that inflammation-induced TG2 release from chondrocytes appears to be a central amplification factor for both OA and cartilage matrix calcifica-

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Figure 3. Functional implications of chondrocyte hypertrophy in calcification and the course of OA. The Figure depicts that chondrocyte hypertrophy is a differentiation state specialized for calcification, in part by alteration of extracellular matrix composition (with attendant dysregulation of matrix repair), as well as by the enhanced release of matrix vesicles to promote mineral seeding, increased generation of PPi and Pi, and increased TG2 and FXIIIA expression. Furthermore, VEGF release by hypertrophic chondrocytes has the potential to promote angiogenesis in the synovium, and may thereby promote synovitis that is observed to a variable degree in the OA joint. The increased susceptibility of hypertrophic chondrocytes to apoptotic death also is partly significant because of the pro-mineralizing effects of chondrocyte apoptosis.

tion [26–28,38]. Specifically, we treated TG2 knockout mouse chondrocytes with IL-1β, CXCL1, the all-trans form of retinoic acid (ATRA) (which promotes endochondral chondrocyte hypertrophy and pathologic calcification), and with C-type natriuretic peptide (CNP) [26]. IL-1β and ATRA induced TG transamidation activity and calcification in wild-type but not in TG2-/- mouse knee chondrocytes. In addition, CXCL1 and ATRA induced multiple features of hypertrophic differentiation, and TG2 was required for these effects. TG2-/- chondrocytes lost the capacity for ATRA-induced expression of Runx2 (cbfa1), a transcription factor necessary for ATRA-induced chondrocyte hypertrophy. In contrast, the essential physiologic growth plate chondrogenic differentiation mediator CNP, which did not modulate TG activity, comparably promoted Runx2 expression and hypertrophy in normal and TG2-deficient chondrocytes. Thus, distinct TG2-independent and TG2-dependent mechanisms promote Runx2 expression, articular chondrocyte hypertrophy, and calcification. Up-regulated TG2 release alone is sufficient to promote chondrocyte hypertrophic differentiation, and TG2 GTP binding, rather than transamidation-catalyzed crosslinking, is essential [27]. TG2 acts as a molecular switch to induce chondrocyte hypertrophy in a beta1 integrin-


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mediated manner, associated with rapid phosphorylation of p38 kinase dependent on TG2 being in the GTP-bound conformational state [27]. Though TG2 transamidation activity is not required for TG2 to induce chondrocyte hypertrophy, TG2 transamidation activity still likely plays a role in modulation of chondrocyte differentiation and function. The capacity of TG2 to directly and rapidly drive chondrocyte hypertrophy when applied to cartilage in organ culture is particularly compelling [27]. It suggests that chondrocytes in OA articular cartilage could rapidly bypass the ordered physiologic progression from resting to proliferative through to hypertrophic differentiation that spatially and temporally controls growth plate differentiation. Mechanisms complementing the effects of TG2 on chondrocyte differentiation driven by inflammation include expression of the Pi co-transporter Pit-1 and sodiumdependent Pi uptake mediated by CXCR1 signaling essential for CXCL8 to induce chondrocyte hypertrophy [25]. Of additional importance is the multiligand receptor for advanced glycation end products (RAGE), which mediates several chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation [40]. RAGE ligands include S100/calgranulins, a class of small, calcium-binding polypeptides, several of which are expressed by chondrocytes [40–42]. Normal human knee cartilages demonstrate constitutive RAGE and S100A11 expression, and both RAGE and S100A11 expression are up-regulated in OA cartilages [40,42]. CXCL8 and TNFα induce S100A11 expression and release in cultured chondrocytes [40]. Moreover, S100A11 induces chondrocyte hypertrophy in vitro [40] and the chondrocyte-expressed calgranulin S100A4 stimulates MMP-13 expression [41]. CXCL1-induced and TNFαinduced but not ATRA-induced chondrocyte hypertrophy are dependent on RAGE, MAPK kinase 3, and p38 MAPK signaling [40]. Taken together, inflammationassociated chondrocyte hypertrophy driven by several cytokines and calcgraulins, TG2, Pi transport, and RAGE signaling can contribute to both chondrocalcinosis and progression of OA.

Role of PTH, PTHrP, and the Calcium Sensing Receptor (CaR) PTHrP is a central regulator of spatial and temporal aspects of chondrocyte development, as well as extent of matrix calcification, in endochondral development, and PTHrP also modulates articular chondrocyte function [22,43]. In normal articular cartilage, PTH/PTHrP receptors are expressed by chondrocytes in all zones [22]. PTHrP expression up-regulation in OA cartilage is robust through all zones, but PTH/PTHrP receptor expression becomes limited principally to the superficial zone in OA [22]. Signaling via the PTH/PTHrP receptor stimulates chondrocyte proliferation, but PTHrP also restrains the progression between prehypertrophic to hypertrophic differentiation in chondrocytes. CPPD crystal deposition disease is some subjects (discussed below) may be partly driven by excess PTH functioning to drive increased chondrocyte proliferation and stimulating chondrocytes to enter an differentiation cascade like that of endochondral maturation. However, hypercalcemia also likely plays a role, as CaR expression is up-regulated in the Hartley guinea pig medial tibial plateau cartilage as early spontaneous knee OA develops. CaR-mediated calcium sensing is known to drive PTHrP release [44], and PTHrP content becomes significantly increased in the medial tibial plateau cartilage as OA develops and progresses in this model. In cultured chondrocytes, CaR-mediated extracellular calcium-sensing, stimulated by the calcimimetic NPS R-467, induces PTHrP and MMP-13 expression and suppresses expression of

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tissue inhibitor of metalloproteinase (TIMP)-3, effects shared by elevated extracellular calcium [44]. Moreover, extracellular calcium-sensing appears essential for PTHrP and interleukin-1 to induce MMP-13 and for PTHrP to suppress TIMP-3 expression.

Potential Translational Relevance of PTH in Chondrocalcinosis Though chondrocalcinosis is highly prevalent in elders in Western countries, there have been scarce data on chondrocalcinosis prevalence in other racial or ethnic populations that might provide clues to pathogenesis. Potential inhibitors of chondrocalcinosis prevalence include high oral calcium intake that suppresses PTH production by the parathyroid. There have been reports of “hard” drinking water in China mediated by high calcium content, and primary hyperparathyroidism is rare in mainland China [45]. A recent study by Zhang et al. recruited a random sample of aged Beijing residents aged > 60 years and assessed radiographic chondrocalcinosis, with comparisons made to Whites in the American Framingham OA Study. In addition, identical methods were employed to collect samples of tap water from Beijing and Framingham and measure levels of calcium and magnesium. Chinese had a much lower prevalence of knee chondrocalcinosis, and wrist chondrocalcinosis was rare in Chinese elderly [46]. Calcium levels in tap water in Beijing were 12–20 fold higher than that in Framingham, whereas no difference was found in levels of magnesium (an inhibitor of CPPD crystal growth). Despite the finding that radiographically detectable knee and wrist chondrocalcinosis were far less common in older Chinese in Beijing than in White counterparts in Framingham, there is an excess of knee osteoarthritis in Beijing [47]. These findings suggest the possibility that chondrocalcinosis is more of an environmentally mediated finding than previously recognized. Given the current lack of effective, rational therapies to prevent or lessen idiopathic CPPD crystal deposition, further study of the potential prophylactic and therapeutic benefits of dietary calcium supplementation on chondrocalcinosis would be compelling.

Dysregulated Chondrocyte ATP Metabolism and PPi Generation in OA and Chondrocalcinosis NO is a central mediator of the pathogenesis of OA, and NO suppresses mitochondrial respiration-mediated ATP generation [48] and stimulates apoptosis in chondrocytes [49]. Treating chondrocytes with mitochondrial ATP synthesis inhibitors, and with sodium nitroprusside (a donor of NO and the mitochondrial complex IV inhibitor cyanide) recapitulate several features of OA chondrocytes in vitro, including decreased matrix synthesis and increased potential for calcification [48,50]. We observed steadily increasing ATP depletion with aging in knee articular chondrocytes in the Hartley guinea pig model of spontaneous OA, in association with up-regulation of ATPscavenging nucleotide pyrophophosphatase phosphodiesterase (NPP) activity and increasing extracellular PPi, comparable to known changes in NPP and PPi in human OA and aging joints [51]. Importantly, NPP1 (formerly known as PC-1) is a major generator of extracellular PPi in chondrocytes via ATP hydrolysis [52]. PPi potently suppresses hydroxyapatite crystal deposition and propagation, and maintenance of a relatively high extracellular PPi concentrations by chondrocytes is a vital physiologic mechanism to prevent articular cartilages from calcifying [53]. Para-


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doxically, aging hyaline articular cartilage and meniscal fibrocartilages create excesses of extracellular PPi in the extracellular matrix. A substantial fraction of extracellular PPi, including that generated by NPP1 [54], appears to be transported from the cell interior via the multiple-pass transmembrane protein ANK and its human homologue ANKH, a known physiologic suppressor of cartilage calcification [55]. Chondrocyte ANKH expression is robustly up-regulated in human knee OA cartilages [54]. Moreover, certain ANKH mutants are associated with autosomal dominant inherited degenerative arthropathy intimately linked with premature-onset CPPD crystal deposition disease, and with autosomal recessive inherited late-onset CPPD crystal deposition disease [reviewed in reference 56]. An ANKH 5'-untranslated region single nucleotide polymorphism also has been linked to increased ANKH mRNA levels and idiopathic chondocalcinosis of aging [57]. Excess extracellular PPi promotes chondrocyte hypertrophy [58], apoptosis [35], and MMP-13 expression [54] as well as CPPD crystal deposition.

Dysregulated Responsiveness to the Chondrocyte Growth Factors TGFβ and IGF-I and Cartilage Calcification Levels of active TGFβ are increased in the OA joint. TGFβ markedly elevates extracellular PPi in chondrocytes [59], mediated partly by induction of NPP1 expression and translocation to the plasma membrane [60]. The capacity of TGFβ to drive up extracellular PPi increases with cartilage aging, whereas TGFβ-induced chondrocyte growth responses decrease [61]. Normally, IGF-I inhibits the capacity of TGFβ to raise chondrocyte extracellular PPi [59]. Thus, decreased IGF-I responsiveness characteristic of chondrocytes in OA may contribute to chronic elevation of PPi generation by cartilage. The pericellular and interterritorial matrix protein Cartilage Intermediate Layer Protein-1 (CILP-1) is one of the inhibitors of IGF-I that is up-regulated in OA cartilage, and CILP-1 promotes increased extracellular PPi in cultured chondrocytes [62].

Summary This review has discussed fundamental mechanisms that converge to actively drive both OA and matrix calcification (Fig. 1). However, it is noteworthy that calcification is not a universally detected feature in joints with OA [1–5]. Conversely, cartilage crystal deposition may occur without advanced cartilage degeneration, particularly in idiopathic CPPD deposition disease of aging. Mechanistic convergence and divergence of OA and cartilage matrix calcification may inform as to predominant operative pathogenic pathways. As discussed above, sustained chondrocyte hypertrophy without progression to apoptosis would be expected to drive both cartilage reparative and matrix calcification responses, and favor CPPD deposition over further progression of OA. In contrast, robust MMP and aggrecanase activation without substantial chondrocyte hypertrophy and derangements in ATP and PPi metabolism would be predicted to favor development of OA without matrix calcification. BCP crystal deposition in cartilage is not easy to detect by standard clinical imaging and synovial fluid analysis tools. Moreover, conventional radiography and synovial fluid analyses have limits in sensitivities to detect CPPD crystal deposition. It is possi-

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ble that more systematic evaluation of joints for evidence and forms of cartilage calcification, done prior to end-stage disease, can provide information to weigh operative pathogenic factors, the phenotype of cartilage repair, and prognosis in degenerative arthritis. Similarly, clinical measurements of one or more of the intra-articular mediators discussed here (such as TG2, S100A11, ANKH, PTHrP, PPi, NPP1, and CILP-1) might provide useful data for these purposes. How practical and cost-effective such measures are may impact on development of rationally targeted and more effective early treatment strategies for both OA and chondrocalcinosis.

Acknowledgements Dr. Terkeltaub’s research on OA is supported by the Department of Veterans Affairs and NIH.

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[41] R.R. Yammani, C.S. Carlson, A.R. Bresnick, R.F. Loeser, Increase in production of matrix metalloproteinase 13 by human articular chondrocytes due to stimulation with S100A4: Role of the receptor for advanced glycation end products, Arthritis Rheum 54 (2006), 2901-11. [42] R.F. Loeser, R.R. Yammani, C.S. Carlson, H. Chen, A. Cole, H.J. Im, L.S. Bursch, S.D. Yan, Articular chondrocytes express the receptor for advanced glycation end products: Potential role in osteoarthritis, Arthritis Rheum 52 (2005), 2376-85. [43] R. Goomer, K. Johnson, D. Burton, D. Amiel, T. Maris, Gujral A, Deftos LJ, Terkeltaub R. A Tetrabasic C-Terminal Motif Determines Intracrine Regulatory Effects of PTHrP 1-173 on PPi Metabolism and Collagen Synthesis in Chondrocytes, Endocrinol 141 (2000), 4613-22. [44] D.W. Burton, M. Foster, K.A. Johnson, M. Hiramoto, L.J. Deftos, R. Terkeltaub, Chondrocyte calciumsensing receptor expression is up-regulated in early guinea pig knee osteoarthritis and modulates PTHrP, MMP-13, and TIMP-3 expression, Osteoarthritis Cartilage 13 (2005), 395-404. [45] J.P. Bilezikian, X. Meng, Y. Shi, S.J. Silverberg, Primary hyperparathyroidism in women: a tale of two cities – New York and Beijing. Int J Fertil Womens Med 45 (2000), 158-65. [46] Y. Zhang, R. Terkeltaub, M. Nevitt, L. Xu, T. Neogi, P. Aliabadi, J. Niu, D.T. Felson, Prevalence of chondrocalcinosis is much lower in Chinese in Beijing than in Whites in the USA: The Beijing Osteoarthritis Study, In Press, Arthritis Rheum (2006). [47] Y. Zhang, L. Xu, M.C. Nevitt, P. Aliabadi, W. Yu, M. Qin, L.Y. Lui, D.T. Felson, Comparison of the prevalence of knee osteoarthritis between the elderly Chinese population in Beijing and whites in the United States: The Beijing Osteoarthritis Study, Arthritis Rheum 44 (2001), 2065-71. [48] K. Johnson, A.S. Jung, A. Andreyev, A. Murphy, J. Dykens, R. Terkeltaub, Mitochondrial Oxidative Phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization, Arthritis Rheum 43 (2000), 1560-70. [49] K. Kuhn, D.D. D’Lima, S. Hashimoto, M. Lotz, Cell death in cartilage, Osteoarthritis Cartilage 12 (2004), 1-16. [50] H.S. Cheung, L.M. Ryan, Phosphocitrate blocks nitric oxide-induced calcification of cartilage and chondrocyte-derived apoptotic bodies, Osteoarthritis Cartilage 7 (1999), 409-12. [51] K. Johnson, C.I. Svensson, D.V. Etten, S.S. Ghosh, A.N. Murphy, H.C. Powell, R. Terkeltaub, Mediation of spontaneous knee osteoarthritis by progressive chondrocyte ATP depletion in Hartley guinea pigs, Arthritis Rheum 50 (2004), 1216-25. [52] K. Johnson, S. Hashimoto, M. Lotz, K. Pritzker, J. Goding, R. Terkeltaub, Up-Regulated Expression of the Phosphodiesterase Nucleotide Pyrophosphatase Family Member Plasma Cell Membrane Glycoprotein-1 (PC-1) is Both a Marker and Pathogenic Factor for Knee Meniscal Cartilage Matrix Calcification, Arthritis Rheum 44 (2001), 1071-81. [53] R. Terkeltaub, Inorganic pyrophosphate (PPi) generation and disposition in pathophysiology, Am J Physiol, Cell Physiol 281 (2001), C1-11. [54] K. Johnson, R. Terkeltaub, Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess, Osteoarthritis Cartilage 12 (2004), 321-35. [55] A.M. Ho, M.D. Johnson, D.M. Kingsley, Role of the mouse ank gene in control of tissue calcification and arthritis, Science 289 (2000), 265-70. [56] R. Zaka, C.J. Williams, Role of the progressive ankylosis gene in cartilage mineralization, Curr Opin Rheumatol 18 (2006), 181-6. [57] Y. Zhang, K. Johnson, R.G. Russell, B.P. Wordsworth, A.J. Carr, R.A. Terkeltaub, M.A. Brown, Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5'-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate, Arthritis Rheum 52 (2005), 1110-7. [58] W. Wang, J. Xu, B. Du, T. Kirsch, Role of the progressive ankylosis gene (ank) in cartilage mineralization, Mol Cell Biol 25 (2005), 312-23. [59] U. Olmez, L.M. Ryan, I.V. Kurup, A.K. Rosenthal, Insulin-like growth factor-1 suppresses pyrophosphate elaboration by transforming growth factor beta1-stimulated chondrocytes and cartilage, Osteoarthritis Cartilage 2 (1994), 149-54. [60] K. Johnson, S. Vaingankar, Y. Chen, A. Moffa, M.B. Goldring, K. Sano, P. Jin-Hua, A. Sali, J. Goding, R. Terkeltaub, Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes, Arthritis Rheum 42 (1999), (9):1986-97. [61] F. Rosen, G. McCabe, J. Quach, J. Solan, R. Terkeltaub, J.E. Seegmiller, M. Lotz, Differential effects of aging on human chondrocyte responses to transforming growth factor beta: increased pyrophosphate production and decreased cell proliferation, Arthritis Rheum 40 (1997), 1275-81. [62] K. Johnson, D. Farley, S.I. Hu, R. Terkeltaub, One of two chondrocyte-expressed isoforms of cartilage intermediate-layer protein functions as an insulin-like growth factor 1 antagonist, Arthritis Rheum 48 (2003), 1302-14.


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IV Leptin, the Prototype of Adipokines: Molecules at the Crossroads of Inflammation and Metabolism Rodolfo GÓMEZ a, Rocío LAGO a, Francisca LAGO b, Juan J. GÓMEZ-REINO a, Miguel OTERO a and Oreste GUALILLO a,∗ a Santiago University Clinical Hospital, Research Laboratory 4 (NEIRID LAB, Laboratory of Neuro Endocrine Interactions in Rheumatology and Inflammatory Diseases), Santiago de Compostela, Spain b Santiago University Clinical Hospital, Research Laboratory 1 (Molecular and Cellular Cardiology), Santiago de Compostela, Spain Abstract. The prevalence of obesity and obesity-related diseases focussed an increasing interest, over the last 10 years, on white adipose tissue and its derived bioactive peptides, being the discovery of leptin in 1994 the trigger of the renaissance of the studies about adipose tissue. Leptin was initially depicted as the most important anorexigenic factor with neuroendocrine actions, but it has been later shown to significantly modulate immune and inflammatory processes. Leptin is a dual molecule: apart from its previously envisaged metabolic activities, increasing evidence frames leptin as a novel pro-inflammatory adipokine and, at present it might be easily considered one of the relevant links among immune system, inflammatory response and neuroendocrine system. Leptin regulates and participates both in immune homeostasis and inflammatory processes by acting as a modulator of cell activity and playing an active role in articular degenerative inflammatory diseases such as osteoarthritis and rheumatoid arthritis, but also in a host of autoimmune inflammatory conditions such as encephalomyelitis, type-1 diabetes, and bowel inflammation. This review will be focussed more on the adipokine facet of leptin, even though its role as metabolic hormone will be also addressed. In addition, the role od other relevant adipokines in inflammation will be covered.

White Adipose Tissue: A Surprising Biochemical Factory Historically, white adipose tissue (WAT) has been viewed as a passive depository of energy and as a protective mechanism for heat loss. For the time being, this vision is still proper but incomplete, and rather insufficient to understand the actual complex functions of WAT. Adipocytes are able to produce and secrete a wide number of mole∗ Corresponding Author: Dr. Oreste Gualillo, Santiago University Clinical Hospital, Research Laboratory 4 (NEIRID LAB, Laboratory of Neuro Endocrine Interactions in Rheumatology and Inflammatory Diseases). Calle Choupana s/n, 15706, Santiago de Compostela, Spain, Phone & Fax: 00+34+981+950905, E-mail: [email protected], [email protected].

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R. Gómez et al. / Leptin, the Prototype of Adipokines

cules, including classical cytokines such as IL-1, IL-6 and TNF-alpha but also novel factors such as adiponectin, resistin, visfatin, vaspin, apelin or leptin among others, the so-called adipokines [1–3]. Thus, WAT is now considered as a true endocrine organ, and probably the largest endocrine organ in the body, whose main actions include regulation of energy homeostasis, metabolism, and immune and inflammatory processes regulation [1–5]. There is a general consensus about the significant contribution of excessive fat accumulation and the so called “low grade inflammatory state”. This cluster of dysfunctions is characterized prevalently by dyslipidemia, insulin resistance, alteration of coagulation cascade and it is associated with increased risk of type 2 diabetes, cardiovascular complications and autoimmune inflammatory diseases. Several concerns support the basis of obesity as a pro-inflammatory condition; indeed, most of the pro-inflammatory cytokines, as well as acute phase molecules, are elevated in obese subjects. For completeness, it is dutiful to mention that WAT in obesity is able to synthesize also anti-inflammatory factors such as IL1-RA, may be as a sort of adaptive response. At any rate, it is reasonable to hipothesize that in obesity the balance between pro-and anti-inflammatory factors is severly altered or at least compromised and that white adipose tissue plays a lead role in the synthesis and secretion of several mediators of inflammatory response which, through a bidirectional way, are involved in the perpetuation of obesity itself and contribute to the mechanisms responsible for the development of the chronic disease associated with obesity.

Leptin and Its Receptors The key event that marked the new glance of WAT as relevant endocrine tissue was the identification of leptin by Zhang et al in 1994 [6]. Leptin is a 16 kDa peptide, encoded by the ob gene and it is mainly produced by adipocytes [6,7], although other organs produce leptin in significant amounts. Leptin expression is prevalently regulated by


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food intake [8], hormones, and cytokines. Leptin levels are directly correlated with insulin [9,10] and negatively correlated with glucocorticoid levels [11,12]. Inflammatory mediators, such as interleukin (IL)-1, tumour necrosis factor (TNF)alpha or leukemia inhibitory factor (LIF), increase leptin synthesis [13–16]. Furthermore, it has been demonstrated a gender-related leptin regulation, based on the observation that testicular steroids inhibits leptin expression [17] whereas ovarian sex steroids, as well as prolactin, increase it [18,19]. Leptin exerts its main action at a central hypothalamic level by increasing energy expenditure and decreasing food intake [8,20]. Leptin has been involved as a modulatory agent in a variety of physiological processes such as the regulation of hypothalamic-pituitary-adrenal axis [21–23], maturation of reproductive system [24–26], hematopoiesis [27] and foetal development [28,29]. Leptin exerts its actions by binding to its specific receptors. Encoded by the diabetes (db) gene, the Ob-R mRNA generates by alternative splicing six different receptor isoforms, but only the long-functional isoform, Ob-Rb, is the functional. Ob-R belongs to the class I cytokine receptors super-family, which typically contains a cytokine receptor homologous domain in the extra-cellular region and includes receptors for IL-6, LIF or gp 130 [30–32]. Leptin receptor long form (Ob-Rb) transduces its signal trough a classic JAK/STAT pathway. Furthermore, Ob-Rb is able to transduce signals by using alternative signalling pathways which involves the SHC/GRB2 pathway as well the IRS-2, PI-3 kinase, MAPK, AMPK pathway and Erk-1/2 SHP-2-dependent activation [33]. It is noteworthy to mention that structural integrity of long form receptor is essential for mediating biological leptin response. Actually, mutations of the STAT binding site of the receptor disrupt the signal trasduction pathway resulting in a phenotype characterised by impaired thermoregulation, obesity and hyperphagia. In addition, the integrity of several tyrosine domains residues are critical for leptin-mediated homeostatic action, but are not for mediating the permissive role of leptin in reproductive function.

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Leptin and Immunity Leptin and its cognate receptor roles in immune-regulation were somehow enlightened by the discovering of thymus atrophy in db/db mice. Since that observation, many results clearly pointed out a wide range of direct leptin’s effects on immune responses [2,34,35–37]. On innate immunity, leptin induces activation of monocytes and increases phagocytosis by macrophages [38–40]. In rodent macrophages, elevated doses of leptin upregulates LPS induced-production of different pro-inflammatory cytokines upregulating inflammatory immune responses [41]. Furthermore, leptin is able to directly modulate the activity and function of neutrophils acting via its receptors activation [38,42,43], or by an indirect way mediated by monocyte- produced TNF in humans [44]. On natural killer (NK) cells, leptin plays an important role in its development, activation and function and it also increases its cytotoxic ability in a dose-dependent way [45–47]. Consistent with these observations, leptin deficiency increases susceptibility to infectious and inflammatory stimuli and it is linked with a marked dysregulation of cytokine production. Regarding leptin actions on adaptive immune responses, it has been proved that leptin induces T-cell activation and proliferation, and protects T lymphocytes from induced-apoptosis [48]; furthermore, leptin is able to polarize T-cell differentiation towards a TH1 response [48], affecting the pattern of cytokines production [36]. Leptin and Endothelial Function Emerging evidences link leptin to cardiovascular disease as an important mediator of inflammatory response at endothelial levels. Indeed, in endothelial cells leptin induces oxidative stress by promoting accumulation of reactive oxigen species and activating endothelial nitric oxide synthase, up-regulates endothelin-1 production, potentiates platelet aggregation, angiogenesis, adhesion molecules expression and expression of monocyte chemo-attractant proteins [49–51]. Leptin: A Novel Pro-Inflammatory Adipokine Several studies demonstrated that numerous inflammatory stimuli modulate both leptin gene expression and circulating leptin levels [13–16]. Serum leptin levels are strongly increased in experimental models of acute inflammation [15], whereas this increase in plasma leptin is not always observed in humans [52]. Modulation of leptin levels during acute inflammatory stimuli suggests that this adipokine is participating in the development of inflammatory processes. This hypothesis has been tested and was confirmed by many observations in different autoimmune-inflammatory models, in which leptin has been postulated to play a relevant role. It was observed that leptin-deficient ob/ob mice were resistant, or at least, less susceptible to the development of different inflammatory diseases. Thus, ob/ob mice were resistant to antigen induced arthritis [53], experimental hepatitis [37,45], colitis [54] or autoimmune encephalomyelitis [55]. In the above mentioned experimental models, leptin deficiency caused a marked reduction in the severity of inflammation, whereas exogenous leptin administration restored the normal secretion pattern of many of the inflammatory modulators and also restored disease susceptibility, making it comparable to wild-type mice.


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Taken together, all these data suggest that leptin plays a main role in the development of the inflammatory response by acting in a pro-inflammatory fashion in a similar way to that observed with other classical and well recognized pro-inflammatory cytokines.

Leptin: A Disrupter for Articular Cartilage The normal joint is a specialized structure consisting on multiple connective tissue elements organized in a manner that permits stability and movement of the skeleton. Among the multiple connective tissues integrated in normal joint, the articular cartilage is, probably, the most affected during rheumatic diseases. Essentially, this connective tissue is composed by an extracellular matrix and only one cell type, the chondrocyte, which is the responsible of its synthesis and homeostasis [56–59]. In a normal situation, chondrocytes are in a delicate balance between matrix synthesis and destruction. Under inflammatory conditions, this balance becomes altered and matrix destruction overcomes synthesis, resulting in a complete joint cartilage loss of structure [56–58]. It was found that chondrocytes expressed Ob-Rb, the functional leptin receptor isoform [59]. So, it is conceivable that leptin could be acting on joint cartilage through these cells. Obesity is a well known risk factor for knee OA. Indeed, the prevalence of knee OA is increased in obese subjects, and conversely, weight reduction decreases this risk. Generally, the influence of obesity to cartilage degradation has been related to an abnormal biomechanical loading. However, it has also been reported that obesity is related to the development of non-weight bearing hip and hand OA [60,61]. So that, it is reasonably conceivable that non mechanical factors but rather obesityassociated proteic factors such as leptin, as well as other adipokines, are likely involved in the development of articular degenerative inflammatory diseases. Two recent studies demonstrated a clear detrimental effect of leptin on articular cartilage. Namely, it has been demonstrated that leptin induces, in a synergistic way with interferon-gamma (IFN-gamma) and interleukin-1 (IL-1), nitric oxide (NO) production via nitric oxide synthase (NOS) type II in chondrocytes [62,63]. In both cases, NOS II synergistic induction is signalled through a common transduction pathway which involves several kinases including Jak-2, PI-3K, MEK-1 and p38K. Nitric oxide has been shown to have a negative effect on chondrocytes physiology and, thereby, over cartilage structures. Indeed, it has been demonstrated that NO increases chondrocyte apoptosis, leads to phenotype loss and induces matrix metalloproteases synthesis [64] by causing complete cartilage degradation. In addition, it has been shown that normal chondrocytes synthesizes leptin and that leptin synthesis is increased in chondrocytes from osteoarthritis patients [65]. Furthermore, it has been demonstrated that leptin expression is increased in articular rat joints injected with exogenous leptin [65], which implies a positive feedback regulation. So, in terms of expression, leptin mimics classic cytokine behaviour under inflammatory conditions. It has also been found that circulating leptin flows from blood to synovial fluid during rheumatoid arthritis [66], as many other classic cytokines do. Furthermore, it has been recently shown that circulating leptin levels, as well as other plasma levels of other adipokines such as adiponectin and visfatin, are signifi-

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cantly increased in patients affected with RA, independently from the amount of white adipose tissue [67] and positively correlates with the C-reactive protein levels.

Adiponectin Adiponectin was discovered more or less in the same period of leptin, but this adipokine did not receive initially the same acclaim of leptin; really, its relevance as a potential protective adipokine in obesity and obesity-related disorders was acknowledged only few years later. Adiponectin, also called gelatin binding protein-28 (GBP28), adipose most abundant gene transcript 1(apM1), adipocyte complement related protein (Acrp) 30 or AdipoQ, is a 244 aa adipose tissue specific protein that has structural homology to collagen VIII and X and complement factor C1q. Adiponectin circulates in the blood in large amounts and constitutes about the 0.01% of total plasma proteins. Adiponectin is present in serum as oligomeric isoforms constituted prevalently by trimers, hexamers but also by high molecular weight isoforms (12–18 mer) [68]. Adiponectin exerts its biological action by mean of two recently described receptors which are expressed prevalently in liver (Receptor 2) and skeletal muscle (Receptor 1) and transduce signals by the activation of AMPkinase, PPAR-alpha and presumably some other unknown signalling pathways leading to an increase of fatty acid oxidation and reducing liver glucose synthesis [69]. Most of the biologic actions mediated by adiponectin receptors involve the activation of AMP kinase as early step of intracellular signal transduction upon receptor activation, in spite of of a classic G-protein coupling and cAMP induction, a quite unusual feature for a 7-transmembrane domain receptor. AMP kinase activation, induced by adiponectin, stimulates phosphorylation of acetyl coenzyme A carboxylase, glucose uptake and fatty acid oxidation in miocytes, whereas in liver induces a clear reduction of gluconoegenesis by limiting the synthesis of specific enzymes. Adiponectin and Inflammation Adiponectin has been described as a potent antiatherogenic factor and a plethora of actions have been described at endothelial and vascular level [70]. Indeed, adiponectin inhibits monocytes adhesion to endothelial cells, reduces the synthesis of adhesion molecules and tumor necrosis factor as well as decreases nuclear factor k beta levels [71]. Adiponectin expression is inhibited by pro-inflammatory cytokines such as IL-6 [72] and TNF-alpha in cultured adipocytes [73]. There is a general consensus about a putative protective role of adiponectin from inflammatory state, at least at endothelial-vascular level. Indeed, circulating adiponectin levels are inversely proportional to obesity and therefore tend to be low in morbid obese subjects. On the contrary, adiponectin levels increase with weight loss and with use of insulin sensitizing drugs [74]. Low levels of adiponectin have been linked to inflammatory atherosclerosis in humans [75]. In addition, animal models have shown that low adiponectin levels increase smooth cell proliferation in response to injury, increase free fatty acid levels and cause insulin resistance [76]. In addition, the pro-diabetic and pro-atherogenic effects of low adiponectin levels, seen in the metabolic syndrome, provide a clear link between inflammation and obesity. However, a recent report by Ehling et al, in contrast to the previously envisaged adiponectin’s protective role in endocrinological and vascular diseases, suggests that adiponectin is in-


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volved in key pathways of inflammation and matrix degradation in the human joint. The effects of adiponectin in human synovial fibroblasts appear to be highly selective by inducing two of the main mediators of rheumatoid arthritis pathophysiology, IL-6 and matrix metalloproteinase-1, via a p38 MAPK pathway [77]. More recently, it has been proposed that cartilage is also a target tissue for adiponectin. Indeed, chondrocytes express functional adiponectin receptors whose activation lead to the induction of nitric oxide synthase type II by a signalling pathway that involve PI3 Kinase (unpublished data from our group). Moreover, adiponectin-challenged chondrocytes are able to increase IL6, TNF-α and MCP-1 synthesis whereas, intriguingly, were unable to modify prostaglandin E2 and leukotriene B4 release. Taken together, all these recent results bind more closely the interactions between adiponectin and articular inflammatory diseases, and suggest that adiponectin is a novel key element in the maintenance of cartilage homeostasis which might be considered as a potential therapeutical target in joint degenerative diseases. It is noteworthy, and somewhat unexpected, that plasma adiponectin in patients with rheumatoid arthritis are higher than those observed in healthy controls [67], the reasons for which are not evident, although increased levels of adiponectin are observed in the synovial fluid of these patients compared with those seen in patients with osteoarthritis [78]. The increased levels of adiponectin, in patients with rheumatoid arthritis, suggest a compensatory mechanism under catabolic or anabolic imbalance. So, it is conceivable that an increase in adiponectin level represents an attempt to antagonise the anorexigenic and well-known pro-inflammatory effect of leptin, suggesting that these two adipokines may act in parallel as opposing metabolic counterparts.

Resistin Resistin is a dimeric protein that received its name from the initial observation that it induced insulin resistance in mice. Resistin belongs to the FIZZ family (found in inflammatory in zone) also known as RELMs (Resistin like molecules). The first identified protein of the family was FIZZ1 (also known as RELM-alpha), a protein that is up regulated in the asthmatic lung in bronchoalveolar fluid of mice with experimentallyinduced asthma [79]. The other homologue FIZZ2 (also known as RELM-beta) was next identified in the proliferating epithelia of intestinal crypt [80]. Finally, the third homologue, FIZZ3 (also known as resistin) was later identified in adipocytes as well as in other cell types such as macrophages. To date, many aspects of resistin biology remain controversial and studies in humans only demonstrate a weak relationship between obesity and diabetes, so its role as a mediator of insulin resistance is at present questionable. Resistin and Inflammation Emerging concepts suggest a role of resistin in inflammatory conditions in humans since a robust expression of resistin is present in monocytes. Some pro-inflammatory cytokines such as TNF-alpha, IL-6 and lipopolysaccharide are able to regulate resistin gene expression. It is noteworthy to stress that resistin regulation in response to proinflammatory stimuli is tissue-dependent. Recent studies have shown the modulation of pro-inflammatory cytokines synthesis by this molecule. Resistin is able to upregulated IL-6 and TNF-alpha in blood mononuclear cells via NF-kB pathway [81]. Conversely,

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LPS was reported to induce resistin gene expression in primary human and murine macrophage via a cascade involving the secretion of pro-inflammatory cytokines [82]. Finally, resistin has been proposed to be involved in the pathogenesis of rheumatoid arthritis in humans. In a rodent experimental model, local injection of resistin is able to induce an arthritis-like syndrom. Indeed, following resistin local joint administration, mice showed leukocyte infiltration of synovial tissues which were associated with hypertrophy of synovial layer and pannus formation [81]. Resistin has been found in the plasma and the synovial fluid of RA patients and in some studies, synovial fluids from RA patients showed higher levels of resistin compared with the serum compartment [83,84]. These findings provide evidence for a specific local dysregulation of adipokines in the joint space and suggest that circulating levels of adipokines do not represent the situation in the joint. Anyway, the high synovial fluid levels of certain adipokines compared to serum may be due to the increased permeability of inflamed synovial membrane. However, plasma resistin levels were not different between RA patients and healthy controls [67,81]. So, the role of resistin is merely apparent, but the precise mechanism of regulation at joint levels needs to be analyzed in depth.

Visfatin Visfatin is an insulin mimetic novel adipokine which was discovered by Fukuhara et al, [85] using a differential display technique to identify genes specifically expressed in abdominal fat. Visfatin was found to be identical to PBEF (pre-B colony enhancing factor), a growth factor for B lymphocytes precursors previously known to be synthesized in liver, skeletal muscle and bone marrow and to be up-modulated in models of acute lung injury and sepsis. Visfatin circulating levels strongly correlates with WAT accumulation and its mRNA expression is dependent from adipocyte differentiation. It is noteworthy that in obesity circulating visfatin levels increases during the development of obesity. Visfatin synthesis is regulated by several factors including glucocorticoids, TNF-alpha, IL-6. Visfatin is produced also by endotoxin-challenged neutrophils and inhibits neutrophils apoptosis through a mechanism mediated by caspase 3 and 8 [86]. In humans, visfatin levels correlates with BMI but not with visceral fat mass or waist-to-hip ratio [87]. In addition, visfatin levels in type II diabetes subjects are higher than normoglycemic counterpart [88]. Interestingly, circulating visfatin is also higher in patients with rheumatoid arthritis than in healthy controls [67]. It is currently unclear what would be visfatin physiological role or relevance in the context of rheumatoid arthritis. Visfatin might be part of a compensatory mechanism that facilitates the accumulation of fat in the intra-abdominal depot, a feedback mechanism preventing the deleterious effects of the rheumatoid cachexia. Moreover, at present it cannot rule out that an increase in visfatin levels may be related to the modulation of inflammatory or immune response or may simply be an epiphenomenon.

Conclusion Obesity and the associated metabolic diseases are the most common and detrimental illness affecting more than 50% of the adult western population. These conditions are associated with a chronic inflammatory response characterized by abnormal cytokine production, increased acute-phase reactants, and activation of inflammatory signaling


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pathways [68]. This association is not inconsequential and is constrained to either obesity itself or closely linked diseases such as insulin resistance, type 2 diabetes, and cardiovascular disease. A very intriguing characteristic of the inflammatory response that emerges in the presence of obesity is that it appears to be triggered, and to reside predominantly, in adipose tissue, although other metabolically critical sites may also be involved during the course of the disease. Leptin, as the prototype of white adipose tissue-produced adipokine, is much more than the initially envisaged anti-obese hormone. Actually, besides its classical role as metabolic hormone, leptin shares many common points, including structural and functional features, with most of the classical pro-inflammatory cytokines. Anyhow, leptin might be considered as a promising therapeutic target in some pathology where this adipokine is thought to promote inflammatory diseases. For instance, soluble receptors, which control the amount of bioavailable leptin, could be used to counteract pro-inflammatory leptin’s action. A similar therapeutic strategy is at present used to antagonize the effect of TNF-alpha in RA. Other potential therapeutic intervention can be achieved by the use of monoclonal humanized antibodies or by leptin mutants with receptor blocker properties, which might be able to bind OB receptor but not to activate it. In conclusion, there are increasing evidences that argue for a role of leptin and other adipokines in the pathogenesis of inflammatory disease, supporting the notions that a pharmacological strategy based on the modulation of adipokines synthesis or action could have attractive therapeutic advantages.

Acknowledgements Some of the research described in this review has been supported by Spanish Ministry of Health, Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI 05/0525, PI030115, PI050419 and G03/152), and Xunta de Galicia. Oreste Gualillo and Francisca Lago are recipient of a contract under the “Programme of stabilization of researchers” co-funded by the Spanish Ministry of Health/Instituto de Salud Carlos III and Xunta de Galicia/SERGAS. Miguel Otero is a recipient of a post-graduate fellowship funded by Fundación Caixa Galicia. Rocío Lago (FI05/01019) and Rodolfo Gómez (PI05/0525) are recipients of pre-doctoral fellowships funded by Instituto de Salud Carlos III. It is reasonable that not all published data were discussed in this review. So, we really apologize to those whose works were not mentioned.

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V The Role of Extracellular Matrix Fragments in the Autocrine Regulation of Cartilage Metabolism Gene A. HOMANDBERG, Lei DING and Danping GUO Department of Biochemistry and Molecular Biology, The University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203

Abstract. The ability of degradation products of the extracellular matrix (ECM) to regulate cartilage homeostasis has now been well documented. There are now numerous observations that different types of products derived from the damaged matrix can provide additional signals that can amplify catabolic processes that serve either to clear tissue components for repair or to initiate reparative signals. These fragments include fibronectin fragments (Fn-f), collagen fragments (Col-f) and hyaluronan fragments (HA-f) and likely link protein fragments (LP-f). Active fragments of other ECM components may be found in the future. ECM fragments can arise during cartilage degeneration with enhanced levels of proteinases and normal rates of matrix synthesis. Ironically and theoretically, fragments might also arise from enhanced synthesis of their native precursors but only with basal levels of proteinases and this might lead to enhanced proteinases. Further, certain types of fragments might arise from synovial tissue. The linkage between catabolic and anabolic pathways in cartilage is amply illustrated by the properties of Fn-fs in that the damage pathways initiated by Fn-fs also initiate anabolic pathways of attempted repair. Observations with Fn-fs show that lower concentrations that initiate the lowest levels of matrix metalloproteinases (MMPs) can initiate anabolic processes while higher concentrations also enhance catabolic protease driven pathways that swamp out the anabolic pathway. Anabolism might be enhanced through post-translational events such as proteolytic activation of ECM bound growth factors although other explanations are possible. Thus, fragment systems may be operative not only during damage, but also during normal metabolism and in either case, may shift metabolism in either direction, depending on the concentration of the fragments. Regulation of the fragment pathways may be through native ligands, since the ECM fragments are likely inhibitors of the native ligands and vice versa. These ECM fragment pathways may define a global pathway in which: (1) one type of fragment, such as a Fn-f, can bind either Fn or type II collage and affect not only Fn integrins but also collagen integrins and (2) one type of fragment may bind one type of integrin proximal to another type and affect integrin complexes or clusters. The signaling pathway of Fn-fs suggest that they bind to receptors and disrupt receptor clusters and this may allow internalization of receptors and initiate new pathways involving MAP kinases, Nf-kB activation and ultimately cytokine and MMP upregulation. It will be important to continue to compare the Fn-f, Col-f and HA-f pathways to determine if there is a single global mechanism that might be subject to therapeutic intervention. There are still some basic questions that need to be addressed such as whether these fragments initiate cartilage degeneration or simply amplify ongoing processes or where they are positioned in the early stages of i.e. osteoarthritis and whether or not partial vs com-

G.A. Homandberg et al. / The Role of Extracellular Matrix Fragments

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plete inhibition of these pathways would be beneficial in a degradative state. More information of the mechanisms is needed especially far upstream at the level of membrane receptors, where re-distribution of integrins may be the key initiating event in the pathway. Keywords. Extracellular matrix, fibronectin fragments, collagen fragments, hyaluronan fragments, integrins

Introduction The ability of degradation products of the extracellular matrix (ECM) to regulate cartilage homeostasis is an ideal means of regulation of the health of cartilage tissue, since the damaged ECM, because of its upstream position, could provide direct signals to communicate with chondrocytes and initiate pathways required for tissue maintenance. Some observations of ECM fragment pathways suggest that the ECM fragments should not be considered only catabolic but may be able to regulate the balance between catabolism and anabolism, based on the relative levels of the fragments. This should not be surprising because tissue damage is known to initiate reparative pathways. This review will attempt to compare and consolidate observations of matrix fragment systems, including fibronectin fragments (Fn-fs), type II collagen fragments (Col-f), hyaluronan fragments (HA-f) and link protein fragments (LP-f) in order to address their physiologic relevance, their potential common mechanism of action, if any, and to conclude with an understanding of their central role. More recent reviews on Fn-fs are [1,2] and for HA-fs [3,4].

ECM Fragments and Their Precursors Are Elevated in Synovial Fluids and Cartilage in RA and OA The cartilage matrix consists of various macromolecules with the ability to contribute degradation products that could theoretically perturb the ECM. These include fibronectin (Fn), type II collagen, various types of proteoglycan, hyaluronan (HA), link protein, COMP and minor amounts of other types of ECM ligands. Naturally, these components are degraded through normal turnover, and thus, a certain level of fragmentation would be expected under normal conditions, but in cartilage degeneration the level of fragmentation likely increases for all. The increases might arise either from basal levels of native ligand with a background of enhanced proteinases in a frank catabolic state or ironically, from elevated levels of native ligand against a background of basal levels of proteinases that might occur in an anabolic repair response or from both. The latter situation suggests that ECM fragments might also represent a secondary effect of attempted repair responses as the tissue responds to mild insults. Thus, it is conceivable, that if the repair response is not tightly regulated, full flown catabolic pathways may result.

Fn and Fn-fs Are Elevated in OA While levels of some proteins decrease in OA, the precursor of Fn-fs, native Fn, is elevated in the cartilage matrix in human OA cartilage [5–7] as well as in canine [8,9] and

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rabbit [9] in vivo models of OA damage. This increase in Fn may be due to both enhanced synthesis and enhanced retention of Fn as shown by Burton-Wurster and Lust [7]. How long Fn levels remain elevated during the course of the disease is not known. It is likely that at some point, Fn synthesis may decrease due to metabolic overload on chondrocytes. Fn levels also increase in synovial fluids in patients with OA and rheumatoid arthritis [RA] [10]. The increase may be up to 3 to 4 fold; the average concentration of Fn in healthy donors increases from 171 μg/ml to 721 and to 568 in RA and OA synovial fluids, respectively [11]. Since initiation of OA bears a strong biomechanical component, it is important to point out that some of the enhanced Fn in OA which likely gives rise to elevated Fn-fs, might be due to altered biomechanical forces. For example, Fn synthesis is increased in canine articular cartilage explants after cyclic impact [12] and in bovine articular cartilage explants after intermittent cyclic loading [13]. Compressive loading and unloading have also been shown to affect Fn synthesis [14]. It is also conceivable that enhanced Fn synthesis may also occur indirectly through the effects of altered biomechanical forces on activation and liberation of growth factors stored in cartilage tissue. With the increases in Fn levels in cartilage and synovial fluids in RA and OA, it would be expected that Fn-f levels would increase also. Griffiths et al. [15] found Fn-fs from 24-kDa to 200-kDa in RA, OA, traumatic arthritis and septic arthritis synovial fluids. These Fn-fs represented a major portion of the total Fn in most cases. Elevated Fn-fs in RA synovial fluids has also been demonstrated [16]. We reported that over half of the total Fn in OA synovial fluids was degraded, resulting in μM concentrations of Fn-fs which ranged in mass from 30-kDa to about 200-kDa [17]. We later confirmed the presence of several amino-terminal Fn-fs of 30 to 230-kDa in extracts of human OA cartilage using an amino-terminal specific antibody [18]. Very recently, others have confirmed our results [19]. It is important to emphasize here that Fn-fs have been reported in many diverse pathologic body fluids and tissues as well and may also play roles in tissue damage/repair in other tissues [20–27]. The Fn-fs found in synovial fluid might be derived from cartilage, synovium and plasma Fn. Some indication has been provided by studies of differentially spliced Fn isoforms that have been found to be relatively tissue specific. However, it appears that synovial fluid contains more than one form. The isoforms in OA synovial fluids include the synovial fluid Fn isoform, the ED-a[+] isoform [28] which is present at only low levels in cartilage of OA patients [29]. In contrast, the population synthesized within cartilage tissue is significantly different than in other tissues and includes relatively high levels of an ED-b[+] form and of a cartilage specific form, [V+C]- which lacks several segments found in the isoforms of other tissues, as reviewed [30]. Interestingly, the ED-b[+] isoform increases throughout the cartilage matrix in a canine model of OA [31] and this isoform is upregulated at the message level in human OA cartilage [29]. Thus, Fn-fs in synovial fluid could theoretically be derived not only from plasma Fn but also from cartilage and synovial fluid isoforms. Since the isoforms have a conserved sequence in the amino-terminus, we cannot deduce, in terms of amino-terminal Fn-f, the source of synovial fluid Fn-fs. To obviate this difficulty, we tested activities of Fn-f solutions from OA synovial fluid or those generated from bovine synovial fluid, bovine cartilage or bovine plasma and found them to all be fully and equally active in cartilage chondrolysis [18]. Thus, the Fn-f activities we have reported are not dependent on the tissue source nor the Fn isoform. Further, we have estimated that cartilage, if moderately degraded, could contribute Fn-fs to a level found in human OA synovial fluids, based on our measurements of Fn content in both synovial fluids and cartilage


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tissue [18]. We have also demonstrated that cartilage damage itself can lead to generation of Fn-fs by demonstraing that IL-1 treated or MMP-3 treated cartilage causes release of enhanced levels of Fn-fs into the culture media [18]. Thus, Fn-f activity as measured by us is independent of the source of the Fn-f isoform. Further, the Fn-f we have focused our studies on is an amino-terminal 29-kDa Fn-f which we have identified in human OA cartilage [18] and which is shared by all Fn isoforms as is the 50-kDa gelatin-binding Fn-f, also studied by us. Our 140-kDa Fn-f likely differs from sequences found in other Fn isoforms since it contains the alternately spliced regions. While we have no proof, we think it highly likely that the 140-kDa Fn-f we study, as isolated from plasma Fn, is as active as its counterpart from cartilage or synovial fluid. A very important question is whether or not Fn-fs could initiate early events in OA or rather arrive on the scene later and amplify the catabolic insult. While there is not yet a clear answer, it should be noted that in an experimental OA animal model, MMP-3 was upregulated in the synovium and subsequently upregulated in cartilage at later stages, contributing further to progression of cartilage lesions [32]. Thus, we could propose that in OA, synovial MMP-3 and other MMPs might be released which act on synovial fluid Fn to generate Fn-fs that might then penetrate cartilage tissue to initate cartilage damage. Thus, Fn-fs could theoretically be an early effector of cartilage damage.

Collagen Fragments Are Elevated in OA The major type of collagen in cartilage, type II collagen, is thought to be elevated in late stage OA [33] and this could lead to elevated type II collagen fragments. Detailed analysis shows that type II collagen synthesis is elevated in OA lesions while the content decreases [34]. Whether or not this correlates with increases in collagen fragments has not been shown. However, it is clear that Col-fs can be found in OA models. For example, Col-fs at a level of 6 µg/ml have been found in synovial fluid of rabbits treated surgically to induce OA [35]. Further, up to 20% of all collagen in human OA cartilage can be partially degraded; suggesting that up to 40 mg of Col-fs per gm may reside or be released from heavily damaged cartilage tissue [36]. While different types of Col-fs from type II collagen can be generated in cartilage degeneration, many observations show that telopeptides become enriched, namely the N and C-telopeptides, the peptides released by MMP-3 attack on the crosslinking regions of type II collagen. N-telopeptides have been shown to be markers of cancer while C-telopeptides are associated with both the prevalence and the progression of radiographic OA at the knee and hip [37]. Interestingly, the C-telopeptide is elevated in urine from OA patients [38], correlates with high cartilage turnover in OA patients [39], increases after joint injury [40] and is elevated in synovial fluid in the rabbit meniscectomy model of OA [41]. Further, C-telopeptides are associated with both the prevalence and the progression of radiographic OA at the knee and hip [42]. It is likely that other types of collagens might also contribute to enhanced levels of collagen fragments that may have chondrolytic activities, however, the major contribution would be expected to be from type II collagen. Thus, it is highly likely that Col-fs such as the N and C telo peptides become enhanced in cartilage degeneration. As will be discussed later, this pool would contribute markedly to cartilage degradation which leads to a question similar to that posed for Fn-fs: Do the Col-fs arise early in OA and help in the early initiating events or simply amplify ongoing processes or both?

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HA-f Are Elevated in OA It is well known that hyaluronan (HA) is also elevated in OA and inflammatory conditions [43]. For example, serum HA and synovium HA synthesis increase in the ACLT canine model [44] and cytokines such as IL-1α have been shown shown to increase HA production in bovine articular chondrocytes [45,46]. The increased production of HA and the inflammatory response should lead to HA fragmentation as shown [47]. Some of the fragmentation may be due to hyaluronidases or free radicals [4]. It has also been shown that CD44, an HA binding receptor, and MMPs can be induced by hyaluronidase treatment of articular chondrocytes [48]. Thus, there is a strong suggestion of enhanced HA fragments (HA-f) in cartilage degeneration and as will be discussed below these HA-f should also contribute to either early events in cartilage degradation or amplify ongoing processes.

Might Other ECM Fragments Be Elevated and Have Activities? During cartilage degeneration, it would be expected that fragments of other ECM macromolecules would also be elevated. It has been shown that a 16-residue synthetic link protein peptide, derived from the amino-terminus of link protein, a component of proteoglycan aggregates, stimulates proteoglycan synthesis in human articular cartilage [49–51]. The peptide was shown to decrease release of IL-1 and to enhance mRNA levels of aggrecan and link protein mRNA [51]. Since link protein can be cleaved near the amino-terminus by enzymes such as MMP-3, such amino-terminal peptides could enhance reparative processes during matrix damage. It should be noted that at low concentrations of Fn-fs, PG synthesis is also stimulated [52,53]. This invites the question or whether or not link peptides might be catabolic at high concentrations. Unfortunately, little is known of the mechanism of action of these LP-f or peptides. It would be interesting to speculate that they may physically perturb the matrix and enhance release of trapped growth factors or indirectly perturb growth factor receptors through ECM interactions.

Fn-f Were the First to Be Studied as Catabolic Mediators Based on the discussion above, it is clear that ECM fragments are elevated in OA and cartilage degeneration, but what are the consequences? Our work with Fn-fs suggests that these ECM fragments play crucial roles in cartilage metabolism. Our laboratory was the first to report that Fn-fs perturb normal signaling through Fn receptors or integrins in chondrocytes to upregulate catabolic processes as well as modulate anabolic processes. We demonstrated that these Fn-fs enhance cartilage damage in vitro [54], penetrate cartilage tissue and bind to the pericellular matrix [55], elevate MMP expression, temporarily suppress proteoglycan (PG) synthesis [56] and enhance rates of PG loss from cartilage tissue in explant cultures [52–54]. The Fn-fs cause the release of half of the total PG from cultured cartilage explants in 10% serum within a few days at concentrations of Fn-f at or below measured concentrations of Fn-fs in OA synovial fluid [55,57]. Interestingly, native Fn is inactive [54] leading to the now accepted notion that the activities of the Fn-fs are liberated from Fn upon proteolysis. The damaging activi-


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Figure 1. Correlation of Cartilage PG Content (A) With MMP-3 Release (B) and Cytokine/Factor Release (C) With High Fn-f. Cartilage explants were treated with 100 nM 29-kDa Fn-f continuously with media changes every other day. In A, cartilage PG content was measured in explants; in B, MMP-3 (stromelysin) was measured by ELISA and in C, cytokines and growth factors in the media were measured by ELISA.

ties of the Fn-fs require mRNA as well as de novo protein synthesis and metabolic energy, suggesting that the Fn-fs are not acting as proteinases [54]. Not only do the Fn-fs enhance proteinase activity, but they also temporarily suppress PG and general protein synthesis, by up to 50% [52,57]. However, the effects of the Fn-fs are not totally reversible. Upon removal of the Fn-fs from cartilage cultures, the PG synthesis rates increase to values up to 140% of control values, however the PG content does not return to normal levels [52,53]. Thus, the in vitro model is an example of “attempted but failing repair” of cartilage. This linkage of damage to repair will be discussed in more detail later. We have studied the effects of Fn-fs on cartilage in both serum-free and serum conditions. Serum-free conditions were initially used to determine rates constants of PG degradation and release into the media [54–56] since these conditions allowed a greater proteolytic response. However, later studies required use of longer term cultures and demonstration of activity in more physiologic conditions, that of serum cultures. The serum slows the rate of PG degradation by several-fold and allows an anabolic response of the cartilage to the damage and provides information on steady state metabolism of PG, rather than simply kinetics of degradation. With these conditions we discovered a very interesting dose response effect of the Fn-fs. A 0.1–1 μM concentration causes a 50% decrease in PG content (Fig. 1A) and marked upregulation of MMP-3 (Fig. 1B) with maximal effects by day 7. These events correlate with enhanced release of IGF-I, TGF-β, TNF-α, IL-1β, IL-1α and IL-6 (Fig. 1C) [additional data in refs 58,59]. However, the lowest concentration of 1 nM Fn-f immediately enhances the PG content and PG synthesis rates (Fig. 2). Further, an intermediate 10 nM concentration shows a lag period for PG depletion (Fig. 2A) and both 10 and 100 nM concentrations, although first suppressing PG synthesis, allow a slow return to normal levels and eventually supernormal levels (Fig. 2C) [52,53].

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Figure 2. Biphasic Effects of Fn-f on PG Content (A) and PG Synthesis (B). Cartilage explants were treated with 1, 10 or 100 nM 29-kDa Fn-f continuously with media changes every other day. At intervals, cartilage was subjected to papain digests and measurement of PG content by DMB assay (left). Similar cultures were subjected to 35S labeling to determine rates of sulfated proteoglycan synthesis (right).

We have proposed that the curves shown in Fig. 1 are due to the summation of both the catabolic and anabolic effects of the Fn-fs. Enhanced anabolic effects occur with lower concentrations of Fn-fs. As the concentration of Fn-f is increased, the catabolic effects predominate. However, after the catabolic phase subsides, the anabolic once again predominates, leading to the supernormal PG synthesis rates and PG contents. We have also suggested that this illustrates that the Fn-fs are true regulators of metabolism. Thus, the 1 nM concentration may reflect repair of mildly damaged cartilage and the higher concentrations may reflect the kinetics of and the linkage of severe damage and attempted repair. These studies also suggested to us the great value of examination of explant cultures over extended periods of weeks. Of course, we would not have discovered these anabolic effects in monolayer cultures or in short term cartilage cultures. In terms of proteinases responsible for the cartilage damage, we have found that several MMPs are upregulated at the protein level including, but likely not limited to, MMP-1, MMP-2, MMP-3 and MMP-9. MMP-3 is a major proteinase in the activity [60]; antibodies to MMP-3 slow damage to Fn-f treated bovine cartilage explants [56]. However, the Fn-fs also increase cleavage of the aggrecanase epitope [61,62]. We have also shown that MMP-3 can degrade intact Fn into small Fn-fs [18]. Thus, MMP-3 induced in OA may generate more Fn-fs, which in turn amplify MMP-3 expression in a positive feed-back loop. Others have also shown that Fn-fs elevate MMP-13 in human cartilage [63]. We have confirmed effects on MMP-13 protein in bovine explants and find that it is elevated in parallel with MMP-3 (unpublished). It is important to note that high concentrations of Fn-fs have many other activities that can be described as catabolic. We also found that Fn-f enhance levels of NO and upregulate iNOS [64] and upregulate activities and levels of IGF-I binding proteins [65]. Subsequently, others showed that Fn-fs upregulate NO production in RA cartilage through the CD44 receptor [66]. Others have shown that a Fn-f containing an alternately spliced domain enhances MMP gene expression [67]. Others have also shown that a gelatin-binding Fn-f induces type II collagen degradation by collagenase [68]. Also, a 45-kDa collagen-binding fragment was shown to upregulate MMP-13 and aggrecanase activity [69]. A 120-kDa Fn-f was shown to upregulate MMP-1, MMP-3 and uPA in fibrocartilaginous cells [70]. It has also been shown that Fn-f upregulate the


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Toll-like receptor 2 in human articular chondrocytes [71]. Fn-fs, as with IL-1, enhance expression of CD44 in bovine chondrocytes [72]. The above listing may not be totally comprehensive, but it does illustrate the broad effects of Fn-fs. Interestingly, Fn-fs also enhance catabolic effects on other types of joint relevant tissues. Fn-fs have also been shown to modulate expression of proteinases and inhibitors in human periodontal ligament cells [73] and to trigger anoikis of human primary ligament cells by suppressing p53 and c-myc [74]. There has been much interest in the role of Fn-f in spine degeneration. Oegema et al. showed elevated Fn-fs in degenerated human intervertebral disc [75]. Subsequently, others showed that Fn-fs stimulated disc degeneration [76,77]. More recently, we showed that Fn-f did not simply enhance degeneration as a generality. When different types of disc cells were examined there were differential effects from enhanced anabolism to enhanced catabolism [78]. The above catabolic activities of Fn-fs could easily be explained if the Fn-fs acted through catabolic cytokines since cytokines are involved in many of these procsses. We showed that Fn-fs enhance protein levels of IL-1α, IL-1β, IL-6 and TNF-α in human cartilage and proved their active role by demonstrating that neutralizing antibodies to these cytokines decrease Fn-f activities [58,59]. Later, it was shown by Northern blotting that Fn-fs upregulate IL-1 [67] and shown by microarray analysis that Fn-f upregulate IL-6 and IL-8 [79]. Others have shown that Fn-fs induce TNF-α in basophilic leukemia cells [80] or in cultured mesangial cells [81]. Thus, it may be a generality that Fn-fs can enhance cytokine pathways in many different types of cells. While we tested the roles of IL-1, IL-6 and TNF-α by use of blocking antibodies, many investigators have tested the role of cytokines in Fn-f pathways by use of IRAP to block IL-1 activities. IRAP decreases induction of MMP-3 and MMP-13 by a heparin-binding Fn-f [82] or partially blocks NO release from human chondrocytes treated with an N-terminal 29-kDa Fn-f [83] or blocks MMP-1 induced release by an extra domain A Fn peptide [67] or decreases MMP-3 upregulation by an RGDS peptide found in the Fn cellbinding domain [84] or blocks activation of MMP-13 in human chondrocytes by a 120-kD cell binding Fn-f [63]. However, there are reports that IRAP failed to block upregulation of MMP-3 and MMP-13 in chondrocytes by a 45-kDa gelatin binding Fn-f [69] or failed to block upregulation of MMP-13 by a 120-kDa Fn-f added to human chondrocytes [63]. These differences may reflect differences in potencies of IRAP, or different cell culture systems or different species of cartilage. It should be noted that in studies where IRAP is tested, the ability of IRAP to block exogenous IL-1 is often used as a control. Yet, this may not be a suitable control since it is likely much more difficult to block IL-1, which after being upregulated may be more highly concentrated around the cell, than exogenously added IL-1 which becomes diluted upon addition and thus easier to block. Further, it would not be expected that blocking of IL-1 would totally decrease MMP upregulation since TNF-α activity is also enhanced by Fn-fs as we have shown [58,59]. Our recent data with bovine cartilage are consistent with an MMP upregulation pattern in which early MMP enhancement is driven by MAP kinases and a later phase beyond 48 hours is augmented through cytokines (unpublished). We have found that IRAP does not totally block Fn-f upregulated MMPs after a 24 hour treatment but is more effective at 48 hours and beyond. We have also found that MAP kinase, PYK2, or PKC inhibitiors can decrease a major portion of the MMPs within the first 24 hours during a time in which we cannot detect significant levels of IL-1 or TNF-α. Thus, it is likely that there is an early cytokine independent pathway as well as a later cytokine driven pathway. Since the most relevant way of studying matrix damage is with

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explants in long term culture beyond a few days, it is likely that cytokines do play a major role in more physiologically relevant conditions beyond 24 hours. The generality of the activities of the Fn-fs has been supported in many ways. We have found that similar cathepsin D and thrombin generated Fn-fs from rabbit plasma Fn, bovine plasma Fn, rat plasma Fn or guinea pig plasma Fn (unpublished) are equally active on bovine cartilage (unpublished). Thus, as a generality, Fn-fs from one species can cause damage to cartilage of another species. Further, the types of proteinases used to generate the Fn-fs may not be important, since regardless of the specific bonds cleaved, similar domains are generated. We have shown that MMP-3 generated human plasma Fn-f mixtures are as active as cathepsin D and thrombin generated Fn-fs on a molar basis [18]. Further, Fn-fs made from Fn from bovine plasma, bovine synovial fluid or bovine cartilage are equally active [18]. We have also shown that removal of Fn-fs from OA synovial fluid decreases the cartilage damaging activity of the resultant fluids [18], demonstrating that OA derived Fn-fs are active. Because of the suitability of the Fn-f cartilage damage model as an OA model, we have tested various agents for their abilities to block the action of Fn-fs. One particular aspect of the model is that Fn-f damaged cartilage does not spontaneously restore PG after the Fn-fs are removed [52]. Thus, this model has given us the capability of testing agents that might be useful in cartilage repair. We have tested synthetic peptide analogs of the cell binding sequence found in Fn [85], anti-oxidants such as N-acetylcysteine, [86,87] and the growth factor, IGF-1, and found that all were partially to fully effective in not only decreasing Fn-f mediated cartilage damage but also promoting restoration of PG in pre-damaged cartilage [88]. In other studies, another growth factor, OP-1, [89] as well as high molecular weight HA [62,90], were tested and also found to both block damage and promote repair. Others have subsequently shown that OP-1 blocks Fn-f mediated MMP-13 upregulation in human cartilage [91]. Since these agents all have different modes of action and some are anabolic and some are anti-catabolic, our data suggest that either attenuation of the catabolic pathways or compensation for the catabolic stress can enhance repair. The data also suggest that agents that block damage also have potential in cartilage repair, perhaps by altering or compensating for pathways of cartilage destruction that continue in the absence of the damage mediator. The Fn-f model has also allowed us to compare the metabolism of human ankle and knee cartilages [92] to address the question of whether or not the lesser susceptibility of ankle joints vs knee joints to OA, which has been supported by numerous cadaveric, radiographic and clinical studies, might have a biochemical basis. We found that addition of Fn-fs to cultured human knee cartilage decreases the PG content comparable to that of bovine cartilage. However, ankle cartilage in most cases is not affected. While MMPs are upregulated in both types of tissue, the knee tissue is more sensitive to Fn-f mediated PG synthesis suppression, suggesting that the difference in cartilage damage between the tissues is due more to effects on matrix synthesis than to upregulation of MMPs. The physiologic significance of the chondrolytic properties of the Fn-fs was supported by our demonstration that that Fn-fs cause a loss of up to 50% of the articular cartilage PG when injected into rabbit knee joints [93]. More recent work shows that this degree of damage can occur within 2 days of injection, that MMP-3 levels plateau within this time and that the Fn-fs temporarily suppress PG synthesis and expose the NITEGE epitope of aggrecan, just as in the in vitro model [94]. PG synthesis, although initially suppressed, slowly increases to supernormal levels, also suggesting a supernormal anabolic response, just as in the in vitro model. This anabolic response leads to


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restoration of PG within 2 weeks in adolescent rabbits, while in skeletally mature rabbits, PG restoration requires months [96]. Thus, this model has potential in studies of aging. One of the most striking observations has been that the non-injected knee cartilage shows evidence of a systemic effect. When a low dose of Fn-fs was injected into the right knee, the noninjected knee showed enhanced PG synthesis rates and PG content. If a higher concentration were injected, the non injected knee showed a rapid decrease in PG synthesis rates and a decline in PG content to the same extent as the injected knee. Much more work will be required to ascertain the mechanism for this systemic effect.

Col-fs Also Have Catabolic Activities Col-fs have also been shown to have potent cartilage damaging activity. These activities should be considered in light of other reports of activities of collagen peptides. Various types of collagen peptides when added to other types of cells have several activities related to inflammation or tissue damage. For example, CNBr peptides of type II collagen stimulate IL-1β release from human monocytes [97] or stimulate collagenase production by synovial cells [98] or modulate processing of MMP-8 by gelatinase [99] or alter types II and IX collagen turnover in bovine articular explants [100]. Perhaps one of the best known Col-f systems is that of endostatin, the C terminal fragment of collagen XVIII, an inhibitor of angiogenesis, that interacts with the Fn receptor, alpha5beta1, and induces clustering and disassembles actin stress fibers and FCs through activation of c-src [101]. Thus, as with Fn-fs, Col-fs likely enhance catabolic states in various types of tissue. The first reports of activities of Col-fs toward cartilage tissue showed that bacterial collagenase digests of type II collagen from bovine articular cartilage generated fragments of 100). PG content was measured every 7 days. In B, cartilage was cultured with 100 nM Fn-f for 4 days prior to start of experiment (4d pre) or with Fn-f at days 7–28 Fn-f (7–28) or with Fn-f 4 days prior to start of experiment and then with 100 nM Fn-f for days 7–28 (4d pre).

pared the effects on PG content to those of 1 nM treatment or 100 nM treatment without pretreaments. Note in the figure that this pretreatment made cartilage more refractory to the catabolic insult as observed with a nearly two week delay in major damage. In the second approach, we tested effects of a high concentration but short pretreatment of 4 days. This short treatment was too short to maximally upregulate MMPs but was sufficient to trigger an anabolic effect. As seen in Fig. 3B, this pretreatment markedly slowed PG depletion as compared with the no pretreatment control. This ability of Fn-f to make cartilage refractory to further damage was also studied by determing the minimal length of time required for Fn-f to cause maximal damage. We found that a single 7 day exposure caused as much damage as a continual exposure for up to 28 days and that a less than 3 day initiated only the anabolic response [53]. We then compared the effects of multiple treatments that summed up to a 7 day exposure and found that even if a second or third exposure added up to 7 days, maximal damage could not be inflicted. Thus, damage required a continual exposure to Fn-f. Otherwise, an anabolic response could occur and make tissue refractory. We further proposed that light to moderate proteolysis of matrix PG, too light to cause a decrease in steady state PG levels, causes the release of IGF-1 and TGF-β. TGF-β is known to be trapped in the matrix [120] and IGF-1 has been shown to be trapped in cartilage tissue [121,122]. Since proteinases have been shown to release TGF-β in other cells [123] and proteinases, including MMP-3, can degrade IGF-1 binding proteins [124] and release IGF-1, the Fn-fs at low or high concentrations may help to mobilize growth factors. To support this proposal, we demonstrated that addition of MMP-3 to cultured cartilage results in the initial suppression of PG synthesis and elevated IGF-1 in the media and after a few days, enhanced PG synthesis, just as do the Fn-fs [65]. Thus, proteases induced during the damage phase might account for a linkage between cartilage damage and attempted repair responses. While we have no evidence of a role for protease induced activation of IGF-1 in the Fn-f system, we do know that one of the IGF-1 binding proteins, BP4, decreases in the presence of the

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Fn-fs [65]. Other work with the Fn-f model has suggested an additional feature that may account for the supernormal anabolic effect. We have found that Fn-fs induce expression of IGF-I binding proteins (BPs) [65]. The induced BPs released from chondrocytes likely bind IGF-I and concentrate it around the cells. Thus, during Fn-f induced proteolysis, the active IGF-I concentration around the cell may be greatly elevated, leading to enhanced effects on PG synthesis. Thus, these studies suggest that Fn-fs might have some role in basal metabolism or light tissue damage where they might amplify anabolic processes. Initially they might also make the tissue more refractory to further damage. However, if the damage progresses to severe damage, catabolic pathways may be additionally activated to assist in tissue clearance. At present, we do not know if Col-fs and HA-fs cause the same effects.

Is There a Single Global Mechanism? Our current studies are focusing on the signaling mechanisms of ECM fragments beginning with receptor and ECM and continuing into activation of intracellular kinases. Our proposed model for our Fn-fs that we think might apply to Col-fs and HA-fs as well is based on an early observation that in fibroblasts, Fn enhances clustering of integrins and activates tyrosine kinases [125]. Thus, Fn-fs might block or disrupt integrin clustering and either block the Fn pathway kinases or activate different kinases. Our unpublished work suggests that Fn-fs do disrupt alpha5 integrin clusters and decreases their areas on the cell surface and that this is associated with enhanced internalization of the receptor subunits and a movement of integrin interacting components from cell membranes into the cytosolic compartments (unpublished). This is consistent with the notion that HA-f may detether the CD44 receptor and also enhance internalization [106]. Our preliminary data also suggest that Col-fs have similar effects and decrease receptor clusters consisting of both Fn and collagen binding integrins. Consistent with this observation, type I Col-fs have been shown to promote disassembly of focal adhesion in smooth muscle cells [126]; thus integrin disruption by type II Col-fs is certainly a possibility. Thus, there may be shared ability among the ECM fragments to disrupt the native receptor clusters at the cell surface, enhance internalization and disrupt the normal interactions with cytosolic accessory or scaffolding proteins and this may initiate new signaling pathways. Thus, the matrix itself might be thought of as a negative regulator of these ECM fragments pathways. When the ECM is degraded, the activities of these fragment pathways may be liberated. We earlier implicated the alpha5 integrin in Fn-f activities by chemical crosslinking of Fn-f to chondrocytes and by antisense oligonucleotide inhibition of the alpha subunit [127,128]. This is the subunit that comprises, with the beta1 subunit, the classical Fn binding integrin. However, more recently we have also observed that Fn-fs can be chemically crosslinked not only to the alpha5 subunit, but also to other subunits, including the alpha1 and alpha2 that comprise collagen binding receptors and alpha3 and alphaV subunits that comprise Fn and thrombospondin binding receptors. Upon further analysis by dual label confocal microscopy we have confirmed that not only do Fn-fs bind in proximity to these integrins but so do Col-fs. This surprising observation prompted us to investigate whether the fragments not only cross interacted with diverse integrins but whether the integrins themselves were proximal to each other. Indeed, we have found by confocal analyis that the alpha 1, 2, 3 or V subunits appear to colocalize with the alpha5 subunit. We consider these observations very preliminary and are


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attempting to devise alternate methods to verify. These observations have prompted us to suggest a global mechanism in which Fn-fs and Col-fs bind to “integrisomes” or integrin clusters through either direct interaction or indirect interactions mediated through their native ligands or the ECM. These interactions would physically alter these “integrisomes” to initiate altered signaling. There is also another possible explanation. Because Fn-fs can bind to native Fn and certain Fn-fs can bind collagen and specific Col-fs might bind both native collagen and Fn, the ECM may establish a platform for both homotrophic and heterotrophic interactions that indirectly disrupt “integrisomes” and alter signaling. Consistent with this proposal, Tuckwell et al. have shown that denatured collagen can bind the alpha5 subunit by binding Fn which bridges to the alpha5beta1 receptor [129]. But what of CD44? Would it communicate within the “integrisomes”? Interestingly, it has been suggested that a C terminal Fn-f utilizes the CD44 receptor [66], so perhaps there is a linkage between all three major ECM fragment systems that occurs at the level of these receptor clusters. In terms of a global mechanism for signaling downstream of receptors, some of the first observations on effects of Fn-fs on signaling showed that Fn-fs activate MAP kinases. Gemba et al. reported that an amino-terminal Fn-f activated MAP kinases in human chondrocytes and that chemical inhibition of the three MAP kinases, ERK1/2, JNK and p38 inhibited Fn-f mediated NO production [83]. Others have reported that MAP kinases are activated by a central cell binding Fn-f and that inhibition of MAP kinase activities decreases MMP-13 upregulation [63]. Others have confirmed the role of MAP kinases in Fn-f upregulation of collagenase [130]. The role of Nf-kB has also been shown in Fn-f upregulation of cytokines and chemokines [79] as well as a role of PYK2 and PKC [131]. Our own unpublished work with bovine cartilage and bovine chondrocytes shows the involvement of MAP kinases, Nf-kB, PYK2 and PKC in MMP-3 and MMP-13 upregulation and cartilage damage (unpublished). However, while NF-kB is often thought to be downstream of MAP kinases in a linear pathway, our work suggests much more complexity. Tests of chemical inhibitors suggest that for MMP upregulation there are separate pathways for PYK2, MAP kinases and Nf-kB. Clearly, our work is showing that the kinase signaling pathways are complex, are not necessarily linear, and may have several arms and that the common points or origin of these arms may be far upstream. Very little has been published on signaling pathways for Col-fs. Our preliminary data suggest that Col-fs are slower at enhancing MMP levels than Fn-fs. This difference is also reflected in a smaller degree of activation of Nf-kB and p-38. Activation of JNK/SAPK is almost undetectable. Further, Col-fs do not appear to greatly upregulate TNF-α or IL-1β even after 72 hours of treatment, yet Col-fs can significantly deplete matrix PG in explants in 7 day cultures. Thus, by comparisons between the Fn-f and Col-f systems, we might conclude that a noncytokine pathway is sufficient for significant cartilage damage but that cytokines do enhance damaging activity. Since these experiments are complex, much more work is needed to clarify the role of cytokines in Col-f signaling. In terms of HA-fs, the most recent observations on signaling activities of HA-fs, suggest that they utilize Nfkb and p38 MAP kinase [114] and upregulate iNOS [115] which would generate NO as a possible mediator. Thus, major points of similarity between the three systems are p38 MAP kinase and Nf-kB. In summary of a global mechanism, based on published data, it appears that all three ECM fragments utilize receptors to which their native precursors bind and that their precursors are largely inactive. Our preliminary data suggest that Fn-fs and Col-fs may utilize similar receptors and that these receptors are proximal to each other in the

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membrane. Our preliminary data also suggest that Fn-fs and Col-fs disrupt integrin clusters and enhance internalization. Observations with HA-fs also suggest a dedethering effect with enhanced internalization. We would propose that the enhanced internalization of receptors disrupts interactions with cytosolic scaffolding proteins and this exposes or creates new signaling pathways. Published data suggest that Fn-fs and HA-f utilize Nf-kb and MAP kinases for various catabolic activities while our preliminary data suggest the same for Col-fs. Our preliminary data with Fn-fs suggest that these pathways are complex and comparison of Fn-fs and Col-fs adds an additional level of complexity in that cytokines may not be necessary for the early catabolic response of Fn-fs and Col-fs but may play an important role over the longer term. These similarities suggest to us that there may be a global mechanism which if attenuation is prudent, is best done upstream closer to receptor.

Summary and Prospectus This review has attempted to describe and compare three ECM fragment systems of cartilage regulation. Their very existence points to the important role of the ECM itself in regulating normal cartilage metabolism and to the concept that regulation by the ECM does not occur through just one component but different components that can interact with each other. Since ECM fragment pathways are derived from the most abundant protein, type II collagen, as well as one of the least abundant proteins, Fn, this suggests that there is a potential for other pathways derived from other relatively minor components. Thus, it is likely that other ECM fragment systems will be discovered. Further knowledge of their mechanisms is important for determining whether or not there is a single global pathway with a single upstream point of intervention. However, further knowledge of the role these systems play in regulation is crucial to our understanding of whether intervention in diseases such as osteoarthritis would be prudent. It is likely that these pathways should be attenuated in the disease process, but complete attenutation, if possible, may not be prudent because of the simultaneous role these pathways likely play in cartilage repair.

Acknowledgments The authors thank past support from the Arthritis Foundation of the Greater Chicago Chapter and the North Central Chapter, the National Institute of Arthritis and Musculoskeletal Diseases Specialized Center of Research (SCOR) Grant, the Dr. Ralph and Marian C. Falk Medical Trust Fund, the North Dakota EPSCoR, and the Eugene W. Cornatzer Chair Trust.

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VI Pathophysiological Relevance of PPAR to Osteoarthritis: From the Control of Inflammation to Cartilage Protection? Arnaud BIANCHI, Mélanie KIRCHMEYER, Marie-Madeleine GALTEAU and Jean-Yves JOUZEAU * Laboratoire de Physiopathologie et Pharmacologie Articulaires (LPPA), UMR 7561 CNRS-UHP Nancy 1, Avenue de la forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France

Abstract. Peroxisome proliferators activated receptors (PPAR) are ligandinducible nuclear transacting factors comprising 3 subtypes, PPARα, PPARβ/δ and PPARγ, which play a key role in lipids and glucose homeostasis. All PPAR subtypes have been identified in joint cells and their activation resulted in a transcriptional repression of pro-inflammatory cytokines (IL-1, TNFα), early inflammatory genes (NOS2, COX-2, mPGES-1) or matrix metalloproteases (MMP-1, MMP-13), at least for the γ subtype. These anti-inflammatory and anti-catabolic properties were confirmed in animal models of joint diseases although much less data are available for experimental osteoarthritis (OA) than for polyarthritis. PPAR agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) production by cytokines-stimulated cells in a subtype-dependent manner. So, PPAR agonists are able to reduce joint inflammation and to prevent cartilage destruction, although many effects were obtained at a higher concentration than required to restore insulin sensitivity or to lower circulating lipids levels. Besides, PPAR agonists were able to modulate the differentiation and/or activity of bone cells, but data are lacking for their effect on OA-associated sclerosis of subchondral bone. Although promising, the therapeutic insight of PPAR agonists in OA warrants additional proofs that could be obtained indirectly from the follow-up of diabetic and/or hyperlipidemic patients with OA treated daily with glitazones or fibrates. Keywords. PPAR subtypes, gene transcription, cytokines, eicosanoids, metalloproteases, animal models, osteoarthritis

1. Peroxisome Proliferators Activated Receptors (PPAR) 1.1. Structure – Overall Expression and Functions Peroxisome proliferators activated receptors, subtypes PPARα (N1RC1), PPARβ/δ (FAAR, NUC1 or NR1C2) and PPARγ (NR1C3), are ligand-inducible nuclear transact* Corresponding Author: Pr. Jean-Yves Jouzeau, LPPA, Phone: +33(3)83683950, Fax: +33(3)83683959, E-mail: [email protected].

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A. Bianchi et al. / Pathophysiological Relevance of PPAR to Osteoarthritis

Figure 1. Schematic representation of the functional domains of PPAR. PPAR are composed of four distinct functional regions: 1) the A/B domain located at the N-terminus, containing a ligand-independent activation function (AF-1) responsible for PPAR phosphorylation; 2) the C domain or DNA binding domain (DBD) promoting the binding of PPAR to the peroxisome proliferator response element (PPRE, a classical direct repeat [DR] separated by one or two nucleotides) in the promoter region of target genes; 3) the D domain is a hinge region responsible for the docking of cofactors, 4) the E/F domain or ligand-binding domain (LBD) located at the C-terminus, responsible for ligand specificity and containing a ligand-dependent activation function (AF-2) which promotes the recruitment of cofactors to assist the gene transcription process and is necessary for the heterodimerization with RXR.

ing factors. They belong to the steroid receptors family including receptors for retinoids, thyroid hormones or corticosteroids [1]. Their overall protein structure is composed by six domains, termed generally from A to F, having key specific functions such as ligand binding (LBD), interaction with DNA (DBD) or control of transactivation (AF domains) (Fig. 1). PPARα is expressed exquisitely in tissues contributing actively to fatty acids catabolism (mainly in liver, and less markedly in brown fat, kidney, heart or skeletal muscle), where it regulates the expression of genes involved in fatty acid uptake and ωor β-oxidation [2]. PPARα is also expressed in endothelial and vascular smooth muscle cells, as well as macrophages/foam cells, and contributes to the control of inflammation, thereby opening insight to the treatment of atherosclerosis [3,4]. PPARβ/δ is the less well-characterized PPAR subtype despite its ubiquitous expression. It takes place in lipids metabolism by favouring the reverse transport of cholesterol and oxidation of fatty acids [5], plays a major metabolic role in muscle and adipose tissue [6] and has been linked to profound anti-obesity and anti-diabetic actions in animal models [7]. Activation of PPARβ/δ has also been linked to cell proliferation or apoptosis depending on the cell type and seems to play a key role in wound healing [8]. PPARγ is highly expressed in white and brown adipose tissue and less intensively in cardiac and skeletal muscle [2]. It plays a pivotal role in adipocytes differentiation and lipids storage [9] and its activation provides insulin sensitizing properties that have entered the clinics [4,10]. PPARγ is also thought to be a negative regulator of inflammation [11] since its activation is able to suppress pro-inflammatory cytokines production [12,13] while being anti-inflammatory in several animal models [14,15]. Of particular note, many metabolic effects of PPARγ and β/δ agonists were supported by their ability to correct the circulating imbalance between leptin and adiponectin [4,10], two adipokines also found at abnormal levels in the synovial fluid of OA patients [16].


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1.2. Mechanism of Action Activation of PPARs leads to the formation of heterodimers with retinoid-X receptors [17] (RXRs). These PPAR-RXR dimers bind to DNA-specific sequences, called peroxisome proliferators response elements (PPRE), to stimulate or dampen the transcription of target genes. The classical PPRE consensus sequence is a direct repeat (AGGTCA) separated by one or two nucleotides (DR1/2) (Fig. 1), which is unfortunately not sufficient to predict the responsiveness to PPAR agonists since a perfect consensus was reported to be possibly non functional [18] whereas a highly degenerated sequence was, on the contrary, demonstrated to be fully responsive [19]. In addition to their differential tissue distribution, PPAR subtypes vary in their selectivity and sensitivity towards agonists and recruit distinct co-activator proteins follow-on in the regulation of different sets of genes [1]. There are several mechanisms by which PPAR activation can regulate the transcriptional machinery (summarized in Fig. 2). Briefly, besides the classical PPRE-mediated effects and possible interference with histone acetylation [20] (Fig. 3), PPAR can develop protein-protein interaction with several transcriptions factors as NF-κB [21–23], AP-1 [24], NF-AT [25], STAT [26] or egr-1 [27], thereby reducing the response to inflammatory cytokines or growth factors. PPAR can also modulate the response to transcription factors by competing for the recruitment of several co-activators [20] or by interfering directly with their DNA binding site, as demonstrated recently for AP-1 in the promoter region of MMP-1 gene [28]. Of particular note, additional PPAR-independent mechanisms have been reported for PPAR agonists, especially the inhibition of NF-κB pathway by the natural PPARγ ligand 15-Δ12,14-Prostaglandin J2 (15d-PGJ2) [29,30], most of which being attributable to the high chemical reactivity of its cyclopentenone ring with substances containing nucleophilic groups, such as cysteinyl thiol group of proteins [31]. 1.3. PPAR Agonists PPAR are considered as lipid sensors that can be activated either by endogenous compounds, generally natural fatty acids and their metabolites (low-binding affinity ligands), or by synthetic agonists (high-binding affinity ligands), including the antilipidemic fibrates [32] and the anti-diabetic thiazolidinediones [33] (see Table 1). 1.3.1. Endogenous Compounds Most endogenous PPAR agonists are eicosanoids derived from arachidonic acid biotransformation by the lipoxygenases or cyclooxygenases pathways. Thus, the 5-lipoxygenase metabolite leukotriene B4 (LTB4) [34] is a potent agonist of PPARα although 8S-HETE (8S-hydroxyeicosatetraenoic acid) has the highest affinity for this subtype [32]. Furthermore, the cyclooxygenases metabolites PGA1 [35] and PGI2 [36] are preferential ligands of PPARβ/δ. Finally, by-products from the 15-lipoxygenase pathway, 9-HODE (9-hydroxy-octadecadienoic acid) and 13-HODE, and the cyclooxygenase-derived dehydration product of prostaglandin D2, 15d-PGJ2, are selective agonists of PPARγ [37,38]. Interestingly, 15d-PGJ2 was demonstrated to be produced in the late resolution phase of experimental acute inflammation and is postulated to be a negative regulator of inflammation [39]. Plenty of fatty acids metabolites,

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Figure 2. Schematic representation of the transcriptional machinery control by activation of PPAR. A) After ligand binding (variable translocation depending on the subtype), the PPAR-RXR heterodimer binds to a peroxisome proliferator response element (PPRE) located in the promoter region to control (generally activate) the transcription of a target gene; B) After ligand binding, the PPAR-RXR heterodimer competes with a transcription factor (TF) for the recruitment of common co-activators, therefore resulting in the suppression of TF-dependent transcriptional activity; C) After ligand binding, the PPAR-RXR heterodimer squelches a transcription factor (TF) by direct protein-protein interaction, therefore resulting in the suppression of TF-dependent transcriptional activity by reduction of its translocation; D) After ligand binding, the PPAR-RXR heterodimer binds to a peroxisome proliferator response element overlapping (PPRE composite) the binding site of a transcription factor (TF) in the promoter region of a target gene, therefore suppressing TF-dependent transcriptional activity by competitive DNA binding.


9-cis retinoic acid

PPAR agonists

PPAR

81

RXR

PPAR

N-CoR SMRT

RXR

N-CoR SMRT

PP

RE

R RX

Gene Transcription

AR PP

CBP/p300 > SRC-1

Gene Repression

Figure 3. General model for transcriptional activation by PPAR. Binding of PPAR agonists and 9-cis retinoic acid to a PPAR-RXR/corepressor complex, which is not bound to DNA, results in the dissociation of the corepressors (Nuclear receptor CoRepressor [N-CoR], Silencing Mediator for Retinoid and Thyroid hormone receptor [SMRT]) and activation of the PPAR-RXR heterodimer. The activated PPAR-RXR heterodimer binds to a peroxisome proliferator response element (PPRE) located in the promoter region of a target gene and recruits co-activators (preferentially CAMP response element Binding Protein [CBP] and the related protein p300 [CBP/p300], or Steroid Receptor Coactivator 1 [SRC-1]) which have intrinsic histone acetyltransferase (HAT) activity. Finally, acetylation of core histones removes the electrostatic attraction between negatively charged DNA and positively charged lysine, allowing the loosening of the nucleosomal structure with a subsequent chromatin remodelling. The subsequent recruitment of large protein complexes, including RNA polymerase II, leads to initiation of gene transcription. In some cases, as the control of COX-2 gene expression in IL-1-stimulated synovial fibroblasts, the recruitment of CBP/p300 can be competitive with other transcription factors, resulting in a decrease in HAT activity and secondary reduction of gene transcription.

including those originating from transcellular metabolism, can activate PPAR with a variable subtype selectivity [40]. However, the theory of an endogenous control of inflammation by PPAR activation suffers from actual limitations. Firstly, most endogenous compounds have a low binding affinity to PPAR (Kd generally over the micromolar range) and, as a consequence, high concentrations of ligands will be necessary to activate the system. Secondly, data are lacking to demonstrate that these metabolites can be produced endogenously in sufficient amounts to activate PPAR, especially for 15d-PGJ2 [41], even if intracellular concentration could overcome plasma levels for

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Table 1. PPAR agonists used in in vitro studies with joint cells PPAR α

PPAR β/δ

PPAR γ

Synthetic high-binding affinity ligands Clofibrate (clofibric acid) Fenofibrate (fenofibric acid) GW9578

+# +# ++ #

– – –

± ± –

Pyrixinic acid (Wy14643)

++ #

–

±

L165041 GW501516

– –

++ # ++ #

± –

Ciglitazone

–

–

+#

GI262570 Pioglitazone

– –

– –

++ # +#

Rosiglitazone

–

–

++ #

Troglitazone

–

–

+#

Compounds

Cell type [References]

C [47,52] C [52] Ocl (differentiation/activity) [59] C [47,48,52] SF [27,60] Obl (maturation) [61] Ocl (differentiation/activity) [59] SF [50] C [62,63] SF [60] Ocl (differentiation/activity) [59,64] Obl (maturation) [61] C [65] C [63] C [28,48,52,66–68] SF [20,50,55,57] Obl (differentiation) [69] C [47,66,68] SF [20,27,57,70] Obl (maturation) [61]

Natural low-binding affinity ligands 15-deoxy-Δ

12,14

-Prostaglandin J2

Prostacyclin

#

–

–

+

–

+#

–

C [47, 48, 55, 62, 63, 65–67] SF [27, 50, 55, 60, 70] Ocl (differentiation) [64] Obl (differentiation) [69,71] Obl [71]

++: very easy binding and/or strong activation in transactivation assays; +: easy binding and/or activation in transactivation assays; ±: weak binding and or low activation in transactivation assays; –: no binding detected and/or no activation in transactivation assays; #: this compound has been determined to have selectivity for this PPAR isoform; C: chondrocytes; SF: synovial fibroblasts; Obl: osteoblasts; Ocl: osteoclasts

some metabolites. Thirdly, most fatty acids metabolites lack any marked selectivity for a given PPAR subtype and the endogenous control of inflammation will depend on whether they are produced concomitantly or sequentially and/or can compete for PPAR binding. Besides these endogenous compounds, dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) could have some therapeutical relevance since they are natural ligands for PPARα and PPARγ [32], but can also modify the endogenous metabolites produced from membrane phospholipids [42]. 1.3.2. Synthetic Ligands Fibrates, as clofibrate, fenofibrate or Wy14643, are agonists of PPARα which were developed as hypolipidemic agents through optimization of their lipid-lowering activity in rodents before the discovery of PPAR [32]. Whereas the active metabolites of clofibrate and fenofibrate are dual activators of PPARα and γ, with an approximately 10-fold selectivity for PPARα, Wy14643 and GW9578 are 50 to 500-fold selective for PPARα [43]. GW501516 is a newly synthesized agonist [44] which is > 1000-fold selective for PPARβ/δ over the other subtypes [45], whereas L165041 is only 10-fold


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more selective for PPARα over PPARγ [43,45]. Agonists of PPARγ include antidiabetic molecules of the thiazolidinediones or “glitazones” family which have been developed initially through empirical screening in rodent models of insulin resistance. Glitazones have an increased selectivity for PPARγ from troglitazone and pioglitazone to rosiglitazone [43,45,46], although non thiazolidinediones derivatives, such as GI262570, have a > 1000-fold selectivity for PPARγ over the PPARα and β/δ subtypes [43]. As for any compound with pharmacological activity, the in vitro concentrations used must be viewed critically to interpret the efficacy of PPAR agonists on joint cells. In several studies, the PPARα agonist Wy14643 was tested above 50 µM [27,47,48] and the PPARγ agonists troglitazone [27,47] or rosiglitazone [47,49,50] above 10 µM, whereas the PPARβ/δ agonist GW501516 was active from 0.1 nM [50] to 100 nM. Most of these concentrations are high compared to the respective binding affinity or activity of PPAR agonists (generally < 1 µM for Wy14643 on PPARα < 10 nM for GW501516 on PPARβ/δ and < 100 nM for rosiglitazone on PPARγ) in cell-based transactivation assays [51] or reporter cell lines expressing human PPAR subtypes [45]. In contrast, PPAR-dependent activities were reported at lower concentrations, namely 1 µM of rosiglitazone [28] or Wy14643 [52] in rabbit chondrocytes monolayers although the selectivity of high concentrations of PPAR agonists was confirmed with a panel of subtype-selective target genes in rat chondrocytes embedded in alginate beads [53]. This discrepancy could be explained, at least in part, by species differences in the response of PPAR to a given agonist [32] and by differences in the methodology (gene reporter technology) or experimental conditions (pericellular environment [54]) used. However, one can also underline that most anti-inflammatory effects were observed only with a high concentrations of PPAR agonists whereas the stimulation of IL-1Ra production occurred from a low agonist’s concentration. This suggests that the sensitivity of genes to a given PPAR agonist could depend on the location of any PPRE in the transcriptional machinery of their promoter or, alternatively, that the anti-inflammatory effects may be rather supported by the titration of transcription factors which requires a higher agonist concentration and/or additional mechanisms. Whatever the PPAR agonist, the time at which it is added relatively to the inflammatory stimulus is critical to interpret its potency in cell culture systems. As PPAR agonists are expected to act as transcriptional regulators, it is not surprising that numerous reports on joint cells were obtained with a pre-treatment time ranging from several minutes [27,55,56] to few hours [57]. However, co-stimulation was also reported to be efficient against cytokines [28,48,50,52,55], whereas the inhibitory effect of 15d-PGJ2 on IL-1-induced MMP-1 expression decreased gradually with time after addition of the stimulus [55]. In some experiments, PPARγ agonists provided an identical inhibition of IL-1-induced responses in pre-treatment or co-incubation [48], but 15d-PGJ2 was reported to be less inhibitory on TGF-β-induced activation of COL1A2 promoter in co-stimulatory conditions [58]. Taken together, these data confirm that the transcriptional activity of PPAR agonists takes advantage from any pre-incubation but suggest that their potency remains obvious when they are added at the same time as cytokine challenge.

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1.4. Expression in Joint Tissues In the last ten years, PPARα, PPARβ/δ and PPARγ were shown to be expressed constitutively in synovial fibroblasts [50,57] as well as chondrocytes from several species [28,47,55,68]. In most cases, activation of PPAR resulted in inhibition of cytokineinduced expression of pro-inflammatory genes or matrix metalloproteases (see below). Nonetheless, contradictory data were reported on the ability of PPARγ agonists to induce chondrocytes apoptosis [62,63,72]. PPAR subtypes were also reported in bone cells where activation of the γ subtype was linked to inhibition of both osteoclastogenesis [64] and differentiation of progenitors into osteoblasts [73], although subtypespecific PPAR agonists had a variable effect on bone resorption [59]. Besides, messenger RNAs for PPARγ were detected in rat articular fat-pad [74] which is a potent source of cytokines [75] and adipokines [16] production within the joints. In acute or chronic inflammatory conditions, activation of PPAR took also advantage from the expression of its three subtypes in monocytes and macrophages [12,23,76] as well as B and T lymphocytes [77,78]. Taken together, these data demonstrate that PPAR subtypes are expressed both in resident and infiltrating joint cells, suggesting that they may have a pleitropic anti-inflammatory effect although in a cell-specific manner. It is worth noting that the expression pattern of PPAR subtypes could be modified in inflammatory conditions since PPARγ was reported to decrease in joint cells in response to bacterial endotoxins [57] or IL-1 [50]. Such inflammation-induced pattern is thought to contribute, at least in part, to the PPARβ/δ-dependent effect of rosiglitazone in IL-1-stimulated chondrocytes [50]. Recently, it was also reported that the mRNA levels of all PPAR subtypes decreased in chondrocytes stimulated with TGF-β [53], further underlining that changes in PPAR levels could be key regulators of the pharmacological responses to their agonists [79].

2. Modulation of Key Inflammatory Genes by PPAR Agonists 2.1. Cytokines In many cell types, the PPARγ agonists 15d-PGJ2 and thiazolidinediones were shown to inhibit the transcriptional induction of genes playing a pivotal role in joint pathophysiology as TNFα [12] or IL-1 [13]. Thus, PPARγ agonists were able to repress LPS-induced TNFα expression [57] and IL-1-induced IL-1β production [50] in synovial fibroblasts. These data were consistent with the ability of 15d-PGJ 2 to suppress pro-inflammatory cytokines production in THP-1 cells stimulated with PMA [80] and synovial fibroblasts from OA or RA patients [81]. A reduction of circulating levels of TNFα, IL-1β and IL-6 was also reported in arthritic mice treated systemically with rosiglitazone [82]. This suppressive effect of PPARγ agonists on cytokines was accompanied by an inhibition of NF-κB pathway in synovial fibroblasts [57,81] and chondrocytes [68] or by a reduced expression of phosphorylated I-κB (pI-κB) in inflamed joint tissues [15]. More recently, the PPARα agonist fenofibrate was also demonstrated to reduce the degradation of I-κB in IL-1-stimulated synovial fibroblasts [83], further suggesting that the modulation of pro-inflammatory cytokines by PPAR agonists may occur, at least in part, by inhibition of the NF-κB pathway.


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Besides, PPAR agonists were reported recently to stimulate IL-1 Ra production in synovial fibroblasts [50], chondrocytes [52] and THP-1 cells [80]. Although there was some discrepancy, due to differences in cell types or experimental conditions [50,80], these data demonstrated that PPARγ agonists were able to correct the imbalance between IL-1β and IL-1Ra towards a less pathological state. Furthermore, the stimulating effect of PPAR agonists on IL-1Ra production was supported by activation of either the β/δ [50] or the α [52] subtype. Finally, the stimulation of IL-1Ra was thought to contribute to the inhibitory effect of several PPARα agonists on IL-1-induced MMPs expression in chondrocytes, although the exact mechanism remains to be elucidated [52]. 2.2. Inducible Arachidonic Acid Cascade Prostaglandins (PG), mainly PGE2, are well known arachidonic acid-derived lipid mediators that are produced in excessive amounts within inflammatory joints [84]. The effects of PGE2 on joint tissues vary with the differentiation status of chondrocytes, but it is generally accepted that PGE2 can contribute to the formation of an altered cartilage matrix [85–88], the maintenance of synovitis [84,89,90] and to an accelerated bone turnover [91,92]. Synthesis of PGE2 is a multi-step process involving a preferential enzymatic coupling between constitutive and inducible isoforms of phospholipases A2 (PLA2), cyclooxygenases (COX) and terminal PG synthases [93]. Besides COX-2, membrane Prostaglandin E synthase-1 (mPGES-1) was shown to play a key limiting role in the stimulated synthesis of PGE2 in synovial fibroblasts [94] and chondrocytes [95]. PPARγ agonists were reported to stimulate the basal expression of COX-2, which has a PPRE consensus site in its promoter [96], but this did not result necessarily in an enhanced production of PGE2 [67]. In contrast, PPARγ agonists were able to suppress cytokine-induced PGE2 production in human [68] and rat [67] chondrocytes, as well as in human synovial fibroblasts [27], with a greater inhibitory potency on mPGES-1 than on COX-2. Although there was some species differences in the contribution of PPARγ to the effect of 15d-PGJ2 [67], this agonist was therefore considered to act as a “dual agent” on arachidonic acid cascade in OA chondrocytes [55]. The transcriptional regulation of human COX-2 and mPGES-1 by pro-inflammatory cytokines involves overlapping, but distinct, signalling pathways [27,97] which were inhibited by interference of PPARγ agonists with histone acetylase p300 activity [20] or the transcription factor early growth response factor-1 (Egr-1) [27], respectively. A major role of NF-κB was suggested in the control of mPGES-1 transcriptional activity in rat chondrocytes [67], but an indirect regulation cannot be ruled out since NF-κB was also shown to regulate the early expression of Egr-1 [98]. These data underline that PPARγ agonists have a multi-step regulatory role on inducible arachidonic acid cascade and provide a rationale for possible negative feedback regulatory loops in joint cells [70] and during the resolution phase of acute inflammation [39]. It is important to recall that, beside their inhibitory property on prostaglandins synthesis, some non selective COX inhibitors were able to activate PPARα and γ in transactivation assays [99], whereas inhibition of PPARβ/δ by sulindac sulfide might contribute to its antiproliferative potency on colon cancer cells [100]. Although providing an additional mechanism to some genomic effects of NSAIDs, as their COX-independent chemopreventive properties [101], the therapeutical relevance of these data remains uncertain in OA for at least three reasons: i) PPAR modulation by NSAIDs was observed at doses which were consistent with adipocytes differentiation

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but far above those required for inhibition of COX isoenzymes in chondrocytes [102] and synovial fibroblasts [103]; ii) loss of PPAR stimulation by COX-derived metabolites [40] could be insufficiently compensated by the weak agonist potency of NSAIDs; iii) increased levels of lipoxygenase-derived metabolites (see § 1.3.1), secondary to COX-inhibition, could compete with NSAIDs for activating PPARs [104].

3. Modulation of Extracellular Matrix Remodelling by PPAR Agonists 3.1. Matrix Metalloproteases (MMPs) Matrix metalloproteases (MMPs) are thought to play a major role in cartilage degradation in OA [105] even if the promising efficacy of their inhibitors in animal models [106,107] has not been confirmed in OA patients [108] or raised safety concern [109]. Amongst MMPs, stromelysin-1 (MMP-3) and aggrecanases (ADAMTS-4 and -5) were implicated in the degradation of proteoglycans, though at different cleavage sites in the interglobular domain of aggrecan [110], whereas collagenase-1 (MMP-1) and -3 (MMP-13) contributed actively to the cleavage of specific collagens [111]. Gelatinases (MMP-2 and -9) may play a secondary role in cartilage breakdown since they have less specificity for a given extracellular matrix component while contributing to the removal of matrix fragments [112]. PPARγ agonists were shown to reduce IL-1-induced MMP-1 expression or activity in synovial fibroblasts [55] and chondrocytes [28] and, in the later case, this was accompanied by a reduced degradation of proteoglycans. The transcriptional down-regulation of MMP-1 gene involved the reduction of activator protein (AP)-1 binding [55] which was ascribed to DNA binding competition on a composite PPRE/AP-1 site in the MMP-1 promoter [28]. More recently, the PPARα agonist clofibrate was also shown to suppress the inducing effect of IL-1β on MMP-1, -3 and -13 expression in rabbit chondrocytes, but this effect was supported by an increase in soluble IL-1Ra production [52]. In human chondrocytes, PPARγ agonists were also reported to inhibit IL-1β-induced MMP-13 production at the transcriptional level, by interfering with the activation of AP-1 and NF-κB [48], a finding consistent with the dual decrease of IL-1β and MMP-13 levels and the reduced severity of cartilage lesions in OA animals treated with pioglitazone [113]. Finally, agonists of PPARγ prevented the expression of the aggrecanase-generated epitope NITEGE in cytokinetreated rat chondrocytes while reducing the expression of MMP-3 and -9 and subsequent occurrence of the MMP-generated epitope VDIPEN [65]. Although there is no firm demonstration that PPAR are playing a role, these data can be brought together with the ability of n-3 PUFAs to prevent glycosaminoglycans release as well as ADAMTS-4, MMP-3 and -13 expression in human OA cartilage exposed to IL-1 [42]. Taken together, these data support that PPAR agonists, mainly of the γ subtype, have an anticatabolic potency towards MMPs and may prevent cartilage degradation in OA. 3.2. Growth Factors Transforming growth factor (TGF-β) is a multifunctional cytokine that plays an important role in tissue repair [114] but has a complex pathophysiological role in OA. On one hand, TGF-β stimulates the synthesis of cartilage-specific components [115] and counteracts the suppressive effect of inflammatory cytokines on them [116], while it


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controls MMPs activity by the synthesis of their natural tissue inhibitors [117]. On the other hand, its intra-articular administration [118] or joint overexpression [119] is inflammatory with the synovial-layer-dependent formation of osteophytes [120], whereas it induces the expression of aggrecanase-1 in joint cells [121,122] with the subsequent occurrence of matrix-derived neo-epitopes. In many other tissues, TGF-β contributes to organ fibrosis by favouring matrix overload but its stimulatory effect on collagens [58,123,124] and fibronectin [125] synthesis is reduced by PPARγ agonists. Such inhibitory potency may contribute to the preventive effect of some PPAR agonists on skin [58], lung [124], kidney [126] or liver [127] fibrosis. It was demonstrated, very recently, that selective agonists of either PPAR subtypes were able to suppress the stimulatory effect of TGF-β on PGs synthesis and deposition by interfering with aggrecan gene expression in chondrocytes maintained in tridimensional culture [53]. Although it remains unclear to what extent such anti-anabolic effect may balance with the respective cytokine suppressive potency of TGF-β and of PPAR agonists in cartilage, these data suggest that PPAR agonists could be deleterious in situation of cartilage repair while being protective in situation of cartilage degradation. Insulin-like growth factor (IGF)-1 is another cytokine sharing in common with TGF-β the ability to stimulate the production of extracellular matrix components [128] and to counteract their degradation [129], although it remains less mitogenic, at least on mature chondrocytes [130]. In OA cartilage, the increased expression of IGF-1 combined with the normal level and functionality of IGF-1 receptors has led to the proposal that chondrocytes become hyporesponsive to IGF-1, because the bioavailability of this growth factor is reduced secondary to an increased production of IGF binding proteins (IGFBP) [131]. In non articular cell types, PPARγ agonists were shown to inhibit several biological responses to IGF-1 [132] while stimulating the production of IGFBP-1 [133,134]. More recently, selective agonists of either PPAR subtypes were demonstrated to stimulate the expression of IGFBP-1, -2 or -5 and to decrease those of IGFBP-6 whereas IGFBP-3 and -4 remained unresponsive in liver and kidney cell lines [135]. Unfortunately, IGFBP-1 and -6 are not secreted by articular cartilage or chondrocytes [131] and IGFBP-3 is expressed predominantly in OA conditions [136] with a maximal content in the more severe lesions [137]. So, PPAR agonists may have a poor theoretical potency to relieve the IGF-1 hyporesponsiveness of OA cartilage although one cannot exclude that they could interfere with the cell-specific proteases that compromise locally the functional activity of growth factor binding proteins [138].

4. Effect of PPAR Agonists in Animal Models of Rheumatic Diseases 4.1. Arthritis Model Agonists of PPARγ were shown to reduce the severity of experimental polyarthritis in rat [83,139] and mice [15,82,140]. A concomitant reduction of synovitis was reported with the natural agonist 15d-PGJ2 [139,140] or several glitazones given preventively or therapeutically [15,82,139], and more recently with the PPARα agonist fenofibrate [83]. Efficacy was associated with an overall reduction of inflammatory genes [15,82,140] and oxidant stress [82,140], although contradictory data were reported for the effect of PPARγ agonists on apoptosis of synovial fibroblasts [15,139]. However, one must consider that the anti-arthritic effect of PPAR agonists was obtained by parenteral route

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[139,140] or with high oral doses [83,139], supporting that their anti-inflammatory effect necessitated much higher doses than required to lower circulating lipids or increase insulin sensitivity. Indeed, the dose ratio between anti-inflammatory and insulin sensitizing properties was estimated to be over 100-fold for troglitazone in rat, although without obvious liver toxicity [139]. 4.2. Experimental Osteoarthritis (OA) There is only one report evaluating the effect of the PPARγ agonist pioglitazone in the menisectomy model of OA in guinea pig [113]. When given at 20 mg/kg/day from 1 day after surgery until necropsy, pioglitazone reduced the severity and extent of cartilage lesions in the knee joint but was ineffective on osteophytes formation. Prevention of macroscopic and morphologic changes was accompanied by a reduction of the percentage of chondrocytes staining positively for MMP-13 or IL-1β in OA cartilage, suggesting that interference with IL-1 signalling in articular chondrocytes may contribute to the protective effect of pioglitazone. As for polyarthritis, cartilage sparing was obtained for a higher dose than used in antidiabetic studies although the circulating levels remained in the range of those reported in humans treated with the highest recommended dose of pioglitazone [113]. As non-insulin dependent diabetes mellitus is also a systemic risk factor for the development of OA [141], there is possibility that glitazones could reduce cartilage destruction by their dual cytokines suppressive and insulin sensitizing properties in diabetic patients with OA.

5. Conclusion and Future Trends Several in vitro and animal studies have demonstrated that PPAR subtypes are expressed and functional in joint cells and that their activation down regulates the transcriptional machinery to reduce pro-inflammatory cytokines and metalloproteases production. From that point of view, PPAR agonists can prevent joint inflammation and cartilage destruction to various degrees. However, one must also consider that most anti-inflammatory effects were reported at concentrations of agonists far above their binding affinity for PPAR subtypes. In addition, PPARγ agonists were able to prevent fibrosis of soft tissues by reducing the synthesis of extracellular matrix components and to cause bone mass loss in some mouse models [142]. This suggests that joint inflammation is less sensitive to PPAR activation than lipids and glucose homeostasis and that PPAR agonists could have a complex interplay with the turn-over of cartilage and bone matrices. So, it seems logical to speculate that the modulation of OA by PPAR activation will come to fruition if we can demonstrate firstly that diabetic patients treated with PPAR agonists are at lower risk of developing OA and/or develop less severe cartilage lesions without significant bone changes.

Note Added to Proof During the processing of this manuscript, an additional work demonstrated that pioglitazone (30 mg/kg/day) reduced the development of cartilage lesions in the anterior cruciate ligament transection model of OA in dog (Boileau C. et al., Arthritis Rheum 56


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(2007), 2288-98). This protective effect was accompanied by a reduced expression of matrix degrading enzymes (MMP-1, ADAMTS-5) and less activation of inflammatory signaling pathways as ERK1/2, p38MAPK or NF-κB in OA cartilage.

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[118] J.B. Allen, C.L. Manthey, A.R. Hand, K. Ohura, L. Ellingsworth, and S.M. Wahl, Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta. J Exp Med 171 (1990), 231-247. [119] A.C. Bakker, F.A. van de Loo, H.M. van Beuningen, P. Sime, P.L. van Lent, P.M. van der Kraan, C.D. Richards, and W.B. van den Berg, Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage 9 (2001), 128-136. [120] H.M. van Beuningen, H.L. Glansbeek, P.M. van der Kraan, and W.B. van den Berg, Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 6 (1998), 306-317. [121] Y. Yamanishi, D.L. Boyle, M. Clark, R.A. Maki, M.D. Tortorella, E.C. Arner, and G.S. Firestein, Expression and regulation of aggrecanase in arthritis: the role of TGF-beta. J Immunol 168 (2002), 1405-1412. [122] N. Moulharat, C. Lesur, M. Thomas, G. Rolland-Valognes, P. Pastoureau, P. Anract, F. De Ceuninck, and M. Sabatini, Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro. Osteoarthritis Cartilage 12 (2004), 296-305. [123] F. Zheng, A. Fornoni, S.J. Elliot, Y. Guan, M.D. Breyer, L.J. Striker, and G.E. Striker, Upregulation of type I collagen by TGF-beta in mesangial cells is blocked by PPARgamma activation. Am J Physiol Renal Physiol 282 (2002), F639-648. [124] H.A. Burgess, L.E. Daugherty, T.H. Thatcher, H.F. Lakatos, D.M. Ray, M. Redonnet, R.P. Phipps, and P.J. Sime, PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis. Am J Physiol Lung Cell Mol Physiol 288 (2005), L1146-1153. [125] B. Guo, D. Koya, M. Isono, T. Sugimoto, A. Kashiwagi, and M. Haneda, Peroxisome proliferatoractivated receptor-gamma ligands inhibit TGF-beta 1-induced fibronectin expression in glomerular mesangial cells. Diabetes 53 (2004), 200-208. [126] Y. Guan, Peroxisome proliferator-activated receptor family and its relationship to renal complications of the metabolic syndrome. J Am Soc Nephrol 15 (2004), 2801-2815. [127] T. Miyahara, L. Schrum, R. Rippe, S. Xiong, H.F. Yee, Jr., K. Motomura, F.A. Anania, T.M. Willson, and H. Tsukamoto, Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 275 (2000), 35715-35722. [128] D.J. McQuillan, C.J. Handley, M.A. Campbell, S. Bolis, V.E. Milway, and A.C. Herington, Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. Biochem J 240 (1986), 423-430. [129] W. Hui, A.D. Rowan, and T. Cawston, Modulation of the expression of matrix metalloproteinase and tissue inhibitors of metalloproteinases by TGF-beta1 and IGF-1 in primary human articular and bovine nasal chondrocytes stimulated with TNF-alpha. Cytokine 16 (2001), 31-35. [130] P.A. Guerne, A. Sublet, and M. Lotz, Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts. J Cell Physiol 158 (1994), 476-484. [131] G. Tardif, P. Reboul, J.P. Pelletier, C. Geng, J.M. Cloutier, and J. Martel-Pelletier, Normal expression of type 1 insulin-like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin-like growth factor binding proteins. Arthritis Rheum 39 (1996), 968-978. [132] A. Aiello, G. Pandini, F. Frasca, E. Conte, A. Murabito, A. Sacco, M. Genua, R. Vigneri, and A. Belfiore, Peroxisomal proliferator-activated receptor-gamma agonists induce partial reversion of epithelial-mesenchymal transition in anaplastic thyroid cancer cells. Endocrinology 147 (2006), 4463-4475. [133] A. Hilding, K. Hall, J. Skogsberg, E. Ehrenborg, and M.S. Lewitt, Troglitazone stimulates IGF-binding protein-1 by a PPAR gamma-independent mechanism. Biochem Biophys Res Commun 303 (2003), 693-699. [134] D. Seto-Young, M. Paliou, J. Schlosser, D. Avtanski, A. Park, P. Patel, K. Holcomb, P. Chang, and L. Poretsky, Direct thiazolidinedione action in the human ovary: insulin-independent and insulinsensitizing effects on steroidogenesis and insulin-like growth factor binding protein-1 production. J Clin Endocrinol Metab 90 (2005), 6099-6105. [135] T. Degenhardt, M. Matilainen, K.H. Herzig, T.W. Dunlop, and C. Carlberg, The Insulin-like Growth Factor-binding Protein 1 Gene Is a Primary Target of Peroxisome Proliferator-activated Receptors. J Biol Chem 281 (2006), 39607-39619. [136] T. Eviatar, H. Kauffman, and A. Maroudas, Synthesis of insulin-like growth factor binding protein 3 in vitro in human articular cartilage cultures. Arthritis Rheum 48 (2003), 410-417.

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[137] T.I. Morales, The insulin-like growth factor binding proteins in uncultured human cartilage: increases in insulin-like growth factor binding protein 3 during osteoarthritis. Arthritis Rheum 46 (2002), 2358-2367. [138] R.C. Bunn and J.L. Fowlkes, Insulin-like growth factor binding protein proteolysis. Trends Endocrinol Metab 14 (2003), 176-181. [139] Y. Kawahito, M. Kondo, Y. Tsubouchi, A. Hashiramoto, D. Bishop-Bailey, K. Inoue, M. Kohno, R. Yamada, T. Hla, and H. Sano, 15-deoxy-delta(12,14)-PGJ(2) induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest 106 (2000), 189-197. [140] S. Cuzzocrea, N.S. Wayman, E. Mazzon, L. Dugo, R. Di Paola, I. Serraino, D. Britti, P.K. Chatterjee, A.P. Caputi, and C. Thiemermann, The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)prostaglandin J(2) attenuates the development of acute and chronic inflammation. Mol Pharmacol 61 (2002), 997-1007. [141] T. Sturmer, H. Brenner, R.E. Brenner, and K.P. Gunther, Non-insulin dependent diabetes mellitus (NIDDM) and patterns of osteoarthritis. The Ulm osteoarthritis study. Scand J Rheumatol 30 (2001), 169-171. [142] S.O. Rzonca, L.J. Suva, D. Gaddy, D.C. Montague, and B. Lecka-Czernik, Bone is a target for the antidiabetic compound rosiglitazone. Endocrinology 145 (2004), 401-406.

Part II Signalling Mechanisms



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VII MAP Kinases Charles J. MALEMUD Department of Medicine, Division of Rheumatic Diseases, and Department of Anatomy, Case Western Reserve University, School of Medicine, Cleveland, Ohio, 44106-5076 (USA) Abstract. Mitogen-activated protein (MAP) kinase activation by cytokines and other soluble mediators in articular chondrocyte cultures was shown to reproduce critical components relevant to cartilage development, synovial joint inflammation as well as human and animal osteoarthritic pathology. MAP kinase activation has been shown to be critical in cartilage formation Cytokines, such as interleukin-1β and tumor necrosis factor-α, soluble mediators, such as nitric oxide, and growth factors, namely fibroblast growth factor, connective tissue growth factor, vascular endothelial growth factor and hepatocyte growth factor activate specific MAP kinases resulting in nuclear factor-κB activation. NF-κB is a transcription factor which regulates matrix metalloproteinase gene expression, induces chondrocyte programmed cell death, up-regulates chondrocyte cytokine gene transcription as well as suppressing extracellular matrix protein biosynthesis, events that are consistent with synovial inflammation and the resultant destruction of articular cartilage in osteoarthritis.

Introduction The mitogen-activated protein (MAP) kinase family is made up of the extracellular signal-regulated protein kinases (ERKs), p38 kinase in its various isoforms (i.e. α, β1, β2, γ, δ) and C-Jun-N-terminal kinase (JNK) [1,2]. Osteoarthritis (OA) is now recognized as a disease ‘process’ that is, in part, characterized by “non-classical inflammation” of synovial joints and a significant imbalance between anabolic and catabolic pathways which ultimately result in the destruction of articular cartilage [3,4]. It is appreciated now more than ever that MAP kinases play a prominent role in both the pathogenesis and progression of OA through their capacity to activate transcription factors such as nuclear factor κB (NF-κB) that result in suppressed extracellular matrix (ECM) protein biosynthesis and up-regulated matrix metalloproteinase (MMP) gene expression. NF-κB activation also regulates pro-inflammatory cytokine gene transcription [5]. Because MAP kinases appear to play an integral role in OA pathogenesis by altering synovial joint allostasis, it has become critical to identify which MAP kinases are specifically activated during the early stages of OA. This information would be crucial for understanding which specific MAP kinases regulate various cellular events such as chondrocyte proliferation, cytokine production, programmed cell death (i.e. apoptosis), ECM protein biosynthesis as well as ECM protein turnover and degradation; processes that are all central to OA [6,7]. In addition, identi-

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fying which MAP kinases are activated in the course of developing gross and microanatomic changes that are consistent with OA pathology may provide the fundamental underpinning for developing small molecule inhibitors (SMI) of MAP kinases for use in the future medical therapy of OA [8]. This review will focus on the substantial amount of compelling evidence that MAP kinases are integral to the initiation and progression of OA by critically examining the relevant literature demonstrating that specific MAP kinase activation occurs in chondrocyte cultures in response to cytokines and other mediators known to be critical to OA pathogenesis and progression. Further, this review will also comment on the few studies of MAP kinase activation in experimentally-induced animal models of OA.

MAP Kinase Activation MAP kinase activation is required in order for MAP kinases to phosphorylate target protein substrates. MAP kinase activation is generally carried out by at least seven upstream protein kinases (PKs) belonging to the MAP kinase kinase (MEK/MKK) protein family [2]. At least 4 MKK genes have been cloned from mammalian cells [9]. MKK activity is regulated by additional upstream MKKs (i.e. MKKKs and MKKKKs) that are either tyrosine- or serine-binding proteins which may also require low molecular weight GTP-binding proteins as co-factors for MKKK activation. In this regard, recent evidence indicated that it is an MKK-MAP kinase complex organized by scaffolding proteins that allows for specific MKKK activation selectivity by GTPases, additional PKs and receptors [10]. Pro-inflammatory cytokine gene transcription, best exemplified by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) gene expression is elevated in OA cartilage and OA synovium [4]. IL-1β and TNF-α gene transcription and their biological activities are regulated by MKK activation and MAP kinases. Thus, in the cellular model proposed by Eder [11] on how MAP kinase activation regulates IL-1β and TNF-α biological activities, MKKKs were proposed to be recruited to either IL-1 or TNF-α signaling pathways where MKKKs became bound to the TNF-α receptor activating factor (TRAF) thus promoting MKK phosphorylation and downstream MAP kinase activation. The Eder model [11] made a case for a common pro-inflammatory cytokine pathway (i.e. ‘pathway redundancy’) so that either IL-1 or TNF-α could result in MKK activation via TRAF with specific pathway bifurcation occurring downstream at the JNK or p38 kinase activation site as well as through NF-κB activation. Additional evidence for the role of MKK activation involving a TNF-α gene expression autocrine loop came from studies that showed that arctigenin, an inhibitor of MKK1 activity, blocked TNF-α mRNA transcription and TNF-α protein production by Raw264.7 cells stimulated with lipopolysaccharide which was also dependent on activator protein-1 (AP-1) activity [12].

ERKs ERK Structure ERKs were originally isolated from a rat brain cDNA library probed with DNA encoding the kinase domain of the insulin receptor-related protein [13]. The ERKs identified


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in that study contained all the amino acid residues conserved within protein-tyrosine kinases. Subsequently, the human ERK gene was localized to chromosome 1p36.1 [14]. This study also provided the first evidence that ERK proteins contained a receptor-like membrane-spanning structure. Of the ERKs that have been extensively studied, ERK1 (i.e. p44) and ERK2 (i.e. p46) are the most abundant ERKs [15,16], but other ERK forms (i.e. ERK-3, -5, -7, -8) have also been isolated [17–19]. Indeed, additional evidence has indicated that specific ERK1 isoforms, namely, ERK1b, a 46kDa protein [20] which like ERK1 or ERK2 was activated by MEK1. However, ERK1b activation did not parallel that of ERK1. ERK1c is a 42kDa protein which is an alternatively spliced form of ERK1b found to be widely distributed in tissues and cells. ERK1c could be activated by MEKs, although ERK1c activation and its slower inactivation by phosphatases distinguished ERK1c from ERK1 [21]. In addition, Eblen et al. [22] showed that Rac (a member of the Ras-oncogene family) in association with p21activated kinase (PAK) enhanced ERK2 signaling. In COS cells, MEK1 was found in association with ERK2. Further, the lack of inducible binding between ERK2 and MEK2 was likely to be a result of that fact that MEK2 is not a suitable substrate for PAK. These results suggested the view that there likely existed a significant degree of specificity in the MEK/ERK activation system [22]. ERK Activation ERKs are rapidly phosphorylated in response to various extracellular stimuli, but especially by growth factors such as epidermal growth factor, nerve growth factor and insulin-like growth factor-1 (IGF-1) [23,24]. When ERK activation is transient, ERK phosphorylating activity rapidly declines and evidence shows that c-Fos, a critical ERK substrate becomes unstable and is not suitably activated [25]. However, if ERK phosphorylation is sustained or prolonged, c-Fos is phosphorylated by ERK and the 90kDa ribosomal S6 kinase resulting in c-Fos becoming fully active. This ERK activation mechanism also appears to be dependent on the ERK targeting DEF domain in c-Fos. Thus, the ERK/DEF domain appears to be dominant in regulating ERK/c-Fos docking which modulates cell cycle progression and cellular transformation [25]. ERK Nuclear Translocation ERK translocation to the nucleus appears to be highly dependent on specific residues in the ERK activation loop since ERK2 mutations created in this domain, especially in residues 176–181, were shown to be responsible for ERK2/MEK1 dissociation upon mitogenic stimulation [26]. In contrast, residues 176-181 as well as residues required for ERK2 dimerization did not appear to play a significant role in ERK2 trafficking through nuclear pores [26]. ERK Inactivation ERKs are inactivated by MAP kinase phosphatases (MKPs). In the case of MKP3, ERK deactivation occurs within a complex of MKP3 and ERK and further, MKP3 may also prevent phospho-ERK homodimerization and any additional ERK activation by MEK [27]. There appears to be significant MKP specificity. For example, when MKP4 was expressed in COS-7 cells, it blocked activation with the selectivity profile of ERK > p38 kinase = JNK [28]. Whereas MKP5 and MKP7 selectively deactivated

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p38α and β kinase, but not p38 γ and p38 δ kinases, MKP7 bound to and inactivated JNK/SAPK, but not ERK [29]. ERKs and Chondrogenesis Studies of in vitro chondrogenesis under basal conditions showed that ERK activity declined as chondrogenesis occurred [30]. However, Murakami et al. [31] showed that FGF-mediated up-regulation of Sox9, an important transcription factor that regulates Type II, IX and Type XI collagen as well as link protein and aggrecan gene expression during chondrogenesis [32,33] was ERK-dependent. In that regard, Sox9 gene expression could be ablated by inhibiting ERK 1/2 with an MEK SMI during in vitro chondrogenesis. By contrast, ERK activity was also shown to be rapid, but transient, when in vitro chondrogenesis was stimulated by growth-differentiation factor-5 (GDF-5) [34]. In addition, the ECM of cartilage-constructs made on polylactic acid-co glycolic acid (PGLA) scaffolds using human mesenchymal stem cells contained aggrecan and Type II collagen when transforming growth factor-β was added to the PGLA scaffold [35]. The emergence of the chondrogenic phenotype appeared, however, to only partially involve ERK signaling pathways as MEK inhibition resulted in down-regulation of Type II collagen, but not aggrecan. These results appear to partially sustain previous studies by Kim et al. [36] and Legendre et al. [37] who showed that maintenance of the chondrogenic phenotype was only partially dependent on ERK 1/2 activity. Additional studies by Yagi et al. [38] using small interference mRNA (siRNA) bcl-2 (an antiapoptosis protein) in the presence of caspase inhibitors have added an additional level of complexity to the previously cited studies relating ERK activity to chondrogenic expression [30–37] because Yagi et al. [38] showed that blc-2 siRNA blocked Sox9 expression in rat chondrocytes via MEK/ERK 1/2, suggesting that bcl-2 could also be a critical regulator of MEK/ERK 1/2-mediated chondrocyte differentiation in vitro. A recent analysis showed that functional cartilage loading in young female Wistar rats induced AP-1 and Runx2 transcription factor activity that were, in part, dependent on ERK activity [39]. These results suggested that ERK was also critical for the differentiation and maturation of cartilage tissue development in vivo. In this regard, Lai et al. [40] showed that β1 integrin interaction with ECM proteins could stimulate both p38 and ERK 1/2 in the CFK-2 chondrocytic cell line. In that study, parathyroid stimulating hormone inhibited both p38 and ERK 1/2 activation, but Indian hedgehog (Ihh) protein expression was down-regulated primarily through ERK 1/2. Ihh has been shown to be an important component in chondrocyte terminal differentiation and endochondral ossification [41]. ERK Activation and Chondrocyte Proliferation ERK activation was shown to play a prominent role in chondrocyte responses to fibroblast growth factor (FGF). Thus, FGF inhibited adult chondrocyte proliferation and an SMI of MEK 1/2 blocked rat chondrosarcoma proliferation as well as ERK 1/2 and p38 signaling which was also dependent on inactivating the retinoblastoma family proteins p107 and p130 [42].


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Events Critical to OA Appear to Be ERK-Dependent At the cellular level, OA pathogenesis and progression resulting in articular cartilage destruction appears to involve various elements of the inflammatory response, including cytokine gene up-regulation, prostaglandin production, MMP gene up-regulation, synthesis and activation, ECM protein gene dysregulation, suppressed ECM protein synthesis, apoptosis, systemic disturbances in the growth hormone/IGF-1 pathway as well as inefficient chondrocyte proliferation and cartilage repair [2–4,43,44]. ERK Activation and OA With regard to the role of ERK activation and its potential role in OA pathogenesis, Martel-Pelletier et al. [6] showed that stimulation of nitric oxide (NO) by the proinflammatory cytokine, IL-17 (which can create a chondrocyte anabolic/catabolic imbalance) was dependent on MEK-1/2 and MEK-3/6 activity in cultured human osteoarthritis chondrocytes. The MEK-1/2 SMI, PD98059 was able to block IL-17 induced inducible nitric oxide synthase (iNOS) and production of NO. ERKs and Cytokine GeneUp-Regulation Cytokine gene up-regulation in OA is best exemplified by the increased levels of IL-1 and TNF-α found in OA synovial fluid. The increase in IL-1 and TNF-α in OA is likely a result of activating both synoviocyte and chondrocyte IL-1 and TNF-α [4]. It is evident, however, that activation of a specific MAP kinase such as ERK 1/2 alone yielded only modest increases in IL-1 and TNF-α gene up-regulation whereas activation of p38, JNK, ERK 1/2 and PI3K resulted in full TNF promoter activity [45]. Anabolic and Catabolic Imbalance in OA Are Partially Dependent on ERK Activity IL-1 plays a central role in chondrocyte dysfunction in OA [6,7]. Active IL-1 was found in synovial membrane isolated from OA joints [46] indicating that activated synoviocytes are a primary source for IL-1 in OA and also critical to IL-6 upregulation. More recently, however, Fan et al. [47] showed that IL-1β induction of IL-6 and leukemia inhibitory factor (LIF), both recognized as playing a role in chondrocyte MMP activity and ECM protein degradation was partially dependent on ERK 1/2 in human osteoarthritis chondrocyte cultures, but neither IL-1 nor LIF were strongly expressed in OA cartilage. Further, Raymond et al. [48] identified an IL-1β-responsive element in the MMP-1 (i.e. collagenase-1) promoter containing a consensus CCAAT enhancer-binding protein (C/EBP) site at threonine-235 which was ERK-dependent since the ERK SMI, PD98059, reduced C/EBP phosphorylation. That study [48] defined a novel role for C/EBP in IL-1β-induced MMP-1 gene transcription in which dominant-negative (dn) ERK1 and ERK2 constructs also suppressed IL-1β-induced MMP-1 promoter transactivation. Selective ERK and NF-κB inhibitors were also shown to partially, but significantly, block MMP-1, MMP-13 (i.e. collagenase-3) and Type II collagen gene transcription in human chondrocyte cultures after IL-1β treatment [49] suggesting that ERKs play a significant role in not only regulating MMP gene transcription but also in maintaining the chondrogenic phenotype as well. Diacerein through its active metabolite Rhein has been tested for its chondroprotective properties. Rhein was shown to protect bovine chondrocytes from IL-1βinduced MMP-1 gene up-regulation and block ECM protein suppression [50] by inhib-

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iting the MEK/ERK pathway as well as by suppressing downstream NF-κB and AP-1 activity. ERKs and Chondrocyte Apoptosis Apoptosis is a significant event in cartilage degeneration because apoptosis results in a reduction in cartilage vitality and chondrocyte proliferation, both of which are required for efficient cartilage repair [51]. In that regard, recent studies showed that BAY117085, a potent anti-inflammatory agent used to treat rat adjuvant arthritis induced human chondrocyte apoptosis [52] which was dependent on sustained ERK 1/2 activation. Thus, ERK 1/2 inhibition might provide chondroprotection and prevent chondrocyte apoptosis induced by IL-1β or TNF-α [8]. Another site for suppressing the apoptosis cascade resides in the induction of proto-oncogenes by external stimuli. Thus, Islam et al. [53] showed that cyclic hydrostatic pressure induced p53 and c-myc gene expression and apoptosis in human osteoarthritis chondrocyte suspension cultures. Based on the finding that myc suppressed ERK signaling in chick embryo fibroblasts it has been suggested that the myc-binding site (BMD) of the protein bin-1 (which negatively regulates myc activity) could be exploited to regulate apoptosis via its ability to stimulate ERK activity [54]. Human chondrocytes treated with IL-1β produced elevated NO and cyclooxygenase-2 (COX-2) levels [55]. Notoya et al. [56] previously showed that human chondrocyte apoptosis induced by NO was COX-2 dependent. In this regard, Nieminen et al. [57] demonstrated that IL-1β transiently activated ERK 1/2 as well as p38 kinase and JNK in immortalized human T/C28a chondrocytes resulting in elevated COX-2 and prostaglandin E2 (PGE2) levels which were suppressed by PD98059. By contrast, SMI p38 kinase and JNK inhibitors, SB203580 and SP600125, respectively, had more variable effects on COX-2 activity and PGE2 production. ERKs and Cartilage Repair Tissue-engineering is a promising approach for stimulating cartilage repair in OA. For cartilage repair employing artificial cartilage implants, tissue-engineered cartilageconstructs would most likely have to be implanted in cartilage where high weightbearing occurs and where significant cartilage degeneration in OA has already been identified. For this reason, it is imperative that the integrity of the tissue-engineered cartilage ECM proteins be optimized so that biomechanical properties of the implants are maintained. Thus, loss of the chondrocyte phenotype by modulation of cartilagespecific gene expression would be expected to significantly compromise potential cartilage repair by these implants. In this regard, Wenger et al. [58] recently demonstrated that human osteoarthritis chondrocytes grown on a scaffold of HYAFF®-11 (Advanced Biopolymers, Termo, ITALY) in mini-bioreactors synthesized significant amounts of sulfated-proteoglycan and Type II collagen, but not Type I or Type X collagen. However, cyclic hydrostatic pressure (CHP) employed at 5MPa using a sinusoidal frequency of 1Hz significantly increased apoptosis in these cartilage-constructs. Studies reported by Schulz-Tanzil et al. [59] also showed that loss of chondrogenic expression by human chondrocytes in culture was characterized by reduced Type II collagen biosynthesis, α3 integrin expression, src-homology collagen (Shc) and ERK activity that eventually resulted in chondrocyte apoptosis. It will therefore be interesting to determine the extent to which key signaling proteins in the ERK and Ras-mitogen-activated PK pathway [59] contribute to CHP-induced apoptosis in tissue-engineered human


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cartilage constructs and whether sustained ERK activity or ERK inhibition alters the chondrogenic phenotype. Finally, IGF-1 appears to be a critical circulating growth factor required for stimulating chondrocyte proliferation and for up-regulating proteoglycan synthesis [60]. In this regard, Starkman et al. [61] showed that PD98059 and another, MEK SMI, namely, U0126 blocked IGF-1-stimulated ERK activation, but failed to alter IGF-1 stimulated proteoglycan synthesis. Instead, IGF-1 stimulated the PI3K/Akt pathway which when blocked by Akt inhibitors suppressed IGF-1 stimulated proteoglycan synthesis. Further, IGF-1 failed to stimulate aggrecan, decorin or biglycan mRNA levels in the presence of PD98059 indicating that IGF-1-mediated proteoglycan synthesis upregulation occurred primarily through its effect on modulating protein translation. P38 Kinase P38 Kinase Structure The most extensively studied p38 kinase isoform structure is that of p38α because of its reported involvement in inflammation. This includes the critical role p38α plays in modulating COX-2 and iNOS gene expression as well as in controlling NO production induced by cytokines [61]. Although the functional distribution of α and β2 p38 kinases are well documented, much less is known about the tissue distribution of β1, γ or δ p38 kinase isoforms [62,63]. A significant amount of p38 kinase structural data emerged from the development of p38 SMIs, such as the diarylimidazole, triarylimidazole and triarylpyrrole SMIs [64,65]. These studies revealed that the molecular basis for the specificity of these SMIs resided in the p38 threonine-106 residue located in the p38 ATP-binding pocket. Further, the peptide-substrate binding site and ATP-binding site for p38 was structurally distinct from ERK2 [66]. Functional studies also revealed several differences between p38 kinase isoforms. Thus, Li et al. [67] showed that in contrast to p38α and β, p38γ did not phosphorylate activating transcription factor-2 or MAPK activated protein kinase-2, but was able to phosphorylate myelin basic protein. Further, p38δ activity was not blocked by pyridinyl imidazole derivative, SB202190, an SMI of p38α, β and γ isoforms. Nonetheless p38δ could be activated by MKK3 and MKK6, which are known activators of the several p38 kinase isoforms [68]. P38 Kinase Activation In a fashion similar to other MAP kinases, p38 kinase activation requires upstream MEK/MKK activity with MKK3, MKK4 and MKK-6 implicated in this process [69,70]. Role of p38 in Chondrocyte MMP Production The current interest in p38 and its role in chondrocyte MMP gene transcription stems from the fact that the pro-inflammatory cytokines that up-regulate chondrocyte MMPs also activate p38 signaling pathways [71–73]. P38 kinase is also prominently involved in MMP-9 (i.e. 92Kda gelatinase) and MMP-13 gene up-regulation stimulated by cytokines, such as IL-1β [74], indicating that MMPs with potent activity towards aggrecan, Type II collagen and denatured collagen are regulated by p38 phosphorylation.

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Geng et al. [75] were among the first to demonstrate that IL-1β and TNF-α caused selective activation of human chondrocyte p38, ERK and JNK. In that regard Geng et al. [75] showed that ERK 1/2 activation brought about by several factors, including, platelet-derived growth factor, IL-6 and IGF-1 contrasted sharply with the more restricted activation of p38 and JNK brought about by p38 and JNK activation. Geng et al. [75] concluded that MAP kinase activation was likely to be early events in cytokine-mediated initiation of the catabolic/anabolic imbalance thought to cause the early surface lesions in OA animal and human cartilage. In a subsequent study, IL-17, was also found to be capable of stimulating the expression of several additional mediators of cartilage ECM protein degradation and apoptosis, including COX-2, iNOS, IL-1β, IL-6 and MMP-3 (i.e. stromelysin-1) in human chondrocyte cultures [76]. The production of these pro-inflammatory mediators was, in part, dependent on p38 activation since the p38 SMI, SB203580, suppressed IL-17-mediated changes in COX-2, iNOS, IL-1β, IL-6 and MMP-3. More recently, additional activators of chondrocyte MMP gene transcription have been reported that also involve p38 activation. Thus, Mengshol et al. [77] reported that IL-1 induced MMP-13 in the SW-1353 chondrosarcoma cell line resulted in p38 and JNK activation via Runx2 transcriptional activity, but the MMP-13 promoter response was primarily regulated by p38 acting via Runx2 and AP-1 activation. The results of the studies reported by Mengshol et al. [77] were recently confirmed by Pei et al. [78] who showed that over-expression of Runx2 stimulated MMP-13 gene transcription but had no effect on MMP-1 in either SW 1353 chondrosarcoma cells or human articular chondrocytes. In that study [78], MMP-13 gene transcription was found to be dependent on Runx2 phosphorylation that resulted from p38 kinase activation. Additional signaling pathways have been explored to determine their significance in chondrocyte MMP gene up-regulation by IL-1. Thus, by employing licofelone, an inhibitor of cyclooxygenases and 5-lipoxygenase, Boileau et al. [79] demonstrated a dose-dependent inhibition by licofelone on IL-1-induced MMP-13 gene transcription in human osteoarthritis chondrocytes. This inhibition correlated with reduced p38 and AP-1 activity which also involved the cyclic AMP response element binding protein. Of note, licofelone failed to inhibit ERK 1/2 or JNK activation. MMP-1 and MMP-13 gene transcription inhibition was also seen in IL-1βstimulated human osteoarthritis chondrocytes after treatment with pomegranate fruit extract which correlated with p38 inhibition, but not ERK or JNK inhibition [80]. Of interest, hyaluronan oligosaccharides (HA-oligos) stimulated chondrocyte MMP-13 gene transcription which was found to be p38 kinase and NF-κB-dependent [81]. Whether hyaluronan depolymerization or fragmentation actually occurs within the synovial joint inflammatory milieu remains to be determined as is the potential relevance of HA-oligos to MMP-13-mediated cartilage degradation. Finally, the novel highly specific p38 SMI R-130823 decreased MMP-1 and MMP-13 gene transcription as well as PGE2 production in IL-1β-stimulated human and bovine chondrocytes further supporting the view that p38 kinase is the predominant activated MAPK regulating chondrocyte IL-1β-stimulated MMP-1 and MMP-13 gene transcription [82]. Potential Role for p38 in Chondrocyte Senescence, ECM Synthesis and Apoptosis Reduced chondrocyte viability and proliferation limits repair of surface defects in aging and OA articular cartilage [3]. Kang et al. [83] employed the p38 SMI SB203580 or dn mutations in MKK6 or p38 to assess the role of p38 in rabbit articular chondrocyte


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proliferation and senescence. They showed that p38 inhibition by any of these three techniques stimulated chondrocyte proliferation during the active growth phase and also extended chondrocyte life span which was telomere-independent [83]. The suppression of chondrocyte ECM protein biosynthesis by IL-1 will also directly affect cartilage repair. Thus, Radons et al. [84] recently showed using PK SMIs that p38 and/or PI3K/JNK were involved in IL-1-induced chondrocyte IL-6 secretion, the latter an important co-factor in regulating anabolic/catabolic balance in cartilage. Apoptosis is also likely to limit cartilage repair in OA and may be responsible for inefficient chondrocyte ECM production in OA [51]. NO is one of the strongest inducers of chondrocyte apoptosis and, indeed, chondrocyte apoptosis and cartilage degradation have been linked in OA [85]. Recent studies have also shown that leptin can act as a pro-inflammatory mediator and working synergistically with IL-1 stimulates iNOS in cultured chondrocytes. IL-1/leptin added to human chondrocytes or the ATDC-5 murine chondrosarcoma cell line increased iNOS activity and was dependent, in part, on p38 activation, but Janus kinase-2, PI3K and MEK-1 were also implicated in iNOS gene transcription in this study [86]. Because articular cartilage oxygen tension levels are likely to be low in the joint, the modulation of the transcription factor, hypoxia-inducible factor-1α (HIF-1α) may also play a critical role in altering chondrocyte allostasis. In a recent study, Coimbra et al. [87] found HIF-1α mRNA in both normal human and OA cartilage. In addition, TNF-α increased chondrocyte HIF-1α mRNA which was partially blocked by p38 SMIs and NF-κB inhibition [87]. As a result of this finding, it will be of interest to determine the extent to which HIF-1α regulates chondrocyte apoptosis under normoxic and hypoxic conditions, since previous studies showed that TNF-α and NO which induce apoptosis in cultured chondrocytes [51] was also found to regulate HIF-1α production not only by a non-hypoxic reactive oxygen species-sensitive mechanism [88] but also as a component of the inflammatory response by its capacity to stimulate iNOS mRNA in activated macrophages [89]. P38 and Chondrogenesis Under basal in vitro conditions, chondrogenesis was accompanied by p38 activation [30], whereas when ACDC-5 cell chondrogenesis was stimulated by GDF-5, p38 activation was slow and sustained [34] indicating that p38 activation was required for chondrocyte terminal differentiation. More recently, Tuli et al. [90] showed that transforming growth factor-β-induced chondrogenesis from adult bone marrow or trabecular bone-derived mesenchymal progenitor cells resulted from p38 and ERK1 activation and to a significantly lesser extent from JNK activation. Activation of p38 and ERK1 was also related to events which modulated N-cadherin and regulation of Wnt-7A gene expression as well as the Wnt/β-catenin/T-cell factor pathway which appears to be critical for N-cadherin-mediated mesenchymal progenitor cell condensation, a critical event in chondrogenesis in vivo [41]. JNK JNK Structure JNK, also known as stress-activated protein kinase (SAPK), is found in 3 forms, namely, JNK1, JNK2 [91,92] and JNK3 [93] which share about 50–80% homology

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with each other, and in particular JNK3 and p38 which share 51% identity in primary sequence [93]. Although JNK1 and JNK2 is found distributed among many tissues, JNK3 appears to have particular significance in nervous system tissue in that it is almost exclusively expressed in brain with low levels expressed in kidney and testes [8]. The mouse JNK/SAPKα gene spans a region of about 36 kilo-bases and contains 13 exons, which represent about 8% of the gene sequence [94]. Splice variants are responsible for several forms of JNK/SAPKα generated by alternative splicing of exons 7 and 8. When a JNK promoter construct contained the activator protein-2 element the JNK promoter activity was increased from 28% to 77% [94]. The Physiologic Significance of JNK The physiological significance of JNK is primarily based on its capacity to bind and phosphorylate the DNA binding protein, c-Jun, which increases c-Jun transcriptional activity [73,95]. The JNK2 form was shown to bind to C-Jun with about a 25-fold greater affinity than JNK1 and had a lower Km for c-Jun than JNK1 [95]. Further, it was JNK2 that was primarily phosphorylated by human osteoarthritis chondrocytes in response to IL-1β [96]. C-Jun is a well recognized component of the AP-1 complex which is critical in cytokine and MMP gene regulation [97]. In addition, JNK has also been reported to bind to and phosphorylate activating transcription factor-2 (ATF-2), Elk-1, nuclear factor of activated T-cells (NFAT) and p53 [63]. JNK Activation JNKs are activated by Rac and Cdc42 via MKK4 and MKK7 by phosphorylating JNK on threonine-183 and tyrosine-185 [98]. MKK4 is activated primarily by IL-1β and TNF-α [91,92] whereas MKK7 is activated by other environmental stressors, including, IL-3, ligation of CD-40, B-cell antigen receptors for Fc, Ras/GTPases, heat, UV irradiation, anisomycin, hyperosmolarity and TNF-α in hemopoietic cells and HeLa cells [98]. The JNK-interacting protein (JIP) group of scaffolding proteins was found to selectively activate MKK7 by causing aggregation of the MMK7/JNK complex [99] and the rat and human islet brain-1 protein was found to be homologous to JIP-1 [100]. In addition, the mouse and human MKK7 protein are highly structurally conserved, the latter being very specific for JNK as evidenced by the fact that MKK7 activated JNK1, but not p38 kinase in co-expression studies [98,101]. Role of JNK in Chondrocyte Cytokine Responses Chondrocyte MMP gene expression induced by TNF-α was shown to be regulated principally by JNK and p38 [102, 103] which resulted in AP-1 activation and MMP gene up-regulation [104]. Phenyl-N-tert-butylnitrone (a spin trap agent) and epigallocatechin-3-gallate (EGCG) were both able to inhibit human osteoarthritis chondrocyte Il-1β-induced JNK and p38 as well as IL-1β-induced MMP-1 and MMP-13 gene transcription [96,105,106] confirming a previous report [107] which showed that IL-1β activated rabbit chondrocyte JNK, ERKs and p38 in an IL-1β concentration- and timedependent manner. However, in that study [107], TNF-α only activated chondrocyte JNK. Our laboratory recently found that TNF-α activated human chondrocyte JNK1,2 and p38 as well as STAT3 but had no effect on JNK, p38 or STAT3 protein levels as determined by Western blotting (unpublished studies). Further, IL-1β was also shown


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to cause a transient elevation in phospho-JNK, phospho-ERK and phospho-p38 in immortalized human T/C28a2 chondrocytes which led to increased COX-2 and PGE2 levels [57], and SP600125 a highly specific JNK SMI [8] but not its negative control compound, namely, N1-methyl-1, 9 pyrazolanthrone, down-regulated COX-2 and PGE2 in a dose-and time-dependent manner suggesting a post-transcriptional regulatory mechanism. Fibronectin (Fnf) fragments but not fibronectin (Fn) have potent biological activity, including the capacity to cause chondrocyte MMP gene up-regulation [108]. In that regard, Forsyth et al. [109] showed that after IL-1β-treatment, a 120-kDa Fnf that binds to the α5β integrin, but not intact Fn, or α2β1 and α5β1 integrin neutralizing antibodies, increased human chondrocyte c-Jun and p38 phosphorylation as well as NF-κB activity which led to a further increase in pro-MMP-13 and activated MMP-13 levels. IL-1 receptor antagonist protein did not suppress JNK or p38 activation, but did partially inhibit MMP-13, the latter finding suggestive of the presence of an IL-1 autocrine feedback loop. In addition to IL-1β, TNF-α and Fnf-mediated activation of JNK, Fanning et al. [110] showed that mechanical static compression loads administered ex vivo stimulated bovine cartilage explant SAPK/ERK-kinase-1 (SEK1) of the JNK pathway with maximum SEK1 activation occurring at 1 hr and with a greater amplitude than for either ERK 1/2 or p38. By contrast, the cartilage explant response to IGF-1 differed considerably in that IGF-1 induced early, but transient ERK 1/2 activation, with no sustained ERK 1/2 activation. Finally, although chondrocyte apoptosis can be induced by IL-1β, TNF-α, and NO as well as by biomechanical stress, [43,51,53], selective activation of JNK was insufficient to induce CC-139 fibroblast apoptosis unless the PI3K pathway was also inhibited [111]. JNK and Chondrogenesis JNK was not required for either basal in vitro or FGF-or GDF-5-stimulated chondrogenesis [30,31,34] which was in contrast to the apparent requirement for p38 and ERK activation under these conditions [30,34] However, Wnt-3a caused c-Jun expression and c-Jun phosphorylation by chicken chondrocyte JNK which resulted in AP-1 activation [112]. In that study [112], AP-1 activation led to Sox9 suppression and reduced Type II collagen synthesis. Thus, it appeared that JNK activation may regulate the modulation of the chondrogenic phenotype by activating Wnt-3a-stimulated betacatenin/T-cell-factor/lymphoid enhancer-factor which is critical for determining the sequence of events under which terminal chondrocyte differentiation occurs in vivo [41]. In addition, the signal for chondrocyte terminal differentiation may also require PI3K activation via protein kinase C (PKC) in CCN2/connective tissue growth factor (CTGF)-stimulated chondrogenesis although a PKC-independent pathway involving JNK was also reported [113].

MAP Kinases, Inflammation and OA In vitro studies of chondrocyte MAP kinase activity as well as transcription factor activation by IL-1β, TNF-α, COX-2, NO and advanced glycation end (AGE) products have led to the postulation that potential novel therapeutic agents directed against MAP

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kinases could be employed to suppress the ‘non-classical’ inflammation of OA as well as cartilage destruction in OA [4,43,114–118]. In addition to the MAP kinase pathways altered by cytokines and growth factors that have already been discussed, 2 additional pathways involving MAP kinases warrant comment. Thus, Qureshi et al. [119] showed that the ERK 1/2 SMI, PD98059 or U0126 were capable of potentiating TGF-β-stimulated production of tissue inhibitor of metalloproteinases-3 (TIMP-3) in IL-1 activated bovine and human primary chondrocyte cultures as well as in the SW-1353 chondrosarcoma cell line. In that study [119], ERK 1/2 inhibition reduced Sp1 activity which is a major transcription factor for regulating TIMP-3 gene expression. The significance of this finding is related to the fact that TIMP-3 is a critical endogenous MMP inhibitor and is the salient inhibitor of MMP-13 and ADAMTS-4 which are the major enzymes implicated in collagen II and aggrecan degradation in OA [43]. Interest in the role of MAP kinases in the neutral sphinogmyelinase-induced hydrolysis of sphinogmyelin to ceramide pathway which can be activated by IL-1 and/or TNF-α first surfaced when Reunanen et al. [120] showed that ceramide-dependent induction of MMP-1 by fibroblasts was inhibited by the MEK1 SMI, PD98059 and by the p38 SMI, SB203580. Activation of C2-ceramide-mediated MMP-1 promoter activity was effectively suppressed by over-expression of MAP kinase phosphatase-1. Follow-up studies showed that ceramide also induced apoptosis in rheumatoid arthritis synovial cells [121], but Gerritsen et al. [122] showed that TNF-α-mediated apoptosis was not due to ceramide activity in human synovial fibroblasts. More recent interest in the ceramide pathway relative to OA pathophysiology resulted from studies showing that the ceramide pathway in rabbit cartilage explants was induced by both IL-1 and TNF-α, that C2-ceramide stimulated MMP-1, -3 and -13 mRNA and that C2-ceramide induced rabbit chondrocyte apoptosis when employed at concentrations ranging from 10–4M to 10–5M [123]. Ceramide was also shown to induce aggrecanase activity as evidenced by ceramide-induced appearance of the C-terminal aggrecan neoepitopes NITEGE373 and DIPEN341 in the culture medium when rabbit cartilage explants were treated with C2-ceramide [124], but in this study no increase in MMP protein levels was detected. Vascular endothelial growth factor (VEGF), an angiogenic peptide has been strongly implicated in synovial neoangiogenesis in rheumatoid arthritis [125]. Recently, VEGF was shown to be increased when osteoarthritis chondrocytes were maintained under hypoxic conditions which also induced HIF-1α [126]. Further, hypoxiainduced VEGF was blocked by p38 SMIs but not by JNK SMIs. By contrast, IL-1-induced VEGF production was blocked by a JNK but not a p38 SMI, suggesting that specific MAP kinase targeting will have to be employed to diminish proinflammatory mediators in OA that are dependent on hypoxia and/or IL-1, respectively. These results are also particularly noteworthy because Reboul et al. [127] showed that OA, but not normal cartilage, expressed hepatocyte growth factor (HGF). HGF stimulated MMP-13 gene transcription by human osteoarthritis chondrocytes that was JNKdependent, and also by an unidentified MAP kinase, which was not p38. Thus, suppressing MMP-13 up-regulation in OA cartilage must take into account that several specific MAP kinase pathways may be simultaneously activated by specific cytokines as well as by inducible growth factors. This viewpoint is particularly relevant in considering any future medical management of OA which must reconcile the bifurcation points whereby after IL-1 stimulation, chondrocyte MAP kinase activation, MMP gene regulation and NF-κB activation are strongly correlated with one another in vitro. This


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is particularly relevant since MAP kinase and NF-κB inhibition resulted in MMP gene down-regulation [128]. Of note, Barchowsky et al. [129] had previously demonstrated that IL-1 was superior to TNF-α in inducing c-Jun and ERK synthesis as well as c-Jun, ERK and NF-κB activation in primary rabbit synovial fibroblasts with all three of these activation events being required to up-regulate MMP-1 transcription. In addition, You et al. [130] showed that IL-17B, a member of the IL-17 family, was strongly expressed by chondrocytes during mouse limb bud development. This finding suggested that IL-17 family proteins could also be expressed by adult chondrocytes so that the IL-17 pathway must be considered pertinent to the present discussion especially if chondrocyte IL-17 or IL-17 over-expression induces chondrocyte anabolic/catabolic imbalance via MAP kinase and NF-κB activation. Indeed, Koenders et al. [131] recently reported that either IL-17A over-expression or local IL-17A gene transfer increased MMP-3, -9, -13 and ADAMTS4 mRNA as well as cartilage damage in IL-1-deficient mice in a streptococcal cell wall-induced arthritis model. Thus, medicinal chemical or genetic strategies designed to suppress NF-κB activation induced by cytokines using either PK SMIs or direct NF-κB inhibition may eventually be made applicable for in vivo use to suppress inflammatory responses in OA and, in fact, this approach is currently being considered for future OA therapeutic intervention [132]. Finally, any strategy based on inhibiting MAP kinase-dependent alterations in chondrocyte metabolism in vitro must also first show some efficacy in OA animal models. In that regard, chondrocyte apoptosis was found to occur early after anterior cruciate ligament transection in the dog. Chondrocyte apoptosis correlated with iNOS and COX-2 induction which was dependent on MEK-1/2 and p38 kinase activity. However, NF-κB inhibition did not alter apoptosis as measured by caspase-3 activity [133]. In addition, a recent study by Takahashi et al. [134] showed that Celecoxib, a selective COX-2 inhibitor used in the clinical management of OA suppressed chondrocyte PGE2 as expected, but Celecoxib also suppressed p38 and ERK 1/2 activity before and after NO induction. More importantly, MEK 1/2 inhibition by the SMI PD198306 partially suppressed cartilage OA pathology which was accompanied by suppression of ERK 1/2 and MMP-1 activity [135]. These results indicated that ERK 1/2 activation correlated with OA pathology, but a recent report by Longobardi et al. [136] showed that TGF-β1-mediated chondrocyte mitogenesis was mediated by ERK 1/2. The IGF-I response was regulated, in part, by ERK 1/2 suggesting that experimental manipulation of ERK 1/2 while dampening OA changes might also limit cartilage repair pathways. In summary, the cell culture and in vivo studies reviewed herein provide the impetus for future investigation in which MAP kinase SMIs with strong specificity for the various p38 kinase isoforms, ERKs and JNKs could be individually employed in OA animal models and then if shown to be efficacious and non-toxic in animals could then be employed in randomized controlled human OA clinical trials [8].

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VIII Transcriptional Control of Chondrocyte Gene Expression Mary B. GOLDRING a,* and Linda J. SANDELL b Research Division, The Hospital for Special Surgery, Weill College of Medicine of Cornell University, New York, New York, USA b Departments of Orthopaedic Surgery and Cell Biology and Physiology, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri, USA a

Abstract. During cartilage formation and maintenance, the expression of chondrocyte-specific genes, such as those encoding type II collagen (COL2A1), aggrecan, and cartilage-derived retinoic acid sensitive protein (CD-RAP), is regulated by both activators and repressors that interact with the promoter or enhancer regions of these genes. Cascades of both positive and negative transcription factors have been found to determine developmental events in the embryonic growth plate. The high mobility group protein Sox9, which is required for COL2A1 transcription along with l-Sox5 and Sox6, plays a key role in cartilage formation and maintenance, while Sp1 and the coactivator, CBP/p300, are required for constitutive activity. The bHLH, HOX, SMAD, ETS, and STAT families consist of both positive and negative regulators that directly or indirectly influence COL2A1 and CD-RAP during chondrogenesis and chondrocyte hypertrophy. In osteoarthritis, activation of mature articular chondrocytes may result in recapitulation of these developmental events and phenotypic modulation by the associated transcription factors. Cytokine-induced transcription factors, including NF-κB, C/EBP, ETS, and AP-1 family members that activate catabolic and proinflammatory genes, may then suppress chondrocyte phenotype and cartilage repair mechanisms by inhibiting expression of cartilage-specific genes. This review will focus on the transcriptional regulation of COL2A1 and CD-RAP genes by factors involved in cartilage formation and homeostasis, as well as in inflammatory and catabolic events that adversely affect cartilage integrity. Keywords. Gene regulation, chondrogenesis, chondrocyte, transcription factors, cytokines

Introduction As the unique cellular components of adult articular cartilage, chondrocytes are responsible for maintaining the structural and functional integrity of the cartilage extracellular matrix in physiological conditions [1,2]. The articular cartilage matrix is a complex * Corresponding Author: Mary B. Goldring, PhD, Hospital for Special Surgery, Caspary Research Building, Room 528, 535 East 70th Street, New York, NY 10021, USA, E-mail: [email protected].

M.B. Goldring and L.J. Sandell / Transcriptional Control of Chondrocyte Gene Expression

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mix consisting primarily of type II collagen (COL2A1) and other cartilage-specific collagens, type IX (COL9) and type XI (COL11). In addition, the large aggregating proteoglycan aggrecan makes up approximately 50% of the matrix, as well as a large number other collagens, small proteoglycans, and other non-collagenous proteins [1]. In the absence of joint inflammation or other pathology, the turnover and remodeling of the matrix components is very low, the half-life of collagen having been estimated at greater than 100 years [3,4]. The glycosaminoglycan constituents on the aggrecan core protein are more readily replaced and the half-life of aggrecan has been estimated to be in the range of 3 to 24 years [4]. In osteoarthritis (OA), there is a loss of the steadystate equilibrium between synthetic (anabolic) and resorptive (catabolic) activities resulting in increased metabolic activity and progressive destruction of the cartilage matrix (reviewed by Sandell [5]). Local loss of proteoglycans and cleavage of type II collagen occur initially at the cartilage surface resulting in an increase in water content and loss of tensile strength in the cartilage matrix as the lesion progresses. It is generally agreed that the cartilage damage is associated with increased production of proteinases, including the metalloproteinases (MMPs), MMP-1, MMP-3, MMP-8, MMP-13, and MMP-14 [6,7] and the aggrecanases, ADAMTS-4 and -5 [8,9]. Synovial fluids from patients with OA contain both aggrecanase- and MMP-generated aggrecan fragments [10]. MMP-13-specific type II collagen cleavage products and MMP-13 itself have been detected in OA cartilage [11,12]. In addition to the increased production of matrix-degrading proteinases in the early stage of OA, there is a transient increase in chondrocyte proliferation, as well as evidence of a general increase in synthetic activity and an alteration of the pattern of extracellular matrix synthesis, which is often interpreted as a repair response. Genomic and proteomic analyses of global gene expression have detected increased expression of the type II collagen gene (COL2A1) in early OA cartilage [13,14], possibly associated with the increased levels of anabolic factors such as bone morphogenetic protein (BMP) 2 and inhibin βA/activin [13,15]. These and other transforming growth factor (TGF)-β family members may stimulate aggrecan synthesis at the same time as promoting the formation of fibrocartilage and osteophytes, bony structures at the periphery of the joint surface. Type III collagen and type VI collagen, which are present at low levels in normal cartilage: the chondroprogenitor splice variant of the type II collagen gene, type IIA: and type X collagen, a marker of the hypertrophic chondrocyte that is normally absent in adult articular cartilage, have been detected during certain stages of OA or at atypical sites within OA cartilage [16,17]. Other genes associated with growth plate development, such as MMP-9 and Indian hedgehog (Ihh) are detected in the vicinity of early OA lesions, although the expression of Sox9, the master regulator of cartilage formation, is decreased and does not correlate with active type II collagen gene expression [7,18]. These observations have lead to the concept that the chondrocyte responds to early activation by attempting to revert to a progenitor phenotype and to recapitulate developmental events [16,19]. Multiple mechanisms are likely involved in the disturbance of chondrocyte remodeling activities in OA. It has been proposed that mechanical disruption of chondrocytematrix associations may lead to alteration of metabolic responses in the chondrocyte [20]. More rapid matrix turnover may occur in the immediate pericellular zones compared to the interterritorial zones of cartilage [12,21,22]. This suggests roles for chondrocyte cell surface receptors such as integrins and DDR2 in the response to mechanical stress that may result in the disruption of normal remodeling of matrix components [23–26]. In addition to acquired or age-related alterations in chondrocyte function and

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the effects of excessive mechanical loading, inflammation and accompanying dysregulated cytokine activities may also contribute to cartilage catabolism [27,28]. OA is not considered a classical inflammatory arthropathy, due to the absence of neutrophils in the synovial fluid and systemic manifestations of inflammation. However, synovitis is common in advanced OA involving infiltration of mononuclear cells, and expression of proinflammatory mediators is observed in early and late OA [29]. Evidence from numerous studies in vitro and in vivo indicates that interleukin-1 (IL-1) and tumor necrosis factor (TNF)-α are the predominant proinflammatory cytokines involved in the induction of cartilage-degrading proteinases. The balance of these cytokines in relation to anabolic factors may have profound effects on the ability of the chondrocyte to repair the degraded matrix. The following review will compare and contrast the transcriptional regulation of genes involved in cartilage matrix synthesis (COL2A1 and CDRAP) by anabolic and catabolic factors. 1. Transcription Factors Involved in the Regulation of Cartilage-Specific Genes During the past two decades, many of the transcription factors that control the expression of cartilage-specific genes have been discovered and characterized in studies in vitro and in vivo. Both positive and negative transcription factors have been found to determine developmental events during chondrogenesis, the process by which mesenchymal condensations form the cartilage anlagen, which eventually forms the cartilage of the articular joint or undergoes hypertrophy and endochondral ossification to form bone. These events are controlled by cascades of both activators and repressors that interact with the promoter or enhancer regions of chondrocyte-specific genes, including those encoding type II, type IX, and type XI collagen, aggrecan, and the cartilagederived retinoic acid sensitive protein (CD-RAP). The high mobility group (HMG) protein Sox9 plays a key role in cartilage formation and maintenance by permitting transcription of cartilage-specific genes such as type II and type IX collagens, aggrecan, and CD-RAP [30–34]. Sox9 activates COL2A1 transcription by binding to the first intron enhancer through its high mobility group (HMG) DNA-binding domain and acts cooperatively with L-Sox5 and Sox6 to regulate chondrogenesis in vivo [35,36]. These and other SOX genes are regulated in a dynamic fashion during chondrogenesis by members of the BMP/TGF-β family [37]. Other extracellular mediators that control chondrocyte differentiation include Indian hedgehog (Ihh) via PTHrP, Wnt proteins via β-catenin, and fibroblast growth factors (FGFs) via specific receptors that promote or suppress proliferation (see for review [38]). The anabolic effects of IGF-I, BMP-2, and FGF-2 on differentiated chondrocytes appear to be mediated, at least in part, by Sox9 [35,39–42]. The transcription factors involved in positive and negative regulation of chondrocyte differentiation to be discussed in the following sections are listed in Table 1. 1.1. Sox9, L-Sox5 and Sox6 During skeletal development, COL2A1 expression is regulated in a coordinated fashion by growth and differentiation factors, which modulate a series of transcriptional events requiring elements within both the promoter and first intron regions [36,43,44]. Sox9 plays an essential role during sequential steps of chondrocyte differentiation and the


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Table 1. Transcription factors involved in positive and negative regulation of chondrocyte differentiation Positive* HMG: Sox9, L-Sox5, Sox6 Zinc finger: Sp1

bHLH: USF1, USF2 Scleraxis DEC1 Homeodomain: Hoxa13, Hoxd13 Dlx-2 Cart1 ETS: C-1-1 SMADs: Smad1 Stat1

Sequence (A/T)(A/T)CAA(A/T)G GGGCG

CANNTG (E-box) CANNTG CACNAG TNATNN CNGTAANTG TAATNNNATTA GGAA CAGACA ATTCCTGTAAG

Negative** Runt: Runx2 (Cbfa1) Zinc finger: Sp3 cKrox AP-2α CRYBP1 NT2 Zfp60 Osx bHLH: δEF1 Twist Snail, Slug Homeodomain: Hoxc8, Msx2 Dlx5 ETS: Erg Retinoic acid receptors: (RAR/RXR) NFATp(c2) bZip: JunB, JunD Fra1, Fra2, FosB c-Maf C/EBPβ, C/EBPδ

Sequence TG(C/T)GGT GGGCG GGGAGGGGG GCCNNNGGC GAGAAAAGCC GAGGAGGGGAG

CACCTG CANNTG CAGGTG TAATNN TAATTA

GGAA AGGTCA GAGG TGAC/G

TTGAGAAA (COL2) TTGGGAAA (CD-RAP)

*Designates those factors that promote cartilage-specific gene expression (COL2A1, CD-RAP) and other events during chondrogenesis or in adult chondrocytes; many are induced by BMP/TGFβ family members. **Designates factors that repress cartilage-specific gene expression and may promote transition to chondrocyte hypertrophy prior to endochondral ossification.

Sox9-binding intron enhancer is required for COL2A1 expression during chondrogenesis in transgenic mice in vivo [35,36,45,46]. In mouse chimeras generated with Sox9-/embryonic stem cells, the mesenchymal progenitors lacking Sox9 are excluded from cartilage tissue and are unable to transcribe the COL2A1 gene [46]. L-Sox5 and Sox6 are co-expressed with Sox9 in differentiated chondrocytes and cooperate with Sox9 to fully activate the promoter and maintain of COL2A1 expression both in vitro and in vivo [35,47–49]. Several studies suggest that the proximal promoter may operate at specific times during development when negative regulation is required via promoter regions distinct from those required for positive regulation [36,50]. Sox9 also regulates the transcription of genes encoding type IX collagen (Col9a1) [31], type XI collagen (Col11a1) [32], aggrecan (Agc) [34], and matrilin-1 [51]. The cooperative effects of L-Sox5 and Sox 6, however, are not observed in some situations. For example, L-Sox5 and Sox6 are not required for Sox9-dependent lineage commitment and prechondrocyte differentiation in embryos [52], and the Col9a1 enhancer can be activated by Sox9

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dimers in the absence of L-Sox5 and Sox6 [53]. Our own work on factors controlling transcription of the COL2A1 gene has revealed both positive and negative regulatory domains, many of which are not dependent upon the status of Sox9 binding to the enhancer (Goldring & Sandell, manuscripts in preparation). Thus, the interactions of SOX proteins depend on the promoter context and the differentiation state of the chondrocyte. 1.2. Sp1 and Zinc Finger Proteins Binding sites for the ubiquitous transcription factor, Sp1, were among the first identified in type II collagen genes [54–56]. Sp1, a primary transcriptional regulator of gene expression, is responsible for stimulating or maintaining constitutive COL2A1 promoter activity and a related family member Sp3 represses Sp1-mediated transactivation by binding to the same site [57–60]. The zinc finger protein, cKrox, which also interacts with GC-rich sequences, activates COL2A1 transcription in differentiated chondrocytes but inhibits constitutive activity in subcultured (dedifferentiated) cells [61]. 1.3. Positive and Negative Regulation by Different Transcription Factor Family Members Transcription factors that are members of the homeobox (HOX), basic helix-loop-helix (bHLH), and ETS families may have positive or negative effects on cartilage-specific gene transcription. For example, the inappropriate expression of the C-1-1 variant of the ETS factor Erg can block chondrocyte hypertrophy and endochondral ossification [62–64]. HOX genes, which are important for patterning during limb development, enhance (Hoxa13 and Hoxd13) or suppress (Hoxa11 and Hoxd11) transcription from GC box-dependent promoters that drive expression of, for example, the BMP-4 gene by interacting with GC box-binding proteins such as Sp1 [65]. BMPs also regulate promoter activities via HOX proteins of the Dlx family [66]. Dlx2, which is stimulated by BMP-2, acts via the intron enhancer to increase COL2A1 expression [67], whereas Dlx5 and 6 promote transition to hypertrophy and Dlx5 binds to the Col10a1 promoter and increases its activity [68]. E-box motifs, which are consensus-binding sites for bHLH proteins, are present in promoter and enhancer regions of type II collagen and CD-RAP genes from different species [50,69]. The interaction of δEF1 with conserved E-box sites containing CACCTG or Snail family members Sna and Slugh with CAGGTG represses constitutive activity of the COL2A1 promoter [50,70]. The bHLH protein, scleraxis, can dimerize with other E box-binding proteins and is expressed at early stages of chondrogenesis in regions surrounding Sox9 [71–74]. DEC1 promotes chondrocyte differentiation at early and late stages in response to PTHrP and cAMP [75]. Differential expression of Id1, 2, 3, and 4 may influence chondrogenesis and phenotypic expression in mature chondrocytes and chondrosarcoma cells [76–78]. The nuclear factor of activated T cells NFATp(c2) suppresses chondrogenesis and inhibits aggrecan and COL2A1 gene expression in adult chondrocytes [79], whereas NFAT4 induces chondrogenesis by stimulating BMP expression [80]. Negative transcription factor activity on chondrocyte-specific genes is necessary for two reasons: (1) when required to down-regulate genes expressed in the chondrocyte and (2) to repress transcription in “non-chondrocytes”. The expression of most cartilage genes is very high during maturation of the growth plate and expansion of articular and hyaline cartilages. However, expression is much lower in hypertrophic


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Figure 1. Positive and negative transcriptional regulation during cartilage development.

chondrocytes and in mature cartilage tissue. In fact, the negative regulators, δEF1 and AP-2 are detected by immunohistochemistry in mature tissues and hypertrophic cartilage while the positive factor, Sox-9, is greatly reduced [43]. Factors that are known primarily to suppress chondrocyte differentiation while promoting hypertrophy include the retinoic acid receptor [81,82] and the zinc finger transcription factors, Zfp60 [83], NT2 [84], CRYBP1 [85], AP-2 [86–88], and Osterix (Osx) [89]. Activation of PPARγ, which interacts with retinoid X receptor (RXR), inhibits thyroid hormone-induced chondrocyte hypertrophy [90] and suppresses both chondrogenic and hypertrophic markers, while promoting adipogenesis [91]. This process may be mediated by the PPARγ co-activator 1α (PGC-1α), which acts as a coactivator for Sox9 during chondrogenesis and interacts directly with Sox9 to promote Sox9-dependent transcriptional activity [92]. AP-1 family members that are leucine zipper (bZip) proteins and can form heterodimers, including c-Fos, Fra1, Fra2, FosB, JunB and ATF-2, are important for the expression of Col10a1 and Mmp13 during chondrocyte hypertrophy in the mouse embryo [93–97]. Fra 1 and FosB increase Col10a1 promoter activity by binding to an AP-1 site [98]. MMP-13 is a target of c-Maf, which can form heterodimers with other bZip proteins and regulates the differentiation of hypertrophic chondrocytes [99]. Thus, chondrocyte-specific gene expression depends upon the balance of positive and negative factors interacting with the same DNA elements or with each other [38,100]. A diagram of expression patterns of various transcription factors during endochondral bone development is shown in Fig. 1.

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1.4. Negative Regulators in Tissue-Specific Gene Expression CD-RAP is a secreted protein expressed by chondrocytes with an expression pattern even more restricted than type II procollagen [69,101] and considered the most reliable marker for cartilage [102]. During chondrogenesis, CD-RAP is co-expressed with COL2A1, activated from the beginning of chondrogenesis, and expressed throughout cartilage development. Many regulatory elements characterized thus far in the promoter of the CD-RAP gene play similar roles to those in the COL2A1 or COL11A1 genes. For example, AP-2 at a low concentration is an activator, whereas it represses CD-RAP promoter activity at a high concentration [86]. Sox9 binds the CD-RAP promoter at –410 to –404 bp and activates transcription [103]. Upstream stimulatory factor (USF) and δEF1 with an E-box located at –487 to –482 bp and activate or repress CDRAP gene expression depending on the proportion of the USF to δEF1 in the nuclei (Li and Sandell, unpublished data). Studies in transgenic mice have revealed that a –2251 bp promoter directs tissuespecific expression of E. coli ß-galactosidase gene (LacZ) reporter gene, consistent with the endogenous CD-RAP gene expression pattern. A truncation to a –2068 bp promoter does not reliably express the reporter gene [104]. These results suggest that the 183 bp DNA fragment between –2251 and –2068 bp contains important elements that are responsible for tissue-specific expression of CD-RAP. When the 183 bp fragment is removed from the native –3345 bp promoter, which directs only cartilagespecific expression in vivo, the reporter gene is widely expressed in transgenic mice in muscle, bone, nerve ganglion, lungs and cartilage [105]. This fragment confers tissue specificity by repressing the expression of CD-RAP and COL2A1 in non-cartilage tissues and contains several HMG-like sites, which are targets for binding of L-Sox5, Sox6 and Sox9. Overexpression of these SOX proteins can activate CD-RAP promoter activity via the 183-bp fragment. However, this fragment also contains a negative regulatory site for the transcription factor, CCAAT/enhancer-binding protein (C/EBP). C/EBPβ and C/EBPδ are upregulated in chondrocytes by treatment with IL-1β, an inhibitor of CD-RAP expression, and all C/EBP isoforms repress gene transcription through the C/EBP site within the 183-bp element [106]. Therefore, C/EBP is another negative regulator for cartilage-specific genes. In fact, when the C/EBP binding site is removed from the CD-RAP promoter, the gene is expressed in the muscle cell line, C2C12 [105]. These results indicate that the presence of C/EBP in muscle and in other non-cartilage tissues may contribute to the lack of expression of CD-RAP and other cartilage-characteristic genes. The down-regulation of cartilage gene expression by C/EBPs under the influence of pro-inflammatory cytokines will be explored later in Section 2.4. 1.5. Protein-Protein Interactions and the Coactivator, CBP/p300 An additional control mechanism involves the coactivator, CREB-binding protein (CBP) or its paralogue, p300, which does not interact directly with promoter DNA sequences, but serves as a bridge between DNA-binding proteins and the RNA polymerase II transcriptional machinery. Through intrinsic histone acetylase (HAT) activity, CBP/p300 can directly acetylate the lysine residues of certain transcription factors that are generally activators of gene transcription, including cAMP-responsive binding protein (CREB), NFκB, c-Jun family members, C/EBPs, and SMADs, and thereby serves


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to integrate activities of various factors resulting in transcriptional synergy [107]. With regard to chondrocyte-specific gene expression, CBP/p300 increases transcriptional activities of the cartilage homeoprotein-1 (Cart1) [108] and BMP-responsive Smad1 [109]. CBP/p300 also potentiates transcription by acetylation-dependent loosening of the chromatin structure, and a recent study indicates that its interaction with Sox9 is required for COL2A1 promoter activity [110]. Both CBP and p300 elicit strong positive transcription of the CD-RAP and COL2A1 genes when expressed in chondrocytes. In fact, the expression levels exceed those induced by Sox proteins alone [59,111]. The mechanism for the increase in gene transcription involves both the positive regulator Sox9 and the negative regulator C/EBP. The CBP or p300 acts as a co-regulator by binding to the DNA-binding transcription factors and increasing transcription by different mechanisms depending on the target gene and availability of other factors. After binding to CBP or p300, Sox9 binds to DNA with higher affinity, thereby increasing gene transcription [110]. The binding of p300 or CBP to C/EBP inhibits the binding of this transcription factor to the DNA thereby sequestering and rendering inactive the negative regulator [111]. 1.6. Transcription Factors in the Growth Plate Negative regulators of COL2A1, including Cbfa1/Runx2, δEF-1, C/EBP, and AP-2, are highly expressed in hypertrophic cartilage [43], suggesting that down-regulation of chondrocyte-specific genes is necessary before mineralization can occur. For example, Runx2, together with Runx3 in the embryonic growth plate, stimulates chondrocyte terminal differentiation [112–114] and increases the expression of type X collagen and MMP-13 in hypertrophic chondrocytes [115–117]. Although Sox9 is a dominant transcriptional activator for chondrogenesis, it also acts as a repressor of Runx2 activity during chondrocyte hypertrophy by directly interacting through the HMG domain with the runt domain of Runx2 [118]. Runx 2 partners with SMADs to regulate the type X collagen gene in response to BMPs [119]. However, there is no SMAD site on the Runx2 promoter, and many of the positive and negative regulators of chondrocyte hypertrophy modulate Runx2 transcriptional activity. For example, Dlx3 is an activator when it binds directly to the promoter of a target gene, where interaction with Runx2 can reduce Runx2-mediated transcriptional activation [120]. The homeodomain protein Nkx3.2, which is an early BMP-induced signal required at the onset of chondrogenesis, is a direct transcriptional repressor of Runx2 promoter activity [121,122]. The bHLH factor Twist inhibits chondrogenesis [123] but prevents premature osteoblast differentiation by transiently inhibiting Runx2 function [124]. Twist-1 is expressed exclusively in the perichondrium, where it favors chondrocyte hypertrophy in a Runx2-dependent manner, but paradoxically, it blocks Runx2 activation of the Fgf18 promoter by Runx2 [125]. Cooperation of the Groucho homologue Grg5 or the leucine zipper protein ATF4 with Runx2 promotes chondrocyte hypertrophy [126] or osteoblast differentiation [127], respectively. Histone deacetylase 4 (HDAC4), which is expressed later in prehypertrophic chondrocytes, prevents premature chondrocyte hypertrophy by interacting with Runx2 and inhibiting its activity [128]. The canonical Wnt/β-catenin-induced TCF/Lef transcription factors suppress chondrocyte differentiation in early chondroprogenitors and promote chondrocyte hypertrophy at later stages and subsequent endochondral ossification [129,130] by binding to the Runx2 promoter [131]. Runx2 interacting with BMP-induced Smad1 is required for the induction of GADD45β, a survival factor in hypertrophic chondrocytes that acts as a transcription factor during the induction of

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MMP-13 and COL10A1 [132]. The inhibition of COL2A1 promoter activity and gene expression by GADD45β [133] suggests indirect and unexpected mechanisms by which BMPs and TGFβ may regulate chondrogenesis. The expression patterns of BMP-2, 4-, 6 and 7 contribute to progressive chondrocyte differentiation at different stages of cartilage development, and the local regulation of their overlapping activities is governed by extracellular BMP antagonists, including noggin, follistatin, chordin and twisted gastrulation. Depending upon the differentiation stage of the chondrocyte, BMP-2 can induce COL2A1 or COL10A1 via Sox9 or Runx2, respectively, presumably in cooperation with Smad1, 5, or 8 [134]. In contrast, TGFβ, through phosphorylation of Smad2 and 3, acts as an early mediator of chondrogenesis, but inhibits chondrocyte hypertrophy and COL10A1 expression. TGFβ induces aggrecan gene expression via cross-talk between Smad2 and the ERK1/2 and p38 MAPK pathways during differentiation in the chondroprogenitor ATDC5 cell line [135]. Although BMPs induce type II collagen and proteoglycan synthesis in differentiated articular chondrocytes, direct binding of either TGFβ- or BMP-induced SMADs to gene promoters has been difficult to prove. In fact, TGFβ inhibits COL2A1 promoter activity by decreasing the ratio of Sp1 to Sp3 [58], but increases type II collagen synthesis by a translational or post-translational mechanism involving the TGFβ-activated kinase 1 (TAK1) [136]. Recent findings indicate that TGFβ-induced Smad3 enhances Sox9 transcriptional activity and COL2A1 expression by interacting with Sox9 and enhancing the association between Sox9 and CBP-p300 [137]. The T-box transcription factor, Brachyury, another factor that is induced by BMP-2, is upregulated during early stages of FGFR3-mediated chondrogenesis in the osteochondroprogenitor cell line, C3H10T1/2 [138]. The inhibition of chondrocyte proliferation by FGFR3 prior to hypertrophy involves Stat1 [139] and IFNγ-induced down-regulation of COL2A1 transcription requires Stat1 and its activation by the kinases Jak1 and Jak2 [140]. Together, these and other findings suggest the complexity of the signaling pathways and downstream transcription factors that are involved in regulation of the gene expression in chondrocytes during development and postnatal growth.

2. The Regulation of Chondrocyte Phenotype by Proinflammatory Cytokines The proinflammatory cytokines, IL-1β and TNFα, are in involved in the destruction of the articular cartilage in both rheumatoid arthritis and OA [141,142]. The chondrocyte is the cellular target of cytokine action in cartilage, and IL-1β and TNFα and their receptors colocalize with MMP production in superficial regions of OA cartilage [11]. IL-1β suppresses the expression of a number of genes associated with the differentiated chondrocyte phenotype, including COL2A1 and CD-RAP [106,143,144]. Early studies in vitro showed that IL-1 and TNFα are capable of inhibiting the synthesis of type II collagen by chondrocytes by suppressing gene transcription and the levels of associated mRNAs [143–146]. IL-1 and TNFβ also stimulate the synthesis of prostaglandin E2 (PGE2), which feedback-regulates COL2A1 transcription in a positive manner [147,148].


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2.1. The Regulation of Chondrocyte Phenotype by Cytokine-Induced Signaling Pathways IL-1 and TNFα share the capacity to activate a diverse array of intracellular signaling pathways, although the cell surface receptors and associated adaptor molecules are distinct (see Chapter 1). In chondrocytes, the JNK and p38 MAPK signaling pathways predominate in the regulation of IL-1 and TNFα-induced genes. The inhibition of COL2A1 expression by IL-1 or TNFα in chondrocytes involves the p38 [149], JNK, and NFκB pathways [150]. Injurious mechanical stress and cartilage matrix degradation products are capable of stimulating the same signaling pathways as those induced by IL-1 and TNFα [151–158]. Since these pathways also induce or amplify the expression of these cytokine genes, it remains controversial whether inflammatory cytokines are primary or secondary regulators of cartilage damage and defective repair mechanisms in OA [159]. Based on studies in animal models [160,161] and analysis of cartilage samples or body fluids from OA patients [162–166], controversy exists about whether type II collagen, or the type IIA chondroprogenitor variant, is elevated or suppressed, appearing to depend upon the zone of cartilage analyzed and the stage of OA. The early increase in anabolic activity in OA cartilage may be associated with increased expression of BMP-2 induced by IL-1 and TNFα [15]. BMP-2 would, in turn, activate of COL2A1 transcription and permit interaction of the COL2A1 promoter with cytokine-induced factors. Although signaling via the p38 MAPK pathway can regulate gene expression by post-transcriptional mechanisms [167], phosphorylation events that are downstream of ligand binding to cytokine receptors may result in induction and activation of transcription factors that bind to DNA elements of target genes, as discussed below. 2.2. The Regulation of Chondrocyte Phenotype by Cytokine-Induced Transcription Factors Cytokine-activated transcription factors of the NFκB/c-Rel, C/EBP, ETS, and AP-1 families, which mediate the induction of MMPs, cyclooxygenase (COX) 2, and nitric oxide synthase (NOS) 2 by IL-1 and TNFα [168–172], may also be involved in suppressing the transcription of COL2A1 and CD-RAP [59,106] (Table 2). IL-1 may also induce chondrocytes to synthesize other cytokines such as IL-6, which together with the soluble IL-6 receptor that is not present in sufficient amounts in cultured cells, down-regulates COL2A1, aggrecan, and link protein via the JAK/STAT pathway in association with suppression of Sox9 [173]. Studies in knockout or transgenic mice suggest that IL-1 or TNFα are not involved in the formation of articular cartilage in the embryo. However, the cartilage remodeling initiated in response to trauma or inflammation may involve inactivation of the chondrogenic transcription factors, via direct or indirect interactions with cytokine-induced transcription factors. It has been proposed that inhibition of Sox9 expression by IL-1 determines the regulation of COL2A1 gene transcription by these cytokines [41,174,175], although expression of Sox9 and COL2A1 does not always correlate [59,140,176,177]. Recent evidence indicates that Sox9 overexpression in chondrocytes may either increase or decrease COL2A1 transcription depending upon its concentration and the differentiation state of the cells [178]. During chondrocyte hypertrophy, however, the suppression of Sox9 expression and activity by transcription factors such as Runx2 has been proposed as a

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Table 2. Cytokine-induced transcription factors that influence chondrocyte gene expression Transcription factor*

Cytokine

Sequence

NFκB (p65/p55) AP-1 family (AP-1, ATF-2)

IL-1, TNFα, IL-17, IL-18 IL-1, TNFα, IL-17, IL-18 IL-1, TNFα, IL-17, IL-18 IL-1, TNFα, IL-1, TNFα,

GGGRNNYYCC TGACTCA TTG(A/G)GCAAA GGAA GCGGGGGCG

IFN-γ Oncostatin M

TTTCATATTACTCT TTCTGGGAATT

C/EBPβ, C/EBPδ ETS (Ets-1, PEA-3, ESE-1) Egr-1 STATs: Stat1 Stat3

*These transcription factors are generally negative regulators of COL2A1 or CD-RAP expression in adult chondrocytes.

mechanism essential for endochondral ossification [179,180]. Although Runx2 is required for IL-1 induction of MMP-13 gene transcription in articular chondrocytes [169], its role in the suppression of chondrocyte phenotype by inflammatory cytokines has not been defined definitively. 2.3. Early Cytokine-Induced Transcription Factors: NFκB, AP-1, and EGR-1 The understanding of transcriptional regulation by cytokines in chondrocytes and other connective tissue cells is incomplete, even with regard to genes that have been well studied, such as the MMPs. Early work in other systems suggested that NFκB and AP1 are primary response factors for the regulation of IL-1β-induced genes. Further work showed that AP-1 (c-Jun/c-Fos), one of the first transcription factors studied, was insufficient for IL-1β -induced MMP-1 gene expression by fibroblasts, [181]. Furthermore, it has not been possible to attribute the regulation of a significant number of cytokine-responsive genes to direct interaction of NFκB or AP-1 with DNA elements, including those in the COL2A1 promoter, which does not contain functional binding sites for these transcription factors. NFκB binds to and activates the BMP-2 promoter [182], but it also down-regulates Sox9 expression by posttranscriptional destabilization of mRNA [183]. TGFβ expression is also induced by IL-1β through activation of the bHLH factor AP-4, which binds to a site overlapping with an AP-1 site [184]. The Jun activation domain-binding protein 1 (Jab1), which is a coactivator of c-Jun/AP-1, inhibits BMP signaling by binding to Smad5 [185]. These findings suggest alternative mechanisms. Recently, we found that IL-1β-induced and activated EGR-1, an immediate early growth response factor, inhibits COL2A1 promoter activity by binding to the – 131/+125 bp core promoter and displacing Sp1 from at least one of the GGGCG boxes that overlap with the EGR-1 binding site [59]. This mechanism may also account for the increased ratio of Sp3 to Sp1 binding to the Sp1 sites observed in response to IL-1 [60]. Since overexpression of CBP reverses the inhibition, it is probable that EGR-1 acts by disruption of the interactions among Sp1, CBP, and TATA-binding proteins [59]. This early response appears to be transient, suggesting that complete transcriptional repression of COL2A1 promoter activity may be dependent upon the binding of other IL-1β-induced factors to upstream promoter sequences (Fig. 2).


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Figure 2. Mechanism of suppression of COL2A1 transcription by IL-1β in cartilage.

2.4. Inhibition of Chondrocyte Phenotype by C/EBP and ETS Factors Other candidate IL-1-induced transcription factors include C/EBPβ and C/EBPδ, which downregulate the expression of both CD-RAP and COL2A1 [106], and ETS factors. These factors are present at low or undetectable concentrations in the cytoplasm and are part of a cascade of cytokine-induced genes that are normally induced at intermediate and later time points. The subsequent sustained expression suggests involvement in maintenance of the transcriptional response. C/EBP and ETS factors function as activators in the context of cytokine-induced genes such as COX-2 [168,186] and MMPs [169,181,187]. The ETS factors constitute a family of at least thirty members that play central roles in regulating genes involved in development, differentiation and cell proliferation [188]. Several ETS factors, including ETS-1 and PEA3, cooperate with AP-1 in regulating MMP gene expression [169,181,187]. ESE-1, also known as ESX, ELF-3, ERT, and JEN, is a novel ETS factor that is restricted to epithelial tissues under physiological conditions [189,190]. ESE-1 is expressed in non-epithelial tissues undergoing inflammation such as rheumatoid and, to a limited extent, OA synovium and in chondrocytes, as well as glioma cells, smooth muscle cells, synovial fibroblasts, osteoblasts, and monocyte-macrophages, after treatment with IL-1β, TNFα, or lipopolysaccharide (LPS) [172]. This induction relies on the translocation of the NFκB family members, p50 and p65, to the nucleus and transactivation of the ESE-1 promoter via a high affinity NFκB binding site [172,191]. Following induction, ESE-1 can directly activate transcription of NOS2 [191] and COX2 [186] by binding to two or more functional ETS sites in the respective promoters. Together these studies indicate that increased expression of these IL-1β-induced genes is mediated indirectly by NFκB via induction of

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ESE-1, which then serves as a primary transcription factor that binds to and regulates promoter activity of the target gene. ESE-1 acts as a direct repressor of COL2A1 promoter activity by binding to at least two tandem ETS sites upstream of –131 bp and accounts, in part, for the sustained suppression by IL-1β (Fig. 2). Previous studies have shown that IL-1β-induced NFκB inhibits COL2A1 gene expression by suppressing Sox9 promoter activity [174] or by destabilizing Sox9 mRNA [183]. Consistent with those findings, adenoviral overexpression of IκB in chondrocytes blocks the suppression of COL2A1 mRNA by IL-1β [192]. However, the constitutive levels of Sox9, L-Sox5 and Sox6 mRNA are not suppressed by IL-1β, similar to findings for IFN-γ [140], and overexpression of the three SOX proteins, which bind to the intronic enhancer [35], does not reverse the inhibition of COL2A1 activity by IL-1β [193]. Thus, once the promoter is activated, the overexpression of Sox9 with L-Sox5 and Sox6 may further enhance constitutive expression of COL2A1 but the inhibition by IL-1β-induced factors cannot be overcome. A recent study also showed that NFκB is not required for modulation of Sox9-dependent COL2A1 promoter activity by Bcl-2 [194]. Whereas ESE-1 is a potent inducer of events associated with inflammation and tissue destruction [172,186,191,195,196], C/EBP proteins act as negative regulators of chondrogenesis during skeletal development, where IL-1β is not known to play a role [105,111]. Both IL-1β and TNFα increase the expression and protein concentration of the C/EBPs, but the mechanism of action is somewhat different from that of ESE-1. IL-1β stimulates C/EBPβ and δ synthesis in a dose- and time-dependent manner over 48 hours [105]. Significant increases in C/EBPβ and δ and repression of CD-RAP and COL2A1 are observed by 24 hours, suggesting that the C/EBPs are later regulators than ESE-1. Chromatin immunoprecipitation of endogenous DNA shows that the IL-1-responsive binding site in the CD-RAP promoter is within the 183 bp element at –2251/–2068 bp discussed previously [111], in addition to a second element in the 169 bp fragment at –1062 bp. Interestingly, TNFα also induces C/EBP expression and down regulates CD-RAP and COL2A1; however, the -1062 bp site is the only target binding site of the TNFα-induced C/EBP [197] (Fig. 3). Thus, both pro-inflammatory cytokines can act together to further decrease expression of the cartilage-characteristic proteins, but the mechanism of action is somewhat different and, therefore, additive. 2.5. Protein-Protein Interactions Involved in the Suppression of Chondrocyte Phenotype by Cytokine-Induced Transcription Factors Cytokine-induced C/EBPs and ETS factors act as repressors partly by blocking proteinprotein interactions among Sp1, Sox9, CBP, and the basal transcriptional machinery. The early cytokine-activated events are usually associated with positive responses, but in differentiated chondrocytes at a post-developmental stage of low matrix turnover, the COL2A1 promoter cannot respond to negative regulation by cytokines unless it is activated. In cytokine-induced genes, the assembly of higher order nucleoprotein complexes orchestrated by high mobility group (HMG)-I(Y) factors may be important for integrating the responses to the induced signaling pathways [198,199]. Similarly, Sox9 and related HMG factors are architectural proteins that act to maintain the nucleosomes in an open configuration, thereby exposing the endogenous, chromatin-integrated COL2A1 promoter to constitutive factors that interact directly with the promoter [200]. Thus, the transiently transfected promoter, which serves as a convenient model for dis-


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Figure 3. Mechanism of suppression of CD-RAP transcription by TNFα in cartilage.

secting constitutive and regulatory elements, would not be under the same constraints in vitro as in vivo. For example, the COL2A1 promoter constructs express strongly in chondrocytes even in the absence of the Sox9-binding intronic enhancer, suggesting that the promoter is maximally active when expressed ectopically. Constructs spanning the promoter through the first intron express at 10 to 20% of the activities of constructs containing the promoter alone. However, overexpression of combinations of Sox9 with L-Sox5 or Sox6 increases the activity of the complete construct without reversing the inhibition by IL-1β-induced factors [193]. Similar to other ETS factors such as ETS-1, which binds to two cysteine-histidine rich regions of CBP [201], ESE-1 can interact with CBP/p300 [202]. CBP/p300 acts as a positive regulator of chondrocyte-specific gene expression by interacting with the P/Q/S-rich region in the carboxy-terminus of Sox9 [110] and by binding to and sequestering negative factors such as C/EBP [111]. Although CBP overexpression interferes with IL-1β-induced activation of Egr-1 and upregulates constitutive COL2A1 promoter activity [59], it does not reverse the strong suppression by ESE-1. CBP/p300-dependent activity requires HDAC, which may modulate CBP/300 activity directly or influence its interactions with Sox9 [203]. Similarly, the CBP/p300-Ets-1 complex, which exhibits functional HDAC activity, promotes chromatin remodeling and modification of transcription factors and adaptor proteins [204], In endothelial cells, IL-1β upregulates p300 expression [202], possibly accounting for the incomplete inhibition of COL2A1 promoter activity by IL-1β n chondrocytes. In addition to affecting chromatin-DNA structure, HMG proteins also act to promote transcription by recruiting transcriptional regulators such as NFκB, ATF-2/c-Jun, and interferon regulatory factor-1 that bind directly to DNA. ESE-1 has two DNA binding domains, a classical ETS domain that would bind the ETS sites in the COL2A1 promoter, and an A/T hook domain that is found also in HMG proteins and recognizes the A/T-rich region of double-stranded DNA [189]. GST-ESE-1 pull-down assays using truncated Sox9 fragments indicate that ESE-1 also interacts with the HMG box

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toward the N-terminus of Sox9, which is distinct from the CBP/p300-interacting site in the C-terminus [110]. These results suggest that ESE-1 does not interfere with Sox9 binding to CBP, but since ESE-1 also binds CBP/p300, it could serve to sequester both proteins and prevent their participation in promoter activation (Fig. 2). Experiments showing that ESE-1 still suppresses COL2A1 promoter activity by ESE-1 in the presence of excess CBP and Sox proteins indicate that it cannot serve as a bridge to enhance complex formation. Other ETS factors, which could serve as positive regulators in the maintenance of constitutive expression, may compete for ESE-1 binding sites. The different roles that ETS factors play in chondrogenesis, as well as in regulating the expression of MMPs and other collagen genes, suggest that the promoter context and the relative concentrations of different factors determine the extent of repression or activation of gene transcription by ETS factors [64,205,206].

3. Conclusion In this review, we have focused on transcriptional regulation of the COL2A1 and CD-RAP genes during cartilage development and in adult articular cartilage. The factors that are involved in the induction and maintenance of chondrocyte phenotype may be similar in both processes, but the balance of transcriptional activators and suppressors is disrupted when the chondrocyte is activated during OA and attempts to respond to the changes in the microenvironment. The involvement of the IL-1-induced transcription factors, such as NF-κB, C/EBPβ and δ, ESE-1, and EGR-1, is consistent with early cytokine-activated events that are usually associated with gene activation but produce a negative response in the context of the COL2A1 or CD-RAP promoter. Although outside the scope of this review, these same transcription factors could also contribute to the expression of non-cartilaginous genes such as types I and III collagens, which are increased by IL-1β in chondrocytes [143], particularly in the superficial zone, and further contribute to the pathogenesis and progression of OA. The differential activation of upstream signaling events that result in induction of these transcription factors could explain the synergy and redundancy in cytokine responses. While proinflammatory cytokines stimulate potent negative transcriptional regulators of COL2A1 promoter activity, they may also stimulate the production of PGE2 and BMP-2, both of which stimulate COL2A1 expression and may blunt the effects of the negative regulators. Therefore, in the context of OA cartilage, the initial events that activate chondrocyte synthetic activity probably result in activation of the normally inactive COL2A1 promoter, which would then be susceptible to transcriptional repression. Thus, we conclude that multiple alternative mechanisms exist for the regulation of chondrocytespecific genes in adult articular cartilage once the synthetic activities of chondrocytes are activated globally. Since transcription factors induced by proinflammatory cytokines also upregulate genes associated with catabolic and inflammatory responses, including COX2, MMP13, and NOS2, and similar signaling pathways may be induced by adverse mechanical stress, the dissection of the molecular mechanisms involved may lead to the development of targeted therapies for blocking destruction of the cartilage matrix and promoting its repair.


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Acknowledgements Dr. Goldring’s research is supported by NIH grant R01-AG022021 and the Arthritis Foundation. Dr. Sandell’s research is supported by NIH grants R01-AR36994 and R01-AR045550.

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IX Gene Expression Profiling of Human Articular Chondrocytes and Osteoarthritis Sergio A. JIMENEZ * and Sonsoles PIERA-VELAZQUEZ Thomas Jefferson University, Jefferson Institute of Molecular Medicine, Philadelphia, PA 19107 Abstract. The recent development of high throughput genomic profiling technologies such as cDNA microarrays combined with advanced computational approaches have provided basic and clinical investigators with the ability to identify and characterize high-resolution expression profiles of numerous disease states and to dissect molecular networks that underlie specific disease phenotypes. In the field of osteoarthritis (OA) and cartilage research, the application of microarray technology holds the promise that it may allow the identification of molecular signatures specific for OA in articular cartilage chondrocytes which could provide clues to the elucidation of the pathogenetic mechanisms involved or responsible for the disease. Some of these molecular signatures may also be of great value in patient management and clinical care by providing potential biomarkers of utility as diagnostic or prognostic tools and as markers of the effectiveness of disease modifying therapies for OA. The aim of this chapter is to provide an overview of the relatively few investigations that applied microarrays to the study of human articular cartilage and OA. Keywords. Functional genomics, gene expression profiling, cDNA microarrays, osteoarthritis, chondrocytes, articular cartilage, cytokines, growth factors

Introduction The recent development of high throughput genomic profiling technologies such as cDNA microarrays [1–3], combined with advanced computational approaches [4–7], have provided basic and clinical investigators with the ability to identify and characterize high-resolution expression profiles of numerous disease states and to dissect molecular networks that underlie specific disease phenotypes. Within a few years following their introduction, microarrays are now routinely used in almost every line of biomedical research with the most impressive examples of the successful utilization of this technology in cancer research [8–13]. In addition to tremendous advances in clinicallyoriented studies, the introduction and widespread application of microarray technology in basic research has allowed the identification and characterization of many molecular * Corresponding Author: Sergio A. Jimenez, MD, Thomas Jefferson University, Jefferson Institute of Molecular Medicine, 233 S. 10th Street, Room 509 BLSB, Philadelphia, PA 19107-5541, Phone: 215-503-5042, Fax: 215-923-4649, E-mail: [email protected].

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pathways and of novel targets for therapeutic intervention. In the field of osteoarthritis (OA) and cartilage research, the application of microarray technology holds the promise that it may allow the identification of molecular signatures specific for OA in articular cartilage chondrocytes which could provide clues to the elucidation of the pathogenetic mechanisms involved or responsible for the disease. Some of these molecular signatures may also be of great value in patient management and clinical care by providing potential biomarkers of utility as diagnostic or prognostic tools and as markers of the effectiveness of disease modifying therapies for OA. The aim of this chapter is to provide an overview of the relatively few investigations that applied microarrays to the study of human articular cartilage and OA, ranging from basic studies in characterization of the chondrocyte phenotype and chondrogenesis to the analysis of the effects of cytokines and growth factors on various aspects of chondrocyte biology and finally to the assessment of human OA tissues.

1. Gene Expression Analysis of Chondrocyte Differentiation and Chondrocyte Phenotype Articular cartilage chondrocytes are highly differentiated cells responsible for the maintenance of the integrity of the tissue extracellular matrix (ECM). To produce and maintain a properly functional articular cartilage matrix the chondrocyte normally displays a specific pattern of gene expression. This pattern changes dramatically in response to structural or mechanical alterations in their surrounding matrix and in response to various growth factors and cytokines [14,15]. The chondrocyte response may lead, in certain situations, to longstanding changes in the phenotype of the cell, and therefore to an inability to properly repair or maintain the cartilage ECM. This is exemplified by the phenotypic changes in the patterns of production or in the temporal or spatial distribution of the synthesis of interstitial collagens, fibroblast-type proteoglycans, and production of ECM proteins that occur during the development of OA, de-differentiation in culture, or in response to cartilage injury [16–19]. Thus, the maintenance of the chondrocyte-specific phenotype plays a crucial role in the preservation of the normal structure and biomechanical properties of articular cartilage and very likely also in the pathogenesis of tissue destruction in OA. Culture of chondrocytes in monolayers for prolonged periods or upon repeated passages leads to the loss of their spherical shape and to the acquisition of an elongated fibroblast-like morphology accompanied by profound biochemical changes including the arrest of the synthesis of the cartilage-specific collagens (types II, IX and XI) and proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III and V), and an increase in the synthesis of fibroblast-type proteoglycans (versican) at the expense of aggrecan [20,21]. The chondrocyte phenotype can be re-expressed in these cells by culturing them in suspension, agarose, alginate beads, or on a hydrogel substrate [22–24]. These changes in the biosynthetic profile of de-differentiated chondrocytes resemble some of the phenotypic changes seen in OA chondrocytes [17–19]. Numerous studies have investigated the mechanisms that underlie the phenotypic instability of chondrocytes employing microarray gene expression analysis of chondrocytes cultured under conditions that allow them to either preserve their differentiated phenotype or under conditions that lead to their de-differentiation. In a pioneering paper, Stokes et al. described studies which examined the gene expression profile of differentiated versus de-differentiated chondrocytes in vitro comparing chondroctyes cul-

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Table 1. Relevant genes that exhibit a 2-fold or greater difference in expression between differentiated and de-differentiated human fetal chondrocytes (HFCs) Higher Expression in Differentiated HFCs Gene Extracellular matrix proteins Matrilin 3 COL11A2 Dermatan sulphate proteoglycan-3 Fibromodulin Col11α1 Col9α2 Aggrecan Chondroitin sulfate proteoglycan-3 Col9α3 Cartilage linking protein-1 COMP Matrilin 1 Transcription factors Hypoxia-inducible factor 1α Sox-9 RING zinc finger protein RZF Hox-B6 Zinc finger protein 35 MADS/MEF2-family transcription factor

Higher expression in De-differentiated HFCs

Fold 27.6 11.3 6.5 5.9 4.9 4.9 4.4 2.5 2.5 2.2 2.1 2.1

Gene

Fold

Extracellular matrix proteins Hexabrachion Chitinase 3-like protein 1 Fibrillin 1 Fibulin 1 Collagen Iα1

4.7 4.4 2.8 2.5 2.1

Transcription factors 3.4 3.5 2.2 2.1 2.0 2.0

Growth factors/cytokines/extracellular mediators Frizzled-related protein 10.2 IGF-II

6.3

Melanoma growth reg protein (CD-RAP) BMP-6 Adhesion proteins Del-1 integrin binding protein Epithelial V-like antigen (EVA)

3.9 2.0

TWIST Freac-4 RXR-β

2.6 2.1 2.0

Growth factors/cytokines/extracellular mediators Insulin-like growth factor-binding protein 4 3.6 precursor Insulin-like growth factor binding protein 3 2.6 precursor

Adhesion proteins 3.5 3.4

Cadherin 11

2.8

tured on polyHEMA, which allows the preservation of the chondrocyte-specific phenotype, with chondrocytes cultured as monolayers on tissue culture plastic to induce the loss of their phenotype [25]. In these studies, the microarray hybridization was performed employing the UniGEM Human V Microarray (Genome Systems, Inc.) which contains approximately 5000 known genes and 3000 ESTs. Table 1 lists the genes which showed a 2-fold or greater difference in expression grouped in the following four categories: (1) extracellular matrix proteins, (2) transcription/gene regulatory factors, (3) growth factors/cytokines/extracellular signaling molecules, and (4) cell adhesion proteins. A large number of chondrocyte-specific ECM protein genes were down-regulated whereas numerous genes encoding ECM proteins associated with the fibroblastic phenotype including COL1A1 and tenascin were upregulated in de-differentiated chondrocytes. Among the gene regulatory factors whose mRNA levels were differentially regulated were the chondrogenic factor SOX9, the O2-regulated hypoxia-inducible factor 1α (HIF-1α), the basic-helix-loop-helix transcription factor Twist, the winged-helix transcription factor Freac-4 and the retinoic acid receptor RXR-β. In the category of

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Figure 1. Northern analysis of mRNA isolated from differentiated and de-differentiated human chondroctyes hybridized with cDNA probes for human COL2A1 and TWIST (A); IGF-II, IGFBP-4, RXR-β and BMP-6 (B); and HIF-1α (C). Hybridization with GAPDH was performed to control for differences in sample loading. pH: polyHEMA (differentiated); Pl: tissue culture plastic (de-differentiated). Adapted from ref. [25]. (Reproduced with permission from the publisher).

growth factors/cytokines/extracellular signaling molecules, differentiated chondrocytes expressed higher levels of transcripts for the frizzled-related protein, FRZB, and for insulin-like growth factor-II (IGF-II), cartilage derived-retinoic acid induced protein (CD-RAP) and bone morphogenetic protein-6 (BMP-6). Interestingly, higher levels of the transcripts for two insulin-like growth factor binding-proteins, IGFBP-3 and 4 were observed in the de-differentiated chondrocytes. In the category of cell adhesion proteins there was an increase in the levels of transcripts for the bone-associated adhesion protein, cadherin-11, in the de-differentiated chondrocyte cultures. Northern-blot analyses of transcripts from selected genes that showed substantial differential expression were performed to validate the microarray results. Figure 1 shows the results of Northern hybridization analyses for expression of COL2A1, TWIST, IGF-2, IGFBP-4, BMP-6, RXR-β, and HIF-1α transcripts in differentiated vs. de-differentiated chondrocytes. In summary, these extensive microarray studies documented the dramatic change in phenotype of chondrocytes during differentiation and de-differentiation in vitro as evidenced by the down-regulation of numerous genes associated with the ECM of cartilage and the up-regulation of ECM genes associated with an undifferentiated mesenchymal cell phenotype. Transcription and regulatory factors that showed differential expression between the two cell states such as SOX9, TWIST, and HIF-1α were of substantial interest as it is likely that they might play a role in controlling the phenotypic difference. The validity and applicability of these results to human OA was documented by the demonstration by PCR that many of the differentially expressed transcripts could be detected in fresh normal and OA articular cartilage or in freshly isolated chondrocytes prepared from these tissues (Fig. 2). For example, it was notable that TWIST expression was substantially increased in articular cartilage samples from patients with OA, providing strong confirmation of the functional relevance of the microarray results obtained during de-differentiation of human chondrocytes in culture.


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Figure 2. RT-PCR analysis of the expression of selected genes in intact adult human normal and OA cartilage and in chondrocytes isolated from these tissues. Total cellular RNA was isolated directly from cartilage samples of OA patients or from freshly isolated OA chondrocytes. (A) RT-PCR analysis for the expression of HIF-1α, IGFBP-3, IGFBP-4, IGF-2, TWIST, Del-1 and β-Actin in RNA directly isolated from the cartilage of four different patients with OA. (B) RT-PCR analysis for the expression of cadherin-11 as in (A). (C) RTPCR analysis for expression of TWIST in RNA from freshly isolated chondrocytes from adult human normal (N) and OA cartilage (OA). From ref. [25]. (Reproduced with permission from the publisher).

In a similar study, Finger et al. [26] performed DNA microarray analysis to investigate the molecular phenotype of a human chondrocyte cell line derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen (Line C-20/A4) cultured as de-differentiated chondrocytes (monolayer) or as differentiated cells (cultured on alginate beads). In these studies, the Clontech Human Cancer Array 1.2 was employed and the results were validated employing quantitative PCR. These investigators found that in monolayer cultures, numerous genes involved in cell proliferation were strongly upregulated. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated when the cells were cultured in alginate beads, findings consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. These results indicated that although immortalized chondrocyte cell lines were not identical to primary chondrocytes in their patterns of gene expression, they may nevertheless serve as valuable models for examining chondrocyte function and pathophysiology when the scarcity, lack of availability and loss of the chondrocyte-specific phenotype with serial passages limit the use of primary chondrocytes. Gene expression profiles during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patients undergoing an autologous chondrocyte transplantation therapy were studied by Tellheden et al. [27]. Monolayer expanded human articular chondrocytes from four donors were cultured in a pellet system and the redifferentiation was investigated by microarray analysis. The culture expanded chondrocytes redifferentiated in the pellet model and the gene expres-

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sion pattern changes included an increase in expression of genes encoding type II collagen and other cartilage-specific matrix proteins, a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX9 genes, suggesting that redifferentiation closely mimicked some aspects of the signaling processes involved in early chondrogenesis. Thus, these data showed that adult human articular chondrocytes expanded from the cells remaining following autologous chondrocyte transplantation in monolayer cultures retain the ability to redifferentiate under certain permissive culture conditions and form cartilage like matrix in vitro. The microarray data further suggested that this process involves the participation of genes known to be expressed in early chondrogenesis. In a more recent study of gene expression during chondrocyte differentiation and de-differentiation, Goessler et al. [28] investigated the expression of distinct markers during the de-differentiation of human chondrocytes in cell culture using microarrays focusing on transforming growth factor β (TGFβ) pathways. In chondrocytes undergoing de-differentiation, the gene for TGFβ1 was consistently expressed, while the gene for TGFβ2 was not expressed under any conditions. The genes for TGFβ3, TGFβ4 and TGFβi were activated with ongoing de-differentiation. TGFβ-receptor 3 was constantly expressed, while the genes for TGFβ-receptors 1 and 2 were not expressed at any time. The genes for LTBP1 and LTBP2 were activated with ongoing de-differentiation, whereas the gene for LTBP3 was constantly expressed. Immunohistochemical staining was employed for validation of the microarray results. The results suggested that TGFβ3, TGFβ4, TGFβi, LTBP1 and LTBP2 participate in the process of dedifferentiation, while TGFβ1 and TGFβ2 might not be involved in this process. Of the TGFβ-receptors, only the TGFβ receptor 3 appeared to be involved in dedifferentiation. In other studies, the same group of investigators [29-31] examined the changes in the expression patterns of various collagens and various regulatory proteins during ongoing culture and de-differentiation of nasal septum human chondrocytes maintained in primary cell culture for 1, 6, and 21 days employing a microarray which allowed to examine the expression of more than 9,000 individual human genes. After 6 and 21 days, collagen IX and X genes were downregulated, whereas collagen XI genes were activated. Collagens I and II genes were downregulated initially but were reactivated after 21 days. The results with analysis of TGFβ1 gene expression were grossly similar to those reported in their previous study [28]. The genes encoding integrins β1, β5, and α5 were upregulated from day 1 to day 21; integrin β3 was downregulated. Although all data were not shown, the authors concluded that these studies suggested that genes for collagens III, IV, VII, IX and XI might be new markers for the de-differentiation of chondrocytes. Collagen II gene expression might reflect more closely the synthetic activity of the cells rather than their de-differentiation and that integrins β1, β5, and α5 might be involved in signal transmission involved in the de-differentiation process. An interesting study performed a comprehensive analysis of gene-expression profiles in human articular hyaline cartilage presumably representing fully differentiated chondrocytes and meniscus fibrocartilage presumably representing fibrous dedifferentiation of chondrocytes by means of a cDNA microarray consisting of 23,040 human genes [32]. The profiles of the two types of cartilage were compared with each other and with those of 29 other normal human tissues. Remarkable similarities were found between the patterns of gene expression of the two cartilaginous tissues. It should be mentioned, however, that the normal articular cartilage employed for these


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studies was obtained from the patello-femoral joint of patients undergoing total knee replacement for OA and although the tissues had a “normal” macroscopic appearance, there were no histologic studies to confirm their normality, thus, it is likely that the gene expression profile of these tissues may have reflected some early OA changes or early changes of de-differentiation which rendered them more similar to the meniscus fibrocartilage. Given the remarkable lack of differences in the gene expression profiles of hyaline cartilage and meniscus fibrocartilage, the authors then compared the patterns of gene expression of the two types of cartilage to those of 29 other non-cartilaginous normal tissues. They identified 24 genes that were specifically expressed in both cartilaginous tissues. Among these, the most important genes expressed specifically in cartilaginous tissues (both hyaline and fibrocartilage) compared to non-cartilage tissues were WNT7A (a member of the WNT family of secreted signaling molecules), COMP, GLG1 (a membrane bound sialoglycoprotein involved in chondrogenesis), and GPS2 (a polypeptide which regulates RAS and MAPK pathways). A high level of expression of SOX9 was found in both cartilages, however, SOX9 was also substantially expressed in 9 of the other non-cartilaginous normal tissues. The cartilage profiles were also compared with the profiles of gene expression in human mesenchymal stem cells. Twenty-two genes that were differentially expressed in cells representing the two cartilaginous lineages (11 specific to each type) were identified. These could serve as markers for predicting the direction of chondrocyte differentiation.

2. Gene Expression Analysis of Chondrogenesis Chondrogenesis, the process by which uncommitted pluripotent mesenchymal stem cells evolve into the specific cells that populate all cartilaginous tissues in the body, is one of the most crucial events during the embryonic development of all vertebrate species. Chondrogenesis leads to the formation of permanent cartilaginous tissues that undergo endochondral bone formation in the skeleton as well as the cartilage of the growth plate and other organs such as the upper respiratory tract and the structural components of the inner ear. Chondrogenesis is also responsible for the formation of all articular surfaces and, therefore, even minor alterations in this highly organized and extremely complex process are likely to lead to the development of serious disorders ranging from chondrodysplasias to OA. Although remarkable progress has been accomplished in recent years in the elucidation and understanding of the multiple steps and pathways involved during chondrogenesis and of the crucial molecular participants in this process, there still remains a large body of information that needs to be revealed. The recent application of microarray studies has provided substantial insights and has opened novel pathways of investigation into this crucial process. In one of the earliest studies, Sekiya et al. [33] followed the chondrogenic process induced by recombinant human BMP-2, -4, and -6 in human mesenchymal stem cells (MSC) isolated from the bone marrow from normal adult donors. The cells were cultured in a pellet system for 21 days in chondrogenic medium containing TGFβ-3 and dexamethasone with or without BMP-2, -4, or -6. Microarray analyses were performed employing the HG-U95A array which contains 12,526 genes and the results were confirmed by PCR assays. The results showed that critical regulatory genes for chondrogenesis and numerous chondrocyte-specific genes were expressed in a specific time sequence in response to BMP-2. The most important changes included a greater than 100-fold increase in the expression of COL2A1, COL9A3, COL10A1, COL11A1 and

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A2, COMP, dermatopontin, matrilin-3, aggrecan, fibromodulin and PTHrP-R. Other genes with increased expression, although to a lesser extent, included SOX9, Forkhead, SOX5 and Indian Hedgehog transcription factors and WNT5, IGFB5 and retinol binding protein (RBP). A notable decrease in FRIZZLED 2, cadherins 4 and 13, and integrin α3 genes was also observed. The results from this study displayed numerous similarities to those described by Stokes et al. [26] reviewed in the previous section, thus, confirming the validity of both observations. In another related study, Goessler et al. [34] investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human MSC in comparison with de-differentiating human chondrocytes harvested during septoplasty emphasizing changes in adhesion proteins and their receptors. During chondrogenic differentiation of MSC, the genes for fibronectin-receptor (integrin α5β1), fibronectin and the GPIIb/IIIa-receptor were downregulated. The genes encoding components of the vitronectin-receptor (integrin αvβ3) and CD47 were constantly expressed and the integrin-linked kinase (ILK) gene was downregulated. In contrast, ILK, CD47, and ICAP genes were activated with ongoing de-differentiation of adult chondrocytes. The authors concluded that a candidate for signal-transmission involved in de-differentiation is the fibronectin receptor (integrin α5β1) in conjunction with its ligand, fibronectin. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Other receptors, e.g., for vitronectin and osteopontin (integrin αvβ3) or their ligands, do not seem to be involved in the signaling events required for dedifferentiation. Osawa et al. [35] performed similar studies to those of Sekiya [33] and Goessler [34] employing instead of human MSC the mouse embryonal carcinoma cell line ATDC5, a cell line which provides an excellent model system for a detailed analysis of the chondrogenesis process in vitro. To understand better the molecular mechanisms of endochondral bone formation, the gene expression profiles during the differentiation course of ATDC5 cells were examined using an in-house microarray harboring 2,913 full-length-enriched cDNAs. The cDNA microarray was constructed from a full-length-enriched cDNA library prepared with mixtures of mRNA obtained from the ATDC5 cells and from mouse cartilage and bone and separately from E14 mouse fetus. The results were validated by real-time RT-PCR and some were verified by Northern hybridization analyses and immunohistology of developing murine growth plates. Following chondrogenic induction, 507 genes were up- or down-regulated by at least 1.5-fold. Genes for growth factor and cytokine pathways were significantly increased in expression during late stages of chondrocyte differentiation and included decorin, osteoglycin and asporin genes, which have been shown to bind to TGF-β and BMPs. The authors emphasized the results suggesting that small leucine-rich proteoglycans and asporin may play an important role in the regulation of chondrogenesis and that the coordinated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage. 3. Gene Expression Analysis of Cytokine and Growth Factor Effects on Chondrocytes and Cartilage The crucial role that cytokines play in the pathogenesis of rheumatoid arthritis, OA, and other rheumatic diseases has been given extraordinary scientific attention since the pioneering and pivotal studies of Saklatvala and Dingle [36] demonstrating the pres-


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ence of a protein from synovium capable of exerting a profound catabolic effect on articular cartilage. The protein termed initially catabolin was subsequently identified as interleukin-1 (IL-1) [37]. It is generally accepted that the discovery of this pleotropic protein is among the most important scientific discoveries of the last three decades and has generated intense research activity particularly within the field of OA and cartilage [38–42]. It is, therefore, not surprising that numerous genomic profiling and microarray studies have been performed in order to increase the understanding of the complex effects cytokines and related growth factors exert on chondrocytes and on articular cartilage structure and function. Vincenti and Brinckerhoff [43] conducted one of the earliest studies applying microarray analysis to identify putative immediate early genes involved in IL-1β effects on articular cartilage employing the SW1353 chondrosarcoma cell line stimulated with IL-1β. The Clontech Atlas Human Cancer 1.2 K array which contains 1,176 unique genes was employed. Although the number of genes in this array is very small and the genes contained had been selected by the manufacturer for their relevance to cancer, this analysis identified alterations in the expression of genes encoding multiple transcription factors, cytokines, growth factors and their receptors, adhesion molecules, proteases, and signaling intermediates that may contribute to inflammation and cartilage destruction in arthritis. Among the numerous transcription factor genes upregulated following IL-1 treatment, those belonging to the NF-κB and AP-1 family were the most prominently increased. In contrast, IL-1 caused significant reduction in expression of HOX-4A, retinoblastoma-like protein 2 and SMAD5 genes along with a substantial decrease in type II collagen transcripts. Among genes for cytokines and growth factors, LIF and IL-6 were increased whereas BMP4 was reduced. A potent stimulation of genes for several MMP family members including collagenase-3, matrilysin and metalloelastase was also observed although that for collagenase-1 was not changed. Of substantial interest was the reduction in FRIZZLED 2 expression which has been also observed in other studies during chondrocyte de-differentiation. Thus, this analysis has identified numerous IL-β-responsive genes that warrant further investigation as mediators of disease in OA. In a similar study, Gebauer et al. [44] investigated the response to IL-1β of the same human chondrosarcoma cell line SW1353 in comparison with primary human chondrocytes (PHC) by gene expression analysis assayed using a custom-made oligonucleotide microarray representing 312 chondrocyte-relevant genes. The expression levels of selected genes were confirmed by real-time PCR. Although gene expression profiling showed only limited similarities between SW1353 cells and PHCs at the transcriptional level, both types of cells showed similar changes with respect to catabolic effects following IL-1β treatment. In similarity to the results obtained by Vincenti and Brinckerhoff [43], MMP-3 and MMP-13 genes were strongly induced by IL-1β in both systems. The gene encoding IL-6 was also found to be up-regulated by IL-1β in both cellular models. However, the MMP-1 gene was found to be increased whereas expression of genes for intercellular mediators such as LIF and BMP-2 was not induced by IL-1β in SW1353 cells. This study also identified NFκB as an important transcriptional regulator of IL-1β-induced genes in both SW1353 cells and PHCs. One important conclusion from these studies was that SW1353 cells are a cell line with only very limited similarities to PHC except for the study of induction of protease expression in response to IL-1β.

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In another related study, Aigner et al. [45] applied cDNA-array technology employing the Clontech Atlas 1.2 K array to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one single donor cultured under anabolic (serum) or catabolic (IL-1β) conditions. Serum and IL-1β significantly altered gene expression levels of 102 and 79 genes, respectively. The anabolic effects of serum supplementation were confirmed by a marked stimulation of expression of various collagen genes (COL2A1, COL11A1, COL6A1, A2, and A3, and COL16A1) (types II, XI, VI, and XVI) as well as some proteoglycan genes including biglycan. The catabolic effects of IL-1β treatment were reflected in substantial downregulation of genes for COL2A1, aggrecan, decorin and biglycan expression and a marked increase in expression of several MMP genes (MMP-1, MMP-3, MMP-13 and MMP-14). In similarity to results from other studies discussed above, IL-1β upregulated the expression of numerous other genes including LIF, IL-6, Rho8 and members of the NF-κB protein cascade. Also in agreement with other studies, a reduction in expression of FRIZZLED 2, vimentin and osteonectin genes was observed upon IL-1β treatment. Comparative gene expression analysis with previously published data from whole normal and OA cartilage showed significant differences compared to the changes detected in OA cartilage indicating that the IL-1β stimulation did not appear to be a good model for the gene expression alterations in OA chondrocytes. In another study, the changes in global chondrocyte gene expression in response to fibronectin fragments (FN-f), were investigated for the expression of various cytokine genes by cDNA microarrays and were confirmed employing a cytokine protein array [46]. Two microarrays were employed; one was a 268 cytokine receptor genes containing array from Clontech and the other the Affimetrix U133A gene chip microarray. Compared with untreated control cultures, stimulation by FN-f resulted in a > 2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene β (GRO-β) gene expression. Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine gene expression was dependent on NF-κB activity, but independent of IL-1. The studies showed the ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokine and chemokine genes suggesting that damage to the cartilage matrix with production of FN-f can induce a potent proinflammatory state responsible for further progressive matrix destruction. To examine the additive or synergistic effects of interleukin-1 (IL-1) in combination with the IL-6/LIF related protein, oncostatin M (OSM), that may be involved in mechanisms of cartilage repair and degradation, Barskby et al. [47], employed gene microarray and real-time PCR experiments using RNA from human chondrosarcoma SW1353 chondrocytes and primary human articular chondrocytes. The Affimetrix Human U133A and B microarrays were employed and the results were validated by realtime PCR. The combination of IL-1 and OSM markedly up-regulated either cooperatively or synergistically the expression of numerous genes, including those encoding MMP-1, -3, -10, -12, -13, and -14, cytokines, chemokines, extracellular matrix components, and proteins and mediators involved in signal transduction. Real-time PCR confirmed a synergistic induction of genes for several MMPs, activin A, pentraxin 3 (PTX-3), and IL-8. The results demonstrated that the potent proinflammatory cytokine combination of IL-1 plus OSM synergistically and coordinately up-regulated many genes including those encoding several MMP involved in cartilage degradation. The


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results also showed that the cytokine effects are not solely catabolic since some genes with anabolic functions were also upregulated. Thus, this gene-profiling study emphasizes the complex processes that mediate articular cartilage degradation mediated by members of the interleukin family of cytokines through the coordinated expression of multiple genes.

4. Gene Expression Analysis of OA Although there is a large body of information regarding numerous genes whose expression is either increased or decreased during the development of OA and the participation of their encoded products in the pathogenesis of the disease has been extensively studied, the vast majority of these studies have employed biochemical and molecular analyses of a few genes with putative participation in this process. The introduction of microarray technology, on the other hand, allows the evaluation of the transcriptional expression of very large numbers of genes or even of the entire genome transcriptional activity at a specified point in time or under the influence of a particular stimulus or putative pathogenetic factors/mechanisms. Although there are some important limitations with this approach, this powerful technology has already become successfully applied to the study of human OA and important and valuable information has already been obtained from the few published studies. Most of the work employing microarrays to study human OA was performed by Aigner and collaborators [48–55]. In early studies, these authors utilized samples from single or few OA patients and employed the Clontech Atlas Human Cancer 1.2 array which is a limited microarray containing only 1,176 genes of relevance to cancer research [48–50]. Despite these limitations, these early studies provided valuable information regarding changes in the expression of numerous genes in OA and revealed activation as well as phenotypic instability of articular chondroctyes. However, given the limited number of patient samples studied and the very small number of genes able to be examined employing this array, a broader gene expression profile of OA chondrocytes was performed [51]. This recent study compared normal articular cartilage (18 specimens, from subjects ages 45–88 years), cartilage with early OA (20 specimens, from subjects ages 43–91 years obtained at autopsy), and OA cartilage obtained at the time of total knee replacement (21 samples with mild OA and 19 samples with moderate or severe OA from patients ages 61–84 years). This large gene expression profiling study was performed with 78 normal and disease human articular cartilage samples using a custom-made complementary DNA array covering > 4,000 genes. Several comparisons were made according to the absence or presence of OA lesions and to their degree of severity based on Mankin’s scale. Many differentially expressed genes were identified, including the expected up-regulation of genes encoding matrix macromolecules and anabolic and catabolic mediators. Some of the most relevant genes are listed on Table 2. The genes for types I, II and III collagens were strongly up-regulated in moderate/severe OA cartilage, which is consistent with the findings of numerous previous studies likely reflecting the general metabolic activation of OA chondrocytes rather than de-differentiation. The genes encoding collagen types VI, IX and XI were also found to be significantly up-regulated, but to a much lesser degree. In contrast, genes

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Table 2. Genes up- or down-regulated in cartilage lesions with moderate/severe late-stage OA compared with normal samples [51] Gene Collagen, type I,

1 (COL1A1)

Selenoprotein H (SELH)

Fold change 9.55 9.27

Tenascin C (hexabrachion) (TNC)

8.06

Collagen, type III,

7.37

1 (COL3A1)

Collagen, type II,

1 (COL2A1)

Collagen, type V,

1 (COL5A1)

6.07 5.81

Interleukin-1 receptor, type II (IL1R2)

5.34

Fibronectin 1 (FN1)

4.69

Protease, serine, 11 (IGF binding) (PRSS11)

4.17

Collagen, type I,

3.41

2 (COL1A2)

Secreted protein, acidic, cysteine-rich (osteonectin)

3.34

Cartilage acidic protein 1 (CRTAC1)

3.01

Collagen, type VI,

2 (COL6A2)C2

2.96

Cartilage intermediate-layer protein (CILP)

2.72

Collagen, type XI,

1 (COL11A1)

2.66

Collagen, type VI,

1 (COL6A1)

2.58

Fibromodulin (FMOD)

2.56

Caspase 10, apoptosis-related cysteine

2.53

Chitinase 3-like 2 (CHI3L2)

2.11

Tissue inhibitor of metalloproteinases 1 (TIMP1)

2.05

Glutathione peroxidase 3 (plasma) (GPX3)

0.12

CCAAT/enhancer binding protein (C/EBP), delta

0.15

Thioredoxin-interacting protein (TXNIP)

0.29

Stromelysin 1 (MMP3)

0.29

Stearoyl-CoA desaturase 4 (SCD4)

0.31

Serine hydroxymethyltransferase 2 (mitochondrial)

0.32

Metallothionein 1E (functional) (MT1E)

0.33

Dual-specificity phosphatase 1 (DUSP1)

0.33

Cytokine-like protein C17 (C17)

0.35

BTG family, member 2 (BTG2)

0.36

Metallothionein 1G (MT1G)

0.38

Insulin-like growth factor binding protein 6 (IGFBP6)

0.43

Metallothionein 1X (MT1X)

0.43

Transducer of ERBB2, 1 (TOB1)

0.45

Glutamate-ammonia ligase (glutamine synthase) (GLUL)

0.48

Nicotinamide N-methyltransferase (NNMT)

0.48

Superoxide dismutase 2, mitochondrial (SOD2)

0.48


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Figure 3. Dendrogram and heat map of the top 50 genes selected from the analysis of normal versus latestage OA cartilage. A nearly perfect separation between the normal samples and the late-stage OA samples is seen. Adapted from ref. [51]. (Reproduced with permission of the publisher).

for noncollagenous matrix proteins were generally less up-regulated in OA chondrocytes, except for those encoding fibromodulin, cartilage intermediate-layer protein, fibronectin, tenascin, and osteonectin/secreted protein, acidic and rich in cysteine. Expression of the SOX9 gene which encodes one of the most important cartilage transcription factors involved in chondrogenesis and in the maintenance of chondrocyte phenotypic stability was significantly down-regulated in OA cartilage. One important finding was the down-regulation of the genes for superoxide dismutases 2 and 3 and glutathione peroxidase 3. This observation suggests that continuous oxidative stress to chondrocytes and the cartilage matrix may be a pathogenetic mechanism in OA. Another important observation was that only 15 genes were significantly up- or downregulated between normal and early degenerative cartilage lesions. In contrast, the genes that were differentially expressed between normal and severe OA cartilage were significantly higher. An ontology analysis revealed a broad spectrum of differentially regulated genes and emphasized the up-regulation of numerous genes involved in extracellular matrix formation, the downregulation of genes involved in oxidative damage defense, and changes in expression of numerous genes for cytokines or genes involved in cytokine signaling. One interesting finding was that many genes related to the IL-1 pathway were not up-regulated, but instead down-regulated in OA chondrocytes. This included IL-1β itself as well as IL-6, IL-8, and LIF. Figure 3 shows a biased cluster analysis of the 50 most highly differentially expressed genes in the analysis of normal compared with advanced or severe OA cartilage showing a nearly perfect separation of the normal samples from the OA samples.

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Thus, these findings provide a large reference data set on global gene alterations in OA cartilage and suggest major mechanisms underlying central biologic alterations that occur during OA such as the role of oxidative stress. Another study of global gene expression patterns in OA chondrocytes was performed by Tardif et al. [56] who compared gene expression patterns in normal and osteoarthritic (OA) human chondrocytes using the Atlas Human 1.2 microarray. Of the novel genes identified, the authors focused on follistatin, a BMP antagonist, and three other BMP antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts. The genes for all BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin genes were significantly up-regulated in OA chondrocytes whereas chordin was weakly expressed in normal and OA cells and the gene for noggin was not expressed at all. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. IL-1β had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. Thus, the results of this study show the possible involvement of follistatin and gremlin in OA pathophysiology. In a similar study, Sato et al. [57] analyzed the differences in gene expression profiles of chondrocytes in intact and damaged regions of cartilage from the same knee joint of patients with knee OA. Gene expression profiles in regions of intact and damaged cartilage (classified according to the Mankin scale) obtained from patients with knee OA were examined. Five pairs of intact and damaged regions of OA cartilage were evaluated by oligonucleotide array analysis using a double in vitro transcription amplification technique and the Affimetrix U133A and B high density arrays. The microarray data were confirmed by real-time quantitative PCR. About 1,500 transcripts, which corresponded to 8% of the expressed transcripts, showed > or = 2-fold differences in expression between the cartilage tissue pairs. The expression of some genes related to the wound-healing process, including cell proliferation and interstitial collagen synthesis, was higher in damaged cartilage compared to cartilage from intact regions, similar to the findings for genes that inhibit matrix degradation. Comparisons of the gene expression profile differences with real-time quantitative PCR data supported the validity of the data. Differences between intact and damaged regions of OA cartilage exhibited a similar pattern among the 5 patients examined, indicating the presence of common mechanisms that contribute to cartilage destruction.

Concluding Remarks The extraordinary potential of large-scale gene expression profiling employing microarray technology for arthritis research as discussed by Grant et al. [58] has just begun to be realized in the fields of articular cartilage and OA investigations and an enormous body of valuable information has already been obtained in the relatively limited number of studies which employed this approach. Despite the initial skepticism and serious methodological concerns about the validity and applicability of large-scale genomic profiling [59,60], and the subsequent appreciation of substantial limitations of this novel technology as pointed out in numerous publications [e.g., 61], it has become apparent that rigorous evaluation of the results and prudent, conservative and innovative interpretation of the massive amounts of data obtained can, indeed, provide valu-


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able biological insights into the pathogenesis and disease mechanisms of a variety of complex disorders [62], including OA. The search for specific genes involved in the pathogenesis of OA using cDNA array technology has allowed the identification of several molecules potentially relevant to the disease process, among them SOX9, FRIZZLED and follistatin. Thus, a new era of OA research has arrived with the successful application of large-scale microarrays followed by high throughput validation of the results employing real-time PCR. These studies will undoubtedly advance greatly our current understanding of OA pathogenesis and will provide molecular signatures of OA chondrocytes which will be useful as molecular biomarkers for the diagnosis of OA, identification of its clinical subsets, and evaluation of disease-modifying therapies in the not too distant future.

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[45] T. Aigner, L. McKenna, A. Zien, Z. Fan, P.M. Gebhard, R. Zimmer, Gene expression profiling of serum- and interleukin-1β-stimulated primary human adult articular chondrocytes – a molecular analysis based on chondrocytes isolated from one donor, Cytokine 31 (2005), 227-240. [46] J.I. Pulai, H. Chen, H.J. Im, S. Kumar, C. Hanning, P.S. Hegde, R.F. Loeser, NF-κB mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments, J Immunol 174 (2005), 5781-5788. [47] H.E. Barksby, W. Hui, I. Wappler, H.H. Peters, J.M. Milner, C.D. Richards, T.E. Cawston, A.D. Rowan, Interleukin-1 in combination with oncostatin M up-regulates multiple genes in chondrocytes: implications for cartilage destruction and repair, Arthritis Rheum 54 (2006), 5405-5450. [48] T. Aigner, A. Zien, A. Gehrsitz, P.M. Gebhard, L. McKenna, Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology, Arthritis Rheum 44 (2001), 2777-2789. [49] T. Aigner, A. Zien, D. Hanisch, R. Zimmer, Gene expression in chondrocytes assessed with use of microarrays, J Bone Joint Surg Am 85-A Suppl (2003), 117-123. [50] T. Aigner, J. Saas, A Zien, R. Zimmer, P.M. Gebhard, T. Knoor, Analysis of differential gene expression in healthy and osteoarthritic cartilage and isolated chondrocytes by microarray analysis, Methods Mol Med 100 (2004), 109-128. [51] T. Aigner, K. Fundel, J. Saas, P.M. Gebhard, J. Haag, T. Weiss, A. Zien, F. Obermayr, R. Zimmer, E. Bartnik, Large-scale gene expression profiling reveals major pathogenetic pathways of cartilage degeneration in osteoarthritis, Arthritis Rheum 54 (2006), 3533-3544. [52] T. Aigner, E. Bartnik, A. Zien, R. Zimmer, Functional genomics of osteoarthritis, Pharmacogenomics 3 (2002), 635-650. [53] T. Aigner and J. Dudhia, Genomics of osteoarthritis, Curr Opin Rheumatol 15 (2003), 634-640. [54] T. Aigner, E. Bartnik, F. Sohler, R. Zimmer, Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses, Clin Orthop Relat Res 427 Suppl (2004), S138-143. [55] T. Aigner, A. Sachse, P.M. Gebhard, H.I. Roach, Osteoarthritis: pathobiology-targets and ways for therapeutic intervention, Adv Drug Deliv Rev 58 (2006), 128-149. [56] G. Tardif, D. Hum, J.P. Pelletier, C. Boileau, P. Ranger, J. Martel-Pelletier, Differential gene expression and regulation of the bone morphogenetic protein antagonists follistatin and gremlin in normal and osteoarthritic human chondrocytes and synovial fibroblasts, Arthritis Rheum 50 (2004), 2521-2530. [57] T. Sato, K. Konomi, S. Yamasaki, S. Aratani, K. Tsuchimochi, M. Yokouchi, K. Masuko-Hongo, N. Yagishita, H. Nakamura, S. Komiya, M. Beppu, H. Aoki, K. Nishioka, T. Nakajima, Comparative analysis of gene expression profiles in intact and damaged regions of human osteoarthritic cartilage, Arthritis Rheum 54 (2006), 808-817. [58] E.P. Grant, M.D. Pickard, M.J. Briskin, J.C. Gutierrez-Ramos, Gene expression profiles: creating new perspectives in arthritis research. Arthritis Rheum 46 (2002), 874-884. [59] G.S. Firestein, D.S. Pisetsky, DNA microarrays: Boundless technology or bound by technology? Guidelines for studies using microarray technology. Arthritis Rheum 46 (2002), 859-861. [60] R. Kothapalli, S.J. Yoder, S. Mane, T.P. Loughran Jr, Microarray results: how accurate are they? BMC Bioinformatics 3 (2002), 22. [61] S. Draghici, P. Khatri, A.C. Eklund, Z. Szallasi, Reliability and reproducibility issues in DNA microarray measurements. Trends Genet 22 (2006), 101-109. [62] M. Benson, R. Breitling, Network theory to understand microarray studies of complex diseases. Curr mol Med 6 (2006), 695-701.


Part III Effectors and Different Pathways



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X Prostaglandin E2 and Osteoarthritis: The Role of Cyclooxygenases, Prostaglandin E Synthases and 15-Prostaglandin Dehydrogenases Odile GABAY a,1, Marjolaine GOSSET a,1 and Francis BERENBAUM a,b,* a UMR 7079 CNRS, Physiology and Physiopathology Laboratory, University Paris 6, 7 quai St-Bernard, Paris, 75252 Cedex 5, France b Department of Rheumatology, UFR Pierre et Marie Curie, Saint-Antoine hospital, 75012 Paris, France Introduction Osteoarthritis (OA) is characterized mainly by degenerative changes in joint cartilage resulting in loss of cartilage, alterations of subchondral bone, and a local inflammation [1]. During inflammatory process, membrane phospholipids lead to eicosanoid, products of the acid arachidonic cascade. Among them, Prostaglandin E2 (PGE2) plays an important role. Role of Prostaglandin E2 in Cartilage Homeostasis and in OA The prostaglandins (PG) are a group of fatty acid compounds that have many effects throughout the body, including activity in inflammation, smooth muscle contraction, regulating body temperature, and effects on certain hormones. PGE2 is synthesized by many cell types and tissues. It has long been considered as the principal prostaglandin in arthritic diseases as well as in age-related diseases such as OA [2]. Notably, Bonner et al. have shown that the concentration of arachidonic acid, the precursor of the prostaglandins, increased markedly with age [3]. PGE2 and Cartilage Degradation PGE2 is highly expressed in arthritis [4]. To understand the mechanism by which prostaglandins could modulate the arthritis-induced cartilage degradation, McCoy et al. 1 The authors participate equaly to the redaction of the manuscript. * Corresponding Author: Pr. F. Berenbaum, UMR 7079 Paris VI-CNRS, Physiology and Physiopathology Laboratory, University Paris 6, 7 quai St-Bernard, Paris, 75252 Cedex 5, France. Phone: +33 144-27-22-83, Fax: +33 144-27-51-40, E-mail: [email protected].

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Figure 1. Role of Prostaglandin (PG)E2 in OA process. Two hypothesis of action are proposed for PGE2 in OA: the decrease of synthesis of glycosaminoglycans and the increase of Matrix Metalloproteases (MMPs) synthesis. PGE2 acts mainly by the binding on the EP2 and EP4 presents on cells membranes of chondrocytes, synoviocytes and osteoblasts.

have studied mice lacking each of the four known PGE2 receptor, named EP1-4 receptors, after generation of collagen-induced arthritis. The deletion of EP1, EP2, EP3 receptors did not affect the development of arthritis but EP4 receptor deficient mice resulted in an absence of cartilage degradation in collagen-induced arthritis. Thus, PGE 2 contributes to cartilage degradation in part by binding to the EP4 receptor [5]. The exact mechanisms of PGE2 actions leading to cartilage degradation are not well understood. However, two main hypothesis are proposed: the first one is a decreased synthesis of glycosaminoglycans [6]. The second one is an increase of MMP synthesis [7]. Pelletier et al. have shown that the synthesis of MMP-1 is eicosanoid–dependent in human OA synovial membrane explants [8]. Moreover, the IL-1β–induced MMP-2 activation and expression were found to be dependent on PGE2 in human chondrocytes [9]. Finally, MMP and TIMP expressions are regulated by PGE2 in equine chondrocytes [10]. However, PGE2 suppressed MMP-1 expression through C/EBP/NF-κB/MEKK1 suppression in synovial fibroblasts [11] (Fig. 1). PGE2 and Apoptosis Chondrocyte death may contribute to the progression of OA: OA cartilage has a higher number of apoptotic chondrocytes than does normal cartilage in animal models [12] and in humans [13]. Nitric Oxide (NO) and PGE2 play a crucial role in chondrocyte death [14]. NO, generated from sodium nitroprusside has been shown to induce apoptosis in human articular chondrocytes, and IL-1β induced NO inhibits chondrocytes proliferation via PGE2 [15,16]. Moreover, Notoya et al. have shown that NO induces


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COX-2 expression through ERK1/2 and p38 kinase pathways resulting in an increase of PGE2 release in human OA chondrocytes [14]. Therefore, PGE2 may sensitize chondrocytes to the cell death induced by NO resulting from an autocrine-paracrine mechanism. Finally, Miwa et al. have shown that PGE2 induces apoptosis through c-AMPdependant pathway in articular chondrocytes [17]. PGE2 and Chondrocyte Differentiation Regulation of chondrogenesis and chondrocyte maturation by prostaglandins has been of interest these last years. Prostaglandins regulate chondrocyte phenotype, but their role in chondrocyte differentiation is not yet clear [18]. Jakob et al. have shown that PGE2 reduced collagen type I expression and doesn’t improved chondrogenesis [19]. Moreover, Li et al. have demonstrated that PGE2 inhibits chondrocyte differentiation through Protein Kinase A and C (PKA and PKC) signalling [20]. However, another study has shown opposite effects of PGE2 depending on its concentration: low levels of PGE2 increase proteoglycans synthesis whereas high doses decrease it [21]. In addition, prostaglandins receptors EP1 to 4 seems to play an important role during the maturation of the chondrocyte but the role of PGE2 seems to be linked to this state of maturation. A full assessment of all subtypes of receptors in chondrocytes need to be better defined [22].

PGE2 Synthesis and Degradation in OA Articular Cells The synthesis of PGE2 is the terminal step of a sequence of enzymatic reactions, including the release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2) and conversion of this substrate to prostaglandin H2 (PGH2) by cyclooxygenase (COX)-1 and COX-2. PGH2 is subsequently metabolized by PGE synthase (PGES) to form PGE2. The COX-1 isoform is expressed constituvely by many cell types, whereas COX-2 requires specific induction by inflammatory mediators such as lipopolysaccharides (LPS) and cytokines [23]. The prostaglandin E synthase catalyse the conversion of PGH2 to PGE2. Three isoforms of PGES have been cloned [24–26] including cytosolic PGES and two microsomal forms: glutathione-specific mPGES-1 and glutathione non-specific mPGES-2. cPGES is constituvely expressed and unresponsive to inflammatory stimuli whereas mPGES-1 is inducible in an inflammatory context [24,25,27]. The coordinate regulation and functional coupling of mPGES-1 and COX-2 have been reported [27] (Fig. 2). After synthesis, PGE2 needs to be released from cells in order to exert their extracellular effect. First, passive diffusion permits a slow exit of PGE 2 from the cells [28]. Second, the transporters MRP2 and 4 (multidrug resistance proteins type 2 and 4) seem to be implicated in PGE2 efflux [29,30]. The specific prostaglandin transporter is implicated in the influx of PGE2 into the cells but not in the efflux [31]. PGE2 usually act on cells as hormones in a paracrine or autocrine way by interaction with specific receptors on cells membranes. PGE2 has 4 distinct receptors, namely E-prostanoid (EP) 1 to EP4, encoded by distinct genes and with an expression depending on the cell type. These receptors belong to the G protein-coupled cell-surface receptor family. Recently, functional EP1-4 receptors were described at the nuclear membrane of many cells [32] but their role in articular cells remain unknown. If PGE 2 does not bind with its specific receptors, this eicosanoid is rapidly converted to an inac-

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Figure 2. The acid arachidonic cascade leading to PGE2 synthesis and its degradation. The conversion of arachidonic acid to prostaglandin (PG)E2 occurs by sequencial enzymatic reactions involving isoformes of cyclooxygénase (COX) and PGE synthase (PGES). COX-1 and cytosolic PGES are constitutive isoforms whereas COX-2 and mPGES-1 isoforms are regulated by pro-inflammatory stimuli. After release from cells, PGE2 interact with specific EP receptors. Its degradation is catalyzed by the cytosolic 15-PG dehydrogenase (15-PGDH) into the inactive metabolite 15-ketoPGE2.

tive metabolite (13-14-dihydro 15-keto PGE2) by the prostaglandin 15-dehydrogenase (15-PGDH) pathway.

A. The COX Enzyme: Structure, Regulation and Role Cyclooxygenase, also known as prostaglandin endoperoxide H synthase, catalyze the conversion of arachidonic acid to PGH2, the common precursor of all prostaglandins synthase. Three isoforms of COX, namely COX-1, COX-2 and COX-3, have been described (Table 1). Whereas COX-1 is constituvely expressed in various cell types to maintain homeostasis, COX-2 is the inducible COX isoform, implicated in PG synthesis in an inflammatory context. COX-2 is implicated in many pathophysiological processes, such as inflammation, pain, Alzheimer’s disease, cancer, angiogenesis and arthritis [33]. COX-3 is a recently described variant of COX-1 as a result of the first intron conservation. COX-3 is also called COX-1 V1 [34]. The focus of this article is to present the expression and the regulation of each COX isoform in articular tissue.

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Table 1. Cyclooxygenase (COX) and PGE synthase (PGES) isoforms Enzymes/ Molecular Chromosome weight/ mRNA size

Cellular location

Expression in joint

Tissue distribution in joint

Functions in joint

Structure

COX-1 9q32-q33,3

72 kDa 3 kb

Nuclear membraneendoplasmic reticulum

constitutive

ubiquitous

unknown

EGF-like, membrane and catalytic with a heme binding site domains

COX-2 1q25,2-25,3

72 kDa 4–4,5 kb

Nuclear membraneendoplasmic reticulum

inducible

Stimulusinduced in tissues

Inflammation, Idem than pain COX-1

COX-3 (variant of COX-1) 9q32-q33,3

65 kDa 5,2 kb

Endoplasmic reticulumNuclear membrane

constitutive

Variant of COX-1

unknown

mPGES-1 9q34,4

15–16 kDa 14,8 kb

Nuclear membrane

inducible

Ubiquitous

Inflammation, MAPEG pain family

mPGES-2 9q33-q34

33 kDa 2 kb

Golgicytoplasm

constitutive

skeletal mus- unknown cle

Thoredoxin homology domain

cPGES 12q13,13

26 kDa 1,9 kb

Cytoplasm – nuclear membrane

constitutive

ubiquitous

Hsp90 cochaperone p23

unknown

Idem than COX-1 &COX-2

Common Features and Differences Between COX-1 and COX-2 COX-1 and COX-2 are encoded by 2 different genes, respectively on the chromosome 9q32-q33.3 [35] and on the chromosome 1q25.2-q25.3 [36]. These enzymes are 72 KDa proteins with 60% homology [37]. They present common crystal structures and are composed of three domains: (1) a N-terminal epidermal growth factor (EGF) domain, (2) a helical membrane binding domain and (3) the large catalytic C terminal domain [38]. This catalytic domain allows COX to converse AA to PGH2 by two sequential enzymatic reactions, (a) cyclooxygenation of AA in PGG2 and (b) reduction of PGG2 to PGH2. The kinetic properties of the two enzymes are quite similar [39,40]. However, COX-1 and COX-2 differentially use the AA substrate pool. Whereas COX1 acts on AA at high concentration (≥ 10µM), reflecting commonly an exogeneous source of AA, COX-2 is biologically active at weak AA concentration (≤ 2.5µM) resulting from an endogeneously release [41]. Moreoever, COX-1 and COX-2 show differences in subcellular localizations. As COX-1 localizes equally in endoplasmic reticulum and nuclear membrane of endothelial cells, COX-2 is preferentially found at the nuclear envelop [42]. Recently, COX-2 have been shown to be localized in the perinuclear region of articular cells. Kojima and colleagues reported that IL-1β induces the localization of COX-2 in the perinuclear region of synoviocytes and chondrocytes [43,44] Moreover, we suggest that, as IL-1β does, mechanical stress induces the localization of the COX-2 enzyme in the perinuclear region of chondrocytes [45].

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Figure 3. Stimuli, signaling pathways and promoter response elements implicated in COX-2 and mPGES-1 expression. COX-2 and mPGES-1 expressions are increased in articular cells by various stimuli including the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, the reactive oxygen species Nitric Oxyde (NO) or the mechanical stress. Moreover, hypoxia has been described as an inhibitor of COX activity. All these stimuli activates two main intra-cellular signaling pathways, the mitogen activated protein kinase (MAPK) and the NF-κB pathway. These signal lead to gene transcription activation of both COX-2 and mPGES-1 genes, implicating promoter response elements.

Regulation of COX Expression COX-1 Many differences exist between COX-1 and COX-2 in their transcriptional regulation. COX-1 is an housekeeping gene. It lacks a CAAT or a TATA box and presents two Sp1 cis-regulatory elements implicating in constitutive expression of COX-1 [46]. In contrast, COX-2 gene presents a variety of response elements which explains, in part, its inducibility by multiple inflammatory mediators, cytokines or growth factors. Stimuli Involved in COX-2 Regulation (Fig. 3) The pro-inflammatory cytokines IL-1β, TNFα, IL-6, the Leukemia Inhibitory Factor (LIF) and LPS are potent activators of COX-2 expression leading to PGE2 increase in all articular cells, ie chondrocytes, synoviocytes and subchondral osteoblasts [23,47–53]. These effects are reversed after a dexamethasone or an NSAID treatment [23,47,51,54]. Accumulating evidences suggest that the nitrated oxygen species and especially nitric oxide (NO) modulate the cyclooxygenase activity in articular tissue. NO plays a role in physiology but also in the OA inflammatory process [2]. This mediator is


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spontaneously released by OA chondrocytes and synoviocytes [55]. Manfield and colleagues reported that the inhibition of the NOSynthase (NOS) activity inhibits the COX activity and therefore the PGE2 release in both bovine chondrocytes and human OA cartilage [49]. However, cartilage explants treated with the NOS inhibitor L-NMMA significantly increased COX-2 expression and subsequent PGE2 synthesis [54]. Therefore, divergent effects of NOS on COX exist, depending on the cell-type and the COX isoform affected [55]. Mechanical stress (MS) is a key regulator of cartilage matrix turn-over but could be deleterious when excessive loading is applied like in obesity. MS is definitely identified as the main risk factor for OA [56,57]. Many authors have described the role of MS on COX-2 expression. Compressive stress applied on cartilage explants or shear stress applied on primary cultured chondrocytes triggers COX-2 mRNA and protein expression [45,58,59]. This effect could be mediated by an increased NO release [59]. As mature articular cartilage is an avascular tissue, the oxygen supply to resident chondrocytes could be a limiting factor for the cyclooxygenase activity. Mathy and colleagues described that an hypoxic environment blocks COX-2 activity in bovine chondrocytes [60] although COX-2 gene is already up-regulated in hypoxic condition by IL-1. In fact, decrease in O2 tension triggers the expression of many factors like the Hypoxia-inducible factor-1 (HIF-1), which is actually known as an activator of COX-2 expression [61]. The molecular mechanisms through which hypoxia modulates COX-2 activity and expression remain to be determined. Finally, the role of estrogens in the development of OA has been suggested. First, estrogens receptors are expressed on cartilage cells [62]. Second, Morisset and colleagues reported that, in bovine chondrocytes, 17-β estradiol is as potent as dexamethasone in preventing basal, but not TNFα− and IL-1β -induced COX-2 mRNA expression [51]. Therefore, 17-β estradiol may play a role in cartilage homeostasis. Signaling Pathways Involved in COX-2 Regulation (Fig. 3) The signaling pathways involved in COX-2 expression are tissue-specific and depend on the stimulus. Among them, MAPK are well described pathways in many cell types, and especially in chondrocytes and synovial cells. p38 MAPK and JNK/SAPK are implicated in ΤΝFα and IL-1β-induced COX-2 expression in human articular chondrocytes [63]. p38 MAPK inhibitor (SB-202190, SB-203580, R-130823) prevented also IL-1β−induced COX-2 expression in human synovials fibroblasts, as in human chondrocytes cell line and in bovine chondrocytes [64–66]. Recently, a study confirm the role of p38 MAPK but describes also, the role of Erk1/2 and Jnk in IL-1β induced COX-2 expression [67]. Moreover, Faour and colleagues observed that IL-17induced COX-2 expression involve a restricted MAPK profile, the MKK3/6/SAPK2/p38 cascade in human chondrocytes and synoviocytes [68]. Finally, we demonstrate the implication of MAPK in mechanical stress-induced COX-2 expression. The p38 MAPK inhibitor SB203580 , the JNK kinase inhibitor SP600125 and the specific ERK 1/2 inhibitor PD98059 significantly inhibit the COX-2 mRNA expression in murine cartilage explants submitted to dynamic loading (personal communication). Regulation of COX-2 Expression at the Promoter Level (Fig. 3) Promoter region of the COX-2 gene contains a TATA box and various putative transcriptional regulatory elements, such as nuclear factor-kB (NF-kB), the CAAT

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enhancer binding protein (C/EBP), the cyclic adenosine monophosphate response element (CRE) and peroxisome proliferator-activated receptors-responsive elements (PPREs) [69]. Two consensus NF-kB binding sites are descibed on the COX-2 promoter. NF-kB is a classical pathway triggered in an inflammatory context [70]. This pathway is involved in the regulation of COX-2 induced by IL-1β in RA synoviocytes [71,72] and induced by TNFα and IL-1β in human chondrocytes cell line [73,74]. The role of NF-kB signalling was confirmed by an antisens strategy [73,75]. In contrast, Thomas and colleagues found a role of NF-kB in the regulation of the human COX-2 promoter, dependent on the binding of C/EBP in cultured rabbit chondrocytes [76]. The CCAAT-enhancer-binding protein (C/EBP) is critical for the stimulation of COX-2 gene. In human synovial fibroblasts, C/EBP is involved in TNFα−induced COX-2 expression [77]. Particularly, the role of C/EBPδ and C/EBPβ factors were highlighted in articular chondrocytes transfected with COX-2 promoter containing mutations in C/EBP cis regulatory elements, and stimulated with IL-1 β [76]. Concerning the ATF/CRE element, there is actually little evidence for its role in the regulation of COX-2 expression in articular tissues. Faour and colleagues described the role of IL-17 on the increased COX-2 gene expression in both human chondrocytes and synovial fibroblasts through the ATF-CRE enhancer site of the promoter. In fact, mutation of this site is sufficient to abrogate induction of COX-2 promoter activity [68]. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcriptions factors, belonging to the nuclear receptor superfamily. After heterodimerisation with retinoid X receptor RXR, they bind to PPAR-responsive elements (PPREs) in the promoter region of gene, like for COX-2. The 15-Deoxy-Δ12. 14–PGJ2 (15d- PGJ2), the end-product metabolite of PGD2 is a potent activator of PPARγ. 15d- PGJ2 acts as a dual agent on the regulation of COX-2 in human osteoarthritic chondrocytes. When cells are stimulated with IL-1β, addition of 15d- PGJ2 partially reduced COX-2 expression whereas 15d- PGJ2 triggers COX-2 expression without PGE2 production in cells in basal condition [78]. In RA synoviocytes, 15d-PGJ2 suppressed IL-1β-induced PGE2 synthesis through the inhibition of cyclooxygenase (COX-2) expression [79]. It seems that 15d-PGJ2 exerts a negative feedback on the AA cascade in both synoviocytes and chondrocytes in an inflammatory context (IL-1). Regulation of COX-2 Expression at a Post-Transcriptional Level COX-1 and COX-2 also show major differences in mRNA splicing, stability and translational efficiency. Post-transcriptional regulations on the 3’-untranslated region (UTR) of COX-2 mRNA resulting in its stabilization, seem to be involved in the regulation of COX-2 mRNA degradation. Such regulation has been reported by several authors in chondrocytes submitted to IL-1α [51] and in both synoviocytes and chondrocytes stimulated by IL-17 [68]. Expression and Role of COX in Articular Tissues Vane first described in 1971 the existence of an enzymatic activity inhibited by aspirin and indomethacin and implicated in the synthesis of prostaglandins. This enzyme was called “cyclooxygenase” ans was thought to be unique [80]. In 1991, three groups


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described the cyclooxygenase type 2 isoform (COX-2) inducible in an inflammatory context [36,81,82]. Then, many authors studied the role of this enzyme in joint inflammation using several models, like adjuvant arthritis, carrageenan-induced paw inflammation or type II collagen-induced arthritis. Therefore an increased expression of COX was described in articular tissues of rats treated with intraperitoneal injection of streptococcal cell wall (SCW) or intradermal injection of Freund’s adjuvant [83]. In 1996, Anderson and colleagues were the first group to report that COX-2 plays a proeminent role in adjuvant arthritis. After adjuvant injection in paws, an increased expression of COX-2 mRNA and protein and a local PGE2 overrelease were found. The use of a selective COX-2 inhibitor, SC58125, rapidly reduced the level of PGE2 in paw tissues [84]. This result was confirmed by Kang and colleagues in 1996 [85]. Recently, COX-2 antisense oligodeoxynucleotide injected after development of adjuvant-induced arthritis in rats significantly suppressed induction of arthritis in a dose-dependent manner [86]. The role of each COX isoform on type II collagen-induced arthritis has been recently assessed. Selective inhibitors of COX-1 (FR122047, SC-560) did not inhibit paw edema and PGE2 release in arthritic model whereas selective COX-2 inhibitor (FR140423) did [87]. Therefore, COX-2 overexpression may be responsible to the increase of PGE2 production implicated in edema, swelling and cellular infiltration in joints. In human OA cartilage and synovial samples, COX-2 mRNA and protein expression were observed [54,85,88–90]. Moreover, OA cartilage samples spontaneously release more PGE2 (50-fold) than normal cartilage and 18-fold higher than normal cartilage stimulated with cytokines or endotoxins [54]. Interestingly, IL-1β triggers a strong increase of COX-2 activity in human OA synovial membrane but a weak increase in articular cartilage. Authors suggest that the induction of COX-2 by synovial cells in response to IL-1β is linked to proteoglycan degradation in OA [88]. The use of specific COX-2 inhibitors confirm the role of this enzyme in overrelease of PGE2 in OA and may result in some beneficial effects in this disease. COX-3 COX-3 is a recently described derivative of COX-1 as a result of the first intron conservation. COX-3 is also called COX-1 V1. At this time, COX-3 mRNA is described in both canine and human cortex and aorta, in the rodent heart, kidney and neuronal tissues [34] and in mouse costal cartilage [45]. But conclusive evidence regarding the existence of a human COX-3 protein is lacking. Conservation of the first intron of COX-1 probably leads to the modification of the active site conformation. COX-3 is more sensitive to acetaminophen than COX-1 and COX-2 suggesting that COX-3 could be the target of this drug [91]. No regulation by inflammatory mediators or mechanical loading [45] has been described yet. Only a COX-3 mRNA expression in a human colon cancer cell line under osmotic stress has been recently described [92]. As COX-1 and COX-3 are derived from the same gene, these enzymes share the same promoter. No regulatory sites of COX-1 promoter by mechanical stress or pro-inflammatory cytokines have been described. This is consistent with the fact that COX-1 is constitutively and ubiquitously expressed.

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B. The Prostaglandin E Synthase (PGES): Structure, Regulation and Roles PGE synthase (PGES) is the last step of the enzymatic reactions of the arachidonic acid cascade leading to PGE2 production. Three isoforms of this enzyme have been cloned. Two of them are membrane-bound enzymes, called membrane-associated-PGES (mPGES) type 1 (mPGES-1) [93] and mPGES-2 [26]. mPGES-1 is inducible and functionally linked with COX-2. The cytosolic PGES (cPGES) is a protein constitutively expressed in a large variety of cells and tissues, linked to COX-1 to promote early PGE2 production during the inflammatory process [25]. PGES are expressed in articular tissues, cartilage, synovium and subchondral bone [94]. 1) mPGES-1 Structure and Properties The human mPGES-1 gene maps to chromosome 9q34.3. It spans 15 kb and is divided into three exons. The primary structure of mPGES-1 from different animal species shows a high degree of sequence homology (≈ 80%) [95]. This enzyme is a gluthatione (GSH)-requiring perinuclear protein, member of the membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG). Two amino acids are conserved in the MAPEG superfamily: Arg110, essential for the enzymatic function, and Tyr117. The mutation of Arg110 abrogates mPGES-1 catalytic function, implying an essential role for this residue [96]. All MAPEG proteins have similar molecular masses of 14–18 kDa and mPGES-1 is a 16 kDa protein. Finally, mPGES-1 is a 10 angström projection structure and constitutes a trimer in the crystal, after electron crystallography [96]. Expression, Function and Regulation of mPGES-1 in Articular Tissues (Fig. 3) mPGES-1 is localized to the superficial layers of human OA cartilage, where OA damages first appear and in which IL-1 β is also present [97]. Accumulating evidences implicate mPGES-1 in the pathogenesis of OA. PGE2 exerts various physiological functions through the EP receptors (EP1, EP2, EP3 and EP4). EP2 and EP4 are detected in synovial fibroblasts from arthritis patients. Selective agonists for the EP2 and EP4 receptors increase mPGES-1 expression, in addition to PGE2, suggesting that PGE2 strongly enhance the expression of mPGES-1 in rheumatoid synovial fibroblasts and OA synovial fibroblasts. The same mechanism is suggested in chondrocytes [98]. Moreover, non steroidal anti-inflammatory drugs, such as selective COX-2 inhibitors, decrease the expression of mPGES-1 in IL-1β stimulated rheumatoid arthritis (RA) synovial fibroblasts [99]. Recently, mPGES-1 inhibitors development have provided evidence of the involvement of this enzyme in the inflammatory process [100]. In OA chondrocytes, osteoblasts and synoviocytes, mPGES-1 is up-regulated by pro-inflammatory cytokines [27,101]. In chondrocytes, IL-1β but not IL-6 and IL-4 are able to increase mPGES-1 [43]. IL-1β stimulates ERK and p38, but not JNK, MAP kinases in chondrocytes leading to PGE2 and mPGES-1 release [102]. Moreover, the cyclopentenone 15d-PGJ2, known to have anti-inflammatory properties, decrease PGE2 synthesis in a dose-dependant manner. mPGES-1 expression is completely abolished with a high dose of 15d-PGJ2 in rat chondrocytes stimulated by


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IL-1β through inhibition of the NF-κB pathway, and in human OA chondrocytes [97,103]. Interestingly, recent findings show that mPGES-1 is also a mechanosensitive gene in cartilage [104]. The putative promoter of the human mPGES-1 gene does not contain transcriptional elements presents in the COX-2 promoter. It reveals the presence of two GC-boxes, Barbie boxes, an aryl hydrocarbon regulatory element (ARE), lacks a TATA box, and contains binding sites for C/EBPα and β, AP-1, two progesterone receptors and three GRE elements [105,106]. It was recently shown that the zinc-finger containing transcription factors Egr-1 binds specifically to GC rich elements in the mPGES-1 promoter region and facilitate the mPGES-1 gene transcription [105]. Further investigations are needed to better define gene regulatory mechanisms that modulate the expression of mPGES-1. mPGES-1 Deficient Mice Involvement of mPGES-1 in pathophysiological events had been clarified by studies with knockout mice mPGES-1 deficient mice are viable and fertile and develop normally [107]. In a collagen induced arthritis model, mPGES-1 deficient mice developed milder arthritis than wild type, with reduced pain and inflammation [108]. Similar phenotype have been observed in mice lacking cPLA2, COX-2 or EP4, revealing a metabolic flow of the cPLA2/COX-2/mPGES-1/EP4 pathway leading to the development of inflammatory arthritis. 2) mPGES-2 Structure and Properties The gene for human mPGES-2 maps to chromosome 9q33-q34, in the vicinity of COX-1 and mPGES-1 genes. It spans 7 kb and consist of 7 exons [109]. Watanabe et al. purified and identified a protein of 33 kDa that possessed a GSHindependent PGES activity from bovine heart tissue [110]. They had previously reported the existence of two separate mPGES enzymes in rat tissues [111]. mPGES-2 does not show a close similarity to mPGES-1, the overall structure of this enzyme being rather distinct from mPGES-1. The catalysis of PGH2 to PGE2 by mPGES-2 does not require the presence of Gluthation, as it does for mPGES-1. The crystallisation of mPGES-2 show a dimer attached to lipid membrane by anchoring the N-terminal section [112]. The amino acid sequence of mPGES-2 was highly conserved among monkey, bovine and human cDNA. Since the evaluation of the amino acid sequence of mPGES-2 did not reveal any homology with any GHS-transferase, it was concluded that mPGES-2 did not belong to the MAPEG family [112]. After analysis of the 377 amino-acid sequence of mPGES-2, it appears that a consensus region, Cys110-x-x-Cys113 carries the enzymatic activity of mPGES-2 [113]. mPGES-2 is first synthetized in the Golgi membrane; then, its terminal hydrophobic domain is removed after a proteolytic process and this enzyme is released into the endoplasmic reticulum.

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Expression, Function and Regulation of mPGES-2 in Articular Tissues mPGES-2 is constitutively expressed in chondrocytes and RA synovial fibroblasts and is not affected by IL-1β stimulation, nor by mechanical stress [45,44]. Although mPGES-2 can be coupled to both COX-1 and COX-2 to produce PGE2 in response to acute and chronic inflammation, it seems that a modest preference for a coupling with COX-2 has been demonstrated [114]. Thus, mPGES-2 may have a role in the production of PGE2 for homeostasis. Nevertheless, a potential role in inflammatory diseases remains to be demonstrated. Little is known about the regulation of this enzyme. Recently, the promoter of mPGES-2 has been cloned. It contains multiple Sp1sites and a GC box, without TATA box motif [109]. mPGES-2 is activated by various thiol reagents and is also stimulated by the addition of gluthatione and 2-mercaptoethanol [26]. 3) cPGES Structure and Properties cPGES is highly conserved among animal species (≈ 95%) and its gene consists in 8 exons. cPGES, a 23 kDa cytosolic protein identical to p23, is a co-chaperone that binds the ATP-dependent conformation of Heat shock protein-90 (Hsp-90). The N-terminus cPGES has a Tyrosine residue, Tyr 9. Mutation of this residue abrogates the enzymatic activity of cPGES. It is a GSH-requiring enzyme, constitutively expressed in a wide variety of cells [25]. Expression, Function and Regulation of cPGES cPGES expression is largely constitutive and ubiquitous, and is not affected by inflammatory stimuli. cPGES activation in cells requires its binding to Hsp90 [115]. This enzyme is functionally coupled to COX-1: it is able to convert COX-1- but not COX-2derived PGH2 to PGE2 in cells [25]. Although PGE2 production by COX-1 is generally considered to be constitutive, more studies are ongoing and suggest that cPGES may undergo a translocation from the cytosol to the nucleus membrane to form an assemblage with COX-1 in order to up-regulate PGE2 production rapidly after cell stimulation [116]. cPGES may physiologically contribute to PGE2 production for maintenance of homeostasis. In activated cells, phosphorylation of cPGES by casein kinase 2 (CK2) occurs in parallel with an increased cPGES activity and PGE2 production. CK2 regulates cPGES by a phosphorylation process. In vitro, phosphorylation of cPGES by CK2 increase the affinity of cPGES for PGH2. CK2 inhibitors decrease cPGES phosphorylation and PGE2 synthesis. This process is facilitated by interaction with Hsp90: these 3 molecules form a complex [25]. Addition of Hsp90 inhibitors resulted in the dissociation of this cPGES/Hsp90 complex and a decrease of PGE2 production [115]. Immediate increase in PGE2 production, as a consequence of cPGES activity, was seen in vivo when rat fibroblasts were stimulated by bradykinin, dexamethasone and p38 MAPKinase inhibitor indirectly suppress cPGES activation [117].


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cPGES Deficient Mice Knockout mice lacking cPGES/p23 are peri-natally lethal. Heterozygotes are viable, fertile, and appear normal, despite a decrease in cPGES/p23 protein level [118].

C. The 15-PGDH Enzyme: Structure, Regulation and Role 15-hydroxyprostaglandin dehydrogenases (15-PGDH) are the key enzymes implicated in the biological inactivation of prostaglandins. 15-PGDH enzymes catalyse the oxidation of the 15(S)-hydroxyl group of prostaglandins to form inactive 15 ketoprostaglandins [119]. Two types of 15-PGDH have been indentified. The 15-PGDH type 1 is also called NAD+ dependent 15-PGDH and exhibit an important specificity for prostaglandins [120]. 15-PGDH type 2, which used NAD+ or NADP+ as cofactors, interacts with more substrates [121] and presents much higher Km values for prostaglandins than 15-PGDH type 1. Therefore 15-PGDH type 1 is considered as the major enzyme involved in the catabolism of prostaglandins and notably of PGE2 which is one of its favourite substrate. The NAD+-dependent 15-PGDH was purified in 1972 in human placenta [122]. This enzyme is ubiquitously expressed in mammalian tissues with highest activities in lung, kidney and placenta, but is also present in mouse costal cartilage [45,123]. The human 15-PGDH gene is localized to 4q34-q35. The cDNA of the enzyme has been cloned and encodes a 266 amino acids protein [124]. The structure of this cytosolic enzyme is a dimeric one composed of two identical subunits with a molecular weight of 29 KDa [125]. Nevertheless it has been proposed that the monomeric enzyme might be active [126]. The N-terminal region of 15-PGDH type 1 contains the binding site for NAD+ and therefore is essential for its enzymatic activity. After its binding to NAD+, 15-PGDH interacts with its substrate at the C-terminal region. Then, the catalytic reaction releases the PG and eventually NADH [127]. The regulation of 15-PGDH expression has been abundantly studied in cancer [128–130]. Moreover, 15-PGDH mRNA and protein expressions were altered in inflammed mucosa from patients with inflammatory bowel disease [131]. Moreover, IL-1β and TNFα reduced 15-PGDH mRNA expression in human colonocytes and trophoblasts cells from chorioamniotitis [132]. These results highly suggest a key role of 15-PGDH in some pathophysiological processes. Actually, the down-regulation of proteins involved in PGE2 inactivation is a largely unrecognized mechanism of inflammation and should be studied in other inflammatory diseases such as OA. Interestingly, a mechanical stress applied on mouse cartilage explants at physiological ranges stimulate 15-PGDH mRNA expression in cartilage but its response is delayed compared to the COX-2 and mPGES-1 induced overexpression [45].

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[102] Masuko-Hongo, K., et al., Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways. Arthritis Rheum, 2004. 50(9): p. 2829-38. [103] Bianchi, A., et al., Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondro-

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XI NO and Other Radicals in the Pathogenesis of Osteoarthritis Martin LOTZ, MD Division of Arthritis Research, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 [email protected] Keywords. iNOS, apoptosis, superoxide, peroxynitrite

Introduction A large body of information supports a role of free radicals in the pathogenesis of arthritis and studies on experimental models of arthritis suggest that inhibitors of their production or radical scavengers are of potential therapeutic value. The most important free radical species in biological systems are derivatives of molecular oxygen, sulphydryl or nitrogen compounds, polyunsaturated fatty acids and quinones and quinone-like compounds. Previously this field was predominantly concerned with the oxygenderived free radicals superoxide and hydroxyl radical. The production of superoxide by intact cells was first demonstrated by Babior in 1973 [1] who showed that leukocytes incubated with latex particles produced an activity that reduced cytochrome c and had the characteristics of superoxide. Since the demonstration that nitric oxide (NO) can be produced by mammalian cells in 1987, interest in this area of research has rapidly expanded. First, because of the diverse physiologic and pathogenetic effects of NO and second, because of the interactions of NO and superoxide to form peroxynitrite, a highly reactive species which may account for much of the free-radical induced toxicity.

Regulation of Nitric Oxide Production The first demonstration of nitric oxide (NO) release from mammalian cells was in the vascular endothelium where it was established as the endothelium-derived factor that causes smooth muscle relaxation. Since then NO has been shown to be produced in many tissues and to regulate diverse cell functions. The production of NO by leukocytes is associated with non-specific defense against certain microorganisms; it acts as a neurotransmitter in the CNS and in non-adrenergic-non-cholinergic peripheral neurons [2]. The role of NO in the pathogenesis of inflammatory diseases varies with the

M. Lotz / NO and Other Radicals in the Pathogenesis of Osteoarthritis

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NOS Citrulline + NO

L-Arginine + O2

NADPH

NADP+

NO + O2

NO 2– / NO3–

NO + O2–

–

–

O-O-N-O + H+

H-O-O-N-O

O-O-N-O

H-O-O-N-O OH + NO 2 / NO3–

The generation of NO is catalyzed by NO synthases (NO) which require L-arginine, NADPH and molecular oxygen as substrates. L-arginine is oxidized at a terminal nitrogen on the guanidino group, resulting in the formation of citrulline. NO is unstable and highly reactive. Nitrite (NO2–) and nitrate (NO3–) are stable and measurable end products. In the presence of nitric oxide and superoxide (O2–) the formation of peroxynitrite occurs (– O-O-N-O). Peroxynitrite can protonate to produce peroxynitrous acid (H-O-O-N-O). Peroxynitrous acid decays rapidly to form the hydroxyl radical (OH) and nitrogen dioxide (NO2) or nitrate (NO3–). Figure 1. NO synthesis and interactions.

type of inflammatory stimulus and the organ involved. A significant function of NO in cartilage and bone is suggested by the high levels of NO production in these tissues. NO and equal amounts of citrulline are enzymatically formed from L-arginine by nitric oxide synthases (NOS) which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors (Fig. 1). Two classes of enzymes are known, the constitutive NO synthases (cNOS) and inducible nitric oxide synthase (iNOS). Tissue-specific subtypes, have been described for cNOS, notably the neuronal and the endothelial cell cNOS [2]. Neuronal and endothelial cell cNOS are encoded by distinct genes but can also be expressed in other cell types. The activities of cNOS are regulated by the intracellular free calcium concentration and the Ca2+ binding protein calmodulin. At resting Ca2+, both isozymes are inactive; they become fully active after an increase in intracellular levels of Ca2+. Besides the conversion of L-arginine, cNOS generates H2O2 and reduces cytochrome p450, activities that are Ca2+/calmodulin-dependent. Other redox activities, the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase activity) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by cNOS appear to be Ca2+/calmodulin-independent. A second class of NOS is represented by the inducible NOS which was originally isolated from mouse macrophages. Only a single gene for iNOS has been identified. cDNA sequences cloned from human hepatocytes, articular chondrocytes and bone cells are identical [2,19]. The inducible enzyme from murine macrophages displays

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only 50% sequence identity to the neuronal enzyme. Like neuronal cNOS, macrophage iNOS has recognition sites for flavin-adenine-dinucleotide (FAD), flavinmononucleotide (FMN), and NADPH and also has a consensus calmodulin binding site. In contrast to cNOS, iNOS binds calmodulin tightly without a requirement for elevated Ca2+. This may explain why iNOS is independent of Ca2+ and elevated calmodulin and appears to be activated simply by being synthesized. Inducible NO synthase activity appears slowly after exposure of cells to cytokines such as IL-1, TNF or IFNγ and bacterial products and its expression is sustained. The inducible NOS can produce much larger amounts of NO than the constitutive forms and it is thought that the release of NO by iNOS accounts for the proinflammatory effects of NO.

NO Production by Chondrocytes iNOS expression has been demonstrated in various cell types. Within the joint, chondrocytes appear to be the major cell source of NO. In chondrocytes the expression of iNOS is readily inducible by a broad spectrum of stimuli. Articular chondrocytes produce increased levels of NO in response to low concentrations of IL-1 [3]. This contrasts with most other cell types where multiple stimuli are required for iNOS induction. Even within the mesenchymal cell lineages the production of NO by chondrocytes is unique. Undifferentiated mesenchymal stem cells do not express iNOS but after chondrocytic differentiation, cells expressed iNOS and produced NO following stimulation with IL-1. Mesenchymal stem cells having undergone adipogenic and osteogenic differentiation did not produce NO after IL-1 stimulation [4]. Moreover, this induction of iNOS expression and in human OA cartilage derives from a glucocorticoidinsensitive mechanism [5]. In addition to IL-1 various other cytokines, extracellular matrix degradation products [6,7], BCP, CPPD and MSU crystals [8–10] stimulate iNOS expression in chondrocytes. Mechanical loading plays a fundamental role in the physiological and pathological processes of articular cartilage and NO production can be induced or inhibited, depending on the type of mechanical stimulation. Application of shear stress upregulated nitric oxide and was associated with increases in chondrocyte apoptosis [11]. Shear stress suppresses collagen II and aggrecan mRNA expression and this is dependent on NO [11]. Mechanical injury also caused a significant loss of viable chondrocytes. Death of cells could be largely prevented by addition of N(G)-monomethyl-L-arginine to inhibit nitric oxide NO [12]. In contrast, dynamic compression to chondrocytes cultured in agarose, downregulates the release of NO and enhances cell proliferation and proteoglycan synthesis [13]. Cyclic tensile strain in chondrocytes inhibits IL-1-induced iNOS expression [14]. Cartilage is avascular and functions under hypoxic conditions. The formation of nitrotyrosine and peroxynitrite are dependent on oxygen tension [15]. A hypoxic environment fully blocks COX-2 activity but favors iNOS gene expression in cultured chondrocytes [16]. In vivo expression of iNOS in arthritis-affected cartilage has been demonstrated in human tissue and in joints from animals with experimental OA [17,18]. The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans [19].


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Molecular NO Effects NO reacts with other radicals, with carbohydrates, proteins, lipids and nucleic acids. NO can bind iron and thus regulate the activity of a large number of enzymes. Well characterized is NO binding to the heme iron in guanylate cyclase which is at the active site of the enzyme. This results in a conformational change and activation of the enzyme. Nitric oxide stimulates the mono-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Associated with ADP-ribosylation is a loss of enzymatic activity. This may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain [20]. N-terminal groups of some proteins can be modified by nitric oxide, perhaps by deamination [21]. Nitric oxide can also cause genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and intact DNA and caused DNA strand breakage [22]. Similar DNA damage can also occur in vivo and observed DNA sequence changes were consistent with a cytosinedeamination mechanism [23]. NO reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. Human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin [24]. By contrast, plasma levels of free nitric oxide are only in the 3-nM range.

Peroxynitrite Many cell types produce both NO and superoxide. The generation of these two radicals can lead to the formation of peroxynitrite [25] (Fig. 1). The formation of this relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide may contribute to inflammatory cell-mediated tissue injury [26]. Because superoxide and nitric oxide can react with each other to form peroxynitrite, they modulate each other’s half life and the quality of their biologic effects. Superoxide can limit the effects of NO by directing it to peroxynitrite and some NO effects such as vasodilation are prolonged in the presence of superoxide scavengers [27]. Conversely, NO can be regarded as a scavenger of superoxide anion and this suggested that NO may provide a chemical barrier to cytotoxic free radicals. Some effects of these radicals are clearly dependent on the formation of peroxynitrite. The inhibition of aconitases is only observed in the presence of peroxynitritie but not by NO in the absence of superoxide. Peroxynitrite is capable of oxidizing a variety of molecules, including sulfides, thiols, deoxyribose, lipids, ascorbate, α1-protease inhibitor. In the case of α1-protease inhibitor peroxynitrite oxidizes the methionine residue to sulfoxide and inactivates the protein. Peroxynitrite also inactivates the tissue inhibitor of metalloproteinase-1 (TIMP-1) activity towards gelatinase-A. High concentrations of peroxynitrite caused protein fragmentation while lower concentrations inactivated TIMP-1 without altering the molecular weight [28]. Peroxynitrite initiates lipid peroxidation and this mechanism contributes to O2- and NO-mediated cytotoxicity [29]. Reactive peroxynitrite anion may also exert cytotoxic effects in part by oxidizing tissue sulfhydryls [30]. Peroxynitrite can decompose to products that nitrate aromatic amino acids. Such nitro-aromatics may be ‘markers’ of NO-dependent oxidative damage. Nitrotyrosine residues were first demonstrated in atherosclerotic plaques and subsequently in synovial fluids. Serum and synovial fluid from patients with the rheumatoid arthritis contain

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3-nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3-nitrotyrosine. The demonstration of nitrotyrosine formation represents evidence for the production of peroxynitrite in vivo since tyrosine is nitrated by peroxynitrite but not by nitric oxide [31].

NO Effects on Cell Growth and Survival NO donors inhibit cell proliferation and endogenous NO production is at least in part responsible for the growth inhibition induced by cytokines or other agents. Conversely, factors that stimulate cell proliferation often inhibit NO formation [32,29]. Blanco et al. first reported that high concentrations of the NO donor sodium nitroprusside (SNP) induced apoptosis-like cell death in cultured human chondrocytes [33]. However, IL-1, an inducer of NO production in chondrocytes did not induce chondrocyte apoptosis [34]. But in combination with an oxygen radical scavenger hypoploidy and DNA fragmentation were observed. It was proposed that the balance between intracellular NO and ROS may determine the type of chondrocyte death, with a low concentration of ROS promoting apoptosis in the presence of NO and a high concentration of ROS promoting necrosis. Del Carlo reported that NO itself is not cytotoxic for human chondrocytes but may be when combined with superoxide. Under certain conditions of oxidative stress NO can even be protective against cell death [35]. In cultured human chondrocytes IL-1β induces binding of annexin V but cell death or a causal relationship between NO generation and annexin V binding was not demonstrated [34]. The NO donor SNP increased caspase-3 activity about 2.5 fold in human OA chondrocytes. A caspase-3 specific inhibitor peptide caused a partial inhibition of nucleosomal DNA fragmentation as analyzed by ELISA suggesting that cell death and cleavage of chromosomal DNA induced by exogenous NO in cultured chondrocytes may depend in part on active caspase-3 [36]. However, caspase-3 processing in response to SNP was not detected by immunoblotting and a caspase-3-specific inhibitor peptide failed to inhibit DNA degradation in cultured human chondrocytes [37]. It was also observed that IL-1β-induced NO can partially inhibit internucleosomal DNA fragmentation and caspase-3 processing induced by CD95 activation and simultaneous treatment with proteasome inhibitors. This effect of endogenous NO was mimicked by SNP. However, cell death was not blocked suggesting that NO specifically interferes with apoptosis execution but does not prevent chondrocytes from undergoing a form of cell death that does not require caspase-3 activation or internucleosomal DNA fragmentation [38]. In rabbit chondrocytes SNP induced p38 mitogen-activated protein kinase-dependent cell death and this was associated with enhanced caspase-3 activity, suggesting apoptosis as the cell death modality [39,40]. However, NO production as a result of adenovirus-mediated overexpression of iNOS did not cause cell death in rabbit chondrocytes [41]. There are no reports on the induction of apoptosis by endogenous NO or NO donors in cartilage explants. An in vivo study in a canine model of OA showed that oral application of the iNOS inhibitor L-NIL significantly reduced the number of apoptotic cells in femoral condyles [42] but it is not clear whether this is directly related to NO effects on cell survival or the result of protective effects of L-NIL against cartilage degradation. In certain cell types NO inhibits apoptosis through S-nitrosylation of cysteine residues present in the catalytic center of caspases as well as through a variety of additional mechanisms while in other cell types exogenous or endogenous NO are proapop-


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totic [43]. The mechanisms responsible for these dual actions of NO in regulating apoptosis are poorly defined. In human chondrocytes the effects of NO-donors on cell death are age-dependent: chondrocytes from older donors show an increased ratio of oxidized glutathione to reduced glutathione when compared to cells from younger donors. This may indicate that the cells from older donors are more susceptible to oxidant stress causing a greater number of chondrocytes from older donors to die in response to a nitric oxide donor [44]. Collectively, these studies illustrate that the impact of NO on cell survival is strongly dependent on the context in which NO is generated. While chondrocyte apoptosis is a feature of OA cartilage and often correlates with the expression of iNOS [45], a causal link has yet to be established.

NO and Extracellular Matrix Most of the currently available data suggest that NO promotes cartilage extracellular matrix degradation. Some discrepancies on the NO effects appear to relate to species differences. Studies with human [46], rat [47] and rabbit cartilage [48] indicate that NO donors and IL-1 induced endogenous NO inhibits proteoglycan synthesis. This was not observed with bovine cartilage [49]. NO also activates matrix metalloproteinases [50] and depolymerizes hyaluronan [51]. NO inhibits the chondrocyte response to the anabolic growth factor IGF-1 [52] and shifts the cytokine balance towards proinflammatory direction by reducing the synthesis of TGFß and IL-1 receptor antagonist [53].

Interaction Between NO and COX Pathways Many of the extracellular stimuli that induce iNOS expression also increase prostaglandin production and in OA-affected joints there is simultaneous increase of COX-2 and iNOS expression. However, the literature is divided with respect to whether NO activates or inhibits PG production and COX activity. In mouse macrophages lipopolysaccharide causes an increase in the release of NO and PGE2. Production of both NO2 and PGE2 was blocked by NOS inhibitors and this was thought to be a direct NO interaction with COX to cause an increase in the enzymatic activity [54]. NO donors also increased cyclooxygenase activity in endothelial cells and intravenous infusion of NO donors in vivo released 6-keto PGF1alpha, the stable metabolite of PGI2 [29]. Besides the activation of COX function, NO donors also amplified IL-1 beta-induced PGE2 production and potentiated IL-1 beta-induced mRNA and protein expression of COX-2 in macrophages and mesangial cells [55]. In chondrocytes NO stimulated NF kB activation and this was required for COX-2 expression [56].

Nitric Oxide Inhibition in Animal Models Development of bones and joints in iNOS knock-out mice appears normal. When experimental OA was induced by ligament transection and partial medial menisectomy iNOS knock out mice showed an unexpected acceleration of OA development [57]. In contrast, experimental rheumatoid arthritis induced by injection of monoclonal antibodies to collagen [58], zymosan-induced arthritis and collagenase-induced osteoarthritis were less severe in iNOS-deficient mice [59].

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Inhibitors of NO synthesis have been evaluated in various models of inflammatory arthritis [59–64] as well as in experimental OA [42,65]. The outcome of NO inhibition in inflammatory arthritis depends on the specific model. Early work in adjuvant arthritis showed antiinflammatory effects of L-NIL and L-NMMA [62,66]. In an acute model of joint inflammation induced by intraarticular injection of carrageenan and kaolin the iNOS inhibitor L-Nil and a selective COX-2 inhibitor inhibited joint swelling and greatest therapeutic benefit was observed with a combination of the two inhibitors. In contrast, in the chronic adjuvant arthritis model L-NIL exacerbated joint inflammation and abrogated the antiinflammatory effect of the COX-2 inhibitor [67]. A profound increase in joint destruction was also seen in response to L-NIL in streptococcal cell wall-induced arthritis [68]. Furthermore, septic arthritis [69] and the acute phase of antigen-induced arthritis [70] were more severe in iNOS-deficient mice. NO can also have certain antiinflammatory effects. NO donors reduce cytokine induced endothelial cell activation [71], inhibit endothelial-leukocyte interactions [72] and attenuate vascular inflammation [73]. These antiinflammatory actions may account for the increased severity seen after iNOS inhibition in some of the animal models. Only one study examined an iNOS inhibitor in experimental OA. In dogs subjected to anterior cruciate ligament transection, the administration of L-NIL reduced the severity of OA lesions and this was associated with reduced chondrocyte apoptosis, MMP and cytokine production [42,65].

Conclusions Increased production of reactive oxygen and nitrogen species in articular cartilage has been documented in human and experimental OA. A large body of literature is available on the in vitro effects of NO and other radicals on chondrocyte survival, extracellular matrix and inflammation. The specific effect of a radical on a given cell function is very much dependent on the experimental context. Specifically, the simultaneous presence of superoxide which leads to the formation of peroxynitrite, a stronger and highly reactive radical is likely to lead to cell and tissue damage. It is important to note that NO does have certain protective effects in cartilage and other tissues. Inhibition of NO in vitro or in vivo can lead potentially lead to undesired exacerbation of inflammation and tissue destruction under certain conditions. The pathophysiological role of peroxynitrite is supported by multiple lines of evidence and consequences of its generation in arthritis-affected tissues have been documented. Selective scavengers of peroxynitrite may thus be more promising candidates for the treatment of osteoarthritis. Compounds such as urate and polyphenolics decrease tissue nitrotyrosine formation. Administration of uric acid but not of and iNOS inhibitor to animals with zymosaninduced arthritis protected against cartilage degradation [74].

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XII Mitochondria and Chondrocytes: Role in Osteoarthritis Francisco J. BLANCO ∗, MD, PhD, María J. LÓPEZ-ARMADA, PhD and Ignacio REGO, PhD Osteoarticular and Aging Research Lab., Biomedical Research Center, Rheumatology Division, C.H. University Juan Canalejo, A Coruña, Spain [email protected]

Abstract. Mitochondria are critical regulators of cell function and cellular survival. Many lines of evidence suggest that mitochondria have a central role in ageing-related diseases. Mutations in mitochondrial DNA and oxidative stress both contribute to ageing. Osteoarthritis (OA) is a rheumatic disease associated to aging and it is characterized by articular cartilage degradation and increases of chondrocyte death. Articular cartilage chondrocytes must survive and maintain tissue integrity in an avascular environment. Then chondrocytes from deep and superficial zones may require adaptively increased anaerobic glucolysis and aerobic respiration respectively to support ATP synthesis. Recent ex vivo studie reported dysfunction of mitochondrial human OA chondrocytes. The analysis of mitochondrial electron transport chain activity in OA chondrocytes shows a significant decrease in Complexes I, II and III compared to normal chondrocytes. This mitochondrial dysfunction can mediate several pathways implicated in cartilage degradation such as, oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, pathologic cartilage matrix calcification and increased chondrocyte death (necrosis or apoptosis). Mitochondrial dysfunction in OA chondrocytes may be originated by somatic mutations in mtDNA or by direct effect of pro-inflammatory mediators (cytokines, prostaglandin, ROS and NO) on mitochondrial activity. Keywords. Mitochondria, chondrocytes, osteoarthritis, apoptosis

Introduction Mitochondrion (plural mitochondria) (from Greek mitos, thread or khondrion, granule) is a membrane-enclosed organelle, found in most eukaryotic cells. Mitochondria are sometimes described as “cellular power plants,” because they convert food molecules into energy in the form of ATP via the process of oxidative phosphorylation [1]. A ∗ Corresponding Author: Francisco J. Blanco. MD, PhD, Osteoarticular and Aging Research Lab., Biomedical Research Center, Rheumatology Division, C.H. University Juan Canalejo, A Coruña, Spain, E-mail: [email protected].

F.J. Blanco et al. / Mitochondria and Chondrocytes: Role in Osteoarthritis

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Figure 1. Structure of Mitochondria.

typical eukaryotic cell contains about 2,000 mitochondria, which occupy roughly one fifth of its total volume [2]. Mitochondria contain DNA that is independent of the DNA located in the cell nucleus. A mitochondrion contains inner and outer membranes composed of phospholipid bilayers and proteins (Fig. 1). The two membranes, however, have different properties. The outer mitochondrial membrane, which encloses the entire organelle, has a proteinto-phospholipid ratio similar to the eukaryotic plasma membrane (about 1:1 by weight). It contains numerous integral proteins called porins, which contain a relatively large internal channel (about 2–3 nm) that is permeable to all molecules of 5000 daltons or less [3]. Larger molecules can only traverse the outer membrane by active transport through mitochondrial membrane transport proteins. The outer membrane also contains enzymes involved in such diverse activities as the elongation of fatty acids, oxidation of epinephrine (adrenaline), and the degradation of tryptophan. The inner mitochondrial membrane contains proteins with four types of functions [3]: 1) Those that carry out the oxidation reactions of the respiratory chain. 2) ATP synthase which makes ATP in the matrix. 3) Specific transport proteins that regulate the passage of metabolites into and out of the matrix. 4) Protein import machinery.The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to generate ATP. In addition, there is a membrane potential across the inner membrane (mitochondrial membrane potential-Δψm). Mitochondria possess their own genetic material, and the machinery to manufacture their own RNAs and proteins. Although most mitochondrial proteins are encoded

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by the nuclear genome, mitochondria contain many copies of their own DNA. Human mtDNA is a circular molecule of 16,569 base pairs that encodes 13 polypeptide components of the respiratory chain, as well as the rRNAs and tRNAs necessary to support intramitochondrial protein synthesis using its own genetic code. Inherited mutations in mtDNA are known to cause a variety of diseases. One hypothesis has been that somatic mtDNA mutations acquired during ageing contribute to the physiological decline that occurs with ageing and ageing-related diseases [4]. Although it is well known that the mitochondria convert organic materials into cellular energy in the form of ATP, mitochondria play an important role in many metabolic tasks, such as apoptosis-programmed cell death, cellular proliferation, regulation of the cellular redox state, heme synthesis and steroid synthesis. Production of ATP is done by oxidizing the major products of glycolysis:pyruvate and NADH that are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited the glycolytic products will be metabolised by anaerobic respiration, a process that is independent of the mitochondria. The production of ATP from glucose has an approximately 15–18 fold higher yield during aerobic respiration compared to anaerobic respiration. Each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane, and into the matrix where it is oxidized and combined with coenzyme A to form CO2, acetyl CoA and NADH. The acetyl CoA is the primary substrate to enter the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or Krebs cycle. The citric acid cycle oxidizes the acetyl CoA to carbon dioxide and in the process produces reduced cofactors (three molecules of NADH and one molecule of FADH2), that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP) (Fig. 1). Protein complexes in the inner membrane (NADH dehydrogenase, cytochrome c reductase and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H+) into the intermembrane space (Fig. 2). This process is efficient but a small percentage of electrons may prematurely reduce oxygen, forming the toxic free radical superoxide. This can cause oxidative damage in the mitochondria and may contribute to the decline in mitochondrial function associated with the aging protein [4]. The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria store calcium, a process that is one important event for the homeostasis of calcium in the cell. Release of this calcium back into the cells interior can initiate calcium spikes or waves. These events coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells. In summary, mitochondria are critical regulators of cell function and cellular survival. Many lines of evidence suggest that mitochondria have a central role in ageingrelated diseases. Mutations in mitochondrial DNA and oxidative stress both contribute to ageing. Osteoarthritis (OA) is a rheumatic disease associated to aging and it is characterized by articular cartilage degradation and increases of chondrocyte death. Mitochondrial function in normal or OA articular chondrocyte has not been studied with detail. However, recent investigations in mitochondria activity and function have opened new exciting knowledge that we will summarize in this chapter.


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Figure 2. Mitochondrial Respiratory Chain.

1. Mitochondria in Articular Cartilage Chondrocytes Considering the structure of articular cartilage, it should be noted that there are no blood vessels, lymphatic channels, or neural elements that enter or pass through adult articular cartilage. Furthermore, the chondrocytes are separated from the blood vessels of the underlying bone by a zone of dense calcification and the mature cortex of the underlying subchondral bony end-plate (Fig. 3). Numerous studies have been performed to assess whether diffusion from the underlying bone can provide nourishment to the cartilaginous surface, and it is now well established that in the adult cartilage transport of nutrients by this route does not occur [5]. Conversely, studies in which nutrients have been injected into the joint cavity have demonstrated rapid transport through the cartilage and the matrix to the cells, suggesting strongly that the major source of nutrients is synovial fluid [6]. Since the synovial fluid is in itself an ultafiltrate of plasma, it is apparent that chondrocytes receive their nutrition through a double diffusion system. Nutrients must first diffuse across the synovial barrier into the synovial fluid and then across the matrix of articular cartilage to reach the cell. It has benn demonstrated that the matrix of articular cartilage is not freely permeable and that diffusion of nutrients is in large measure dependent on size and charge [7]. Detailed information about the metabolic fuels required by cartilage is scanty. The results of a considerable number of experiments indicate that while the TCA is active in cartilage, as much as 80% of glucose is metabolized to lactate by anerobic glycoly-

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Figure 3. Scheme of Joint (Modified from Dinarello C and Moldawer L, A primer for Clinicians, 3rd ed. Thousand Oaks, Ca, USA, Amgen Inc, 2001.

sis [7]. Studies of cells in culture also indicate that isolated chondrocytes are dependent of anerobic glycolysis. In this respect, chondrocytes are similar to other cell types. Results of many investigations clearly document that cultured cells generate much of their ATP through glycolysis and produce lactate as an end-product. However, evidence that cartilage cells can utilize oxidative metabolism to produce chemical energy is supported by the observation that chondrocytes contain mitochondrial dehydrogenases; that isolated mitochondria can utilice TCA cycle substrates; and that the mitochondria contain enzymes required for oxidative phosphorylatoin and electron transport [8,9]. In addition, in cultured human articular chondrocytes, the activity of mitochondrial respiratory chain (MRC) shows an enzymatic activity similar to other mesenchymal cells (Table 1) [10]. Furthermore, it has been reported that mitochondrial oxidative phosphorylation may account for up to 25% of the ATP produce in cartilage [11,12]. In all tissues, oxygen serves as the final electron acceptor for mitochondrial cytochrome oxidase. In addition, oxygen serves as a substrate for a very large number of other enzymes (oxygenases and oxidases). Tissue oxygen tensions vary and the actual value is dependent on a number of factors. These include the number of cells per unit volume and the rate of cellular metabolism, oxygen diffusibility through the extracellular matrix and the vascular supply. Thus chondrocytes in articular cartilage receive oxygen only by diffusion from the synovium (Fig. 3). Moreover, because of the asymmetry of the oxygen supply, a gradient must exist across the tissue [13,14]. As a result of the gradient, it is estimated that cells at the surface experience between 5% and 7%


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Table 1. Values of mitochondrial respiratory chain (MRC) complexes in cultures of normal, OA and normal chondrocytes treated with NO Normal Chondrocytes Age, years Proteins, mg/ml CS enzymatic activity, nmoles/minute/mg protein Mitochondrial complex activity‡ Complex I Complex II Complex III Complex IV

OA Chondrocytes

59.7 ± 21.8 (30) 3.6 ± 1.3 (30) 111.7 ± 29.8 (29)

68.5 ± 7.6 (53) 3.3 ± 0.9 (53) 124 ± 2.6 (51) †

27.9 ± 13.6 (22) 11.5 ± 5.7 (25) 54.2 ± 13.6 (25) 53.6 ± 11.9 (29)

22.5 ± 9.4 (46) 9.2 ± 3.3 (47) † 46.5 ± 9.7 (49) † 53.1 ± 13.2 (49)

Chondrocytes with SNP 59.7 ± 18.9 (11) 4.2 ± 1.4 (11) 106.6 ± 26.2 (11)

22.8 ± 19.1 (11) 10.2 ± 1.81 (11) 6.27 ± 9.7 (11) 40.2 ± 11.3 (11) †

* Values are the mean ± SD (n). CS = citrate synthase. † P ≤ 0.05 versus normal chondrocytes. ‡ CS-corrected complex activity is expressed as (nmoles/minute/mg protein) / (CS specific activity) X 100. Complex I = rotenone-sensitive NADH-coenzyme Q1 reductase; Complex II = Succinate dehydrogenase; Complex III = antimycin-sensitive ubiquinol cytochrome c reductase; Complex IV = cytochrome c oxidase.

O2, compared with 13% in arterial blood [15]. In contrast, cells in the deepest regions of the cartilage are exposed to a low oxygen tension [16,17]. In summary, articular cartilage chondrocytes must survive and maintain tissue integrity in an avascular environment. In addition, oxygen and glucose concentration supply to cartilage is characterized by asymmetry with a gradient from superficial to deep zone. Then chondrocytes from deep and superficial zones may require adaptively increased anaerobic glucolysis and aerobic respiration respectively to support ATP synthesis. 2. Mitochondria and Osteoarthritis Because articular cartilage chondroyctes are traditionally classified as cells highly glycolytic, mitochondrial mediated pathogenesis has not been previously investigated with detail for OA [16,17]. However alteration in some mitochondria functions such as ATP production, apoptosis and redox state could explain some mechanisms involving cartilage degradation during OA. 2.1. Mitochondria in OA Chondrocytes Morphologic studies describe changes in OA cartilage tissue showing nuclear (pyknosis and karyorrhexis) and cytoplasmic changes (fat droplets, glycogen granules, and microfilaments) have been reported [18]. In this study mitochondria were described as “swollen” and authors concluded that the chondrocyte underwent “necrosis”. They noted that these changes increase in extent and degree with an increased severity in the arthroscopic stage classification. Recent ex vivo studie reported dysfunction of mitochondrial human OA chondrocytes [10]. The analysis of mitochondrial electron transport chain activity in OA chondrocytes shows a significant decrease in Complexes II and III compared to normal chondrocytes (Table 1). A dysfunction in Complexes II and III compromises the electron transfer pathway and this defect could be solved by overloading the electron transport via Complex I, a pathway with little oxygen consumption. However, the dimin-

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ished efficiency to transport electrons via Complex II does not enhance the activity of Complex I (rather, activity of Complex I is reduced). On the other hand, mitochondrial mass is increased in OA chondrocytes compared with cells from normal cartilage, as demonstrated by a significant rise in citrate synthase (CS) activity [10]. Therefore, an increase in mitochondrial mass could be a mechanism of OA chondrocytes to compensate the electron transfer deficiency via Complexes II and III and its resulting low production of ATP per mitochondrium. Reduction in chondrocyte respiration causes intracellular ATP depletion by 50–80% [11,19]. Furthermore, OA cells showed a reduction in the Δψm as demonstrated by using the fluorescent probe JC-1. Quantitative studies performed by flow cytometry showed that OA chondrocytes have a lower red/green fluoresecence ratio that normal chondrocytes, indicating depolarization of the mitochondria [10]. 2.2. Mitochondria and OA Pathogenesis Although OA chondrocytes have dysfunction in mitochondria activity, an important aspect to know is the relevance of this finding to explain specific pathogenic pathways implicated in OA. In this sense, some results show that mitochondrial dysfunction can mediate several pathways implicated in cartilage degradation. These include oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, pathologic cartilage matrix calcification and increased chondrocyte death (necrosis or apoptosis). Several studies have reported that the in vitro use of specific inhibitors of mitochondrial electron transport suppressed the synthesis of proteoglycans and collagen by human articular chondrocytes [11,19,20]. In addition, in vitro inhibition of complex I with rotenone also reduced the proteoglycan content of the extracellular matrix in the superficial and middle zones and it increased the release of GAGs from cartilage to supernatant [20]. Several mechanisms can explain the effect of mitochondrial dysfunction on proteoglycans and collagen depletion. For example, the inhibition of complex III or V is able to reduce proteoglycan synthesis and to induce proteases synthesis such as MMP-1, -3, -13 and ADAMS-5 [21,22]. Furthermore, the activity reduction of both mitochondrial complexes increases the synthesis of cytokines (IL-1, IL-6 and IL-18); prostaglandin E2 (PGE2) and chemokynes (IL-8 and MCP-1) [21,22]. Mitochondrial respiratory chain is one of the most important sites of ROS production [23]. In human articular chondrocytes, the inhibition of complex III and V activity with specific MRC inhibitors (antimicyn-A and oligomicyn respectively) induced ROS synthesis [21]. Although increase in ROS formation during hypoxia is difficult to explain, two factors may contribute to an increase mitochondrial ROS formation in hypoxia [23]. Firstly, under hypoxic conditions, low concentrations of NO: may still be produced since the Km for oxygen of the mitochondrial nitric oxide synthase is around 30–40 microM. Secondly, NO may bind and inhibit cytochrome oxidase, resulting in an increase in its Km for oxygen and an increased reduction of electron carriers located upstream from the terminal oxidase, favouring O2- formation at low oxygen concentrations. In this sense, NO production by OA chondrocytes is increased and NO suppresses mitochondrial oxidative phosphorylation by reducing the activity of complex IV and decreases the Δψm [24]. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix [25–27].


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Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Calcium and phosphorus are also clearly present in single mitochondrial granules within growth plate chondrocytes and in certain extracellular particles distinct from MV [25]. Pathologic hydroxyapatite (HA) deposition and calcium pyrophosphate dihydrate (CPPD) crystal deposition are both common in OA, particularly in advanced disease [26]. Significantly, the onset of HA deposition around hyperthophic endochondral cartilage chodrocytes coincides with sharply defined changes in the redox and metabolic states of chondrocytes, in association with focal loss of cell respiration [27]. Moreover, a marked decreases in MV PPi is obligatory for HA crystal deposition to be initiated in the interior of MV in vitro. Direct suppression of mitochondrial respiration promotes MV-mediated mineralization in chondrocytes [11,12]. Previously described changes in mitochondrial function that directly modulate mineralization include regulation of intramitochondrial calcium accumulation and release of intramitochondrial calcium stores. Chondrocytes MRC complexes also regulate mineralization at the levels of the MV content of PPi and the calcium-precipitanting ability associated with MV. Then, the chondrocytes MRC activity could partly regulates differential deposition of HA and CPPD. Regulation of MRC activity may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization [11]. Interestengly, in vivo studies carried out in an animal model support these results. Recently, it has been assessed chondrocytes for ATP depletion and for in situ changes in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee OA in male Hartley guinea pigs [28]. Results showed that spontaneous NO release from knee cartilage samples in organ culture doubled between ages 2 months and 8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels declined by approximately 50%, despite a lack of mitochondrial ultrastructure abnormalities in knee chondrocytes. As ATP depletion progressed with aging in knee chondrocytes, an increased ratio of lactate to pyruvate was observed, consistent with an adaptive augmentation of glycolysis to mitochondrial dysfunction. 2.3. Mitochondria and Chondrocyte Apoptosis Histological studies of OA cartilages show a reduction in the number of chondrocytes compared with normal cartilages [29]. Several authors have suggested that apoptosis is the responsible of the hypo-cellularity in OA cartilage [30–32]. Apoptosis is a distinct mode of cell death that differs morphologically from necrosis [33]. Apoptosis is conceptually an evolutionarily conserved, innate process by which cells systematically inactivate, disassemble, and degrade their own structural and functional components to complete their own demise. “Programmed cell death” is often used interchangeably with apoptosis, reflecting the fact that cell death occurs according to a sequential ‘program’ of cellular, biochemical and molecular events. Apoptosis has been distinguished from necrosis in vivo by the following features: 1) while necrosis is usually accompanied by an acute inflammatory response with exudation of neutrophils and monocytes, this phenomenon is absent in apoptosis; and 2) while necrosis involves groups of neighbouring cells, apoptosis usually appears in discrete individual cells in a tissue. 3) morphologically, nuclear condensation may appear in necrosis, but it has poorly defined edges and is irregularly scattered through the nucleus, while apoptotic nuclear condensation reveals sharply defined masses of uniform texture. The reason that the cellular phenomenon of apoptosis has become the focus of such great interest among

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Figure 4. Mitochondria in the apoptosis of chondrocytes.

researchers is that apoptosis involves a signalling cascade that leads to the organized disintegration of cells that, in contrast to necrosis, is potentially reversible and amenable to therapeutic manipulation. The reported percentages of apoptotic chondrocytes in OA cartilage range from 0–6% [34]. The discrepancy in the percentages by various reports was probably deemed the result of the methodology employed to detect apoptosis [35]. In addition, how it is very difficult to demonstrate typical apoptotic chondrocytes accompanying apoptotic bodies even in advanced human OA articular cartilage; thus, nonapoptotic programmed cell death due to other cell death mechanisms has been postulated (chondroapoptosis or paraptosis) [36]. Taking in mind these comments, abundant literature investigates the stimuli and signalling mechanisms of chondrocyte apoptosis such as the death receptor and mitochondrial pathways (Fig. 4). Apoptosis through the mitochondrial pathway occurs after cellular damage, which causes changes in the conformation and/or activity of the proapoptotic Bcl-2 family members, such as Bak and Bax [37], in the outer mitochondrial membrane. Cytochrome c and other polypeptides are subsequently released from the intermembrane space of the mitochondria and bind to the cytoplasmic scaffolding protein, Apaf-1, causing an ATP-dependent conformational change that allows Apaf-1 to bind to the prodomain of pro-caspase-9 [38]. This interaction enhances the proteolytic activity of procaspase-9, resulting in the activation of executioner caspases, such as caspases-3 and -7. The release of Smac/Diablo inhibits the effect of the inhibitor of apoptosis (IAP), resulting in the inhibition of the interaction of IAP with caspase-9.


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The role of NO has been one focus of interest in the chondrocyte death and apoptosis. Recent studies suggest that NO induces apoptosis in chondrocytes because, it reduces the activity of complex IV and decreases the Δψm [24]. Apart from inhibiting respiration, NO has additional effects on mitochondria such as induction of ROS and induction of mtDNA damage which are related with cell death [39]. However, the precise role of NO in the induction of chondrocyte death is currently debated. Although treatment with NO donors consistently induces apoptosis in cultured chondrocytes [40,41], the production of high levels of endogenous NO by the overexpression of the iNOS gene in transfected chondrocytes was not found to cause cell death [42]. This discrepancy might be the result of using chemical NO donors, which not only generate reactive nitrogen species but also produce various secondary reactions depending on the cellular milieu in in vitro experiments. A recent study that employed diazeniumdiolates, which have been shown to be reliable sources of NO, suggested that exogenous NO is not cytotoxic to cultured chondrocytes per se, and can even be protective under certain conditions of oxidative stress [41]. Nitrite was found to exert a protective effect upon hypochlorous acid-induced chondrocyte toxicity, thus implicating NO in a novel cytoprotective role in inflamed joints [43]. While recent data indicate that NO may not be the sole mediator of chondrocyte death, the role of peroxynitrite, a reaction product of NO and superoxide anions, is postulated [41]. Interestingly, it is therefore proposed that the balance between intracellular NO and ROS may determine the type of chondrocyte death, with a low concentration of ROS promoting apoptosis in the presence of NO and a high concentration of ROS promoting necrosis [40,44]. Finally, a recent study on peroxynitritemediated chondrocyte apoptosis revealed that the predominant mode of cell death involved calcium-dependent cysteine proteases, known as calpains, and that peroxynitrite induced mitochondrial dysfunction in cells that leads to caspase-independent apoptosis [45]. In an “in vitro” experiment using human cartilage, chondrocytes from old donors were found to be more susceptible to cell death induced by an NO donor; this susceptibility was correlated with a higher ratio of oxidized glutathione to reduced glutathione, providing evidence that increased oxidative stress with aging makes chondrocytes more susceptible to oxidant-mediated cell death [46]. In summary, the pathways and patterns of chondrocyte death (apoptosis, chondroptosis, parapotosis) are much more diverse than originally observed. Mitochondria activity plays an important role in chondrocyte survival. Furthermore, it should be noted that the relative contribution of apoptotic cell death in the pathogenesis of OA is still difficult to assess because of the chronic nature of the disease process. However, recently some in vivo studies analyzing the effects of intra-articular apoptosis inhibitors in chondrocytes death and cartilage degradation have showed interesting results [47]. Intra-articular administration of the pancaspase inhibitor Z-VAD-FMK, Caspase-1 inhibitor and the combination of caspase-3 and caspase-8 inhibitors reduced the severity of cartilage lesions in experimental OA (ACLT in rabbits), suggesting that they may have disease-modifying activity in human OA [47].

3. Mechanisms Modifying Mitochondrial Activity in OA Several molecules with catabolic profile (NO, PGE2) and some pro-inflammatory cytokines (TNFα and IL-1β) localized in the synovial fluid of OA joints at high concen-

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trations may modify the mitochondrial activity (Fig. 3). Some results indicate that both cytokines, TNFα and IL-1β, modified mitochondrial function by a mechanism involving decrease in the activity of complex I of CRM and ATP production, as well as a reduction in Δψm in human chondrocyte cells [20]. Interestingly, in human articular chondrocytes, NO suppresses mitochondrial oxidative phosphorylation because it reduces the activity of complex IV causing decrement of ATP synthesis and Δψm [11]. In addition, stimulation of OA chondrocytes with PGE2 decreases Δψm and ATP generation [48]. Mitochondria have been trough to contribute to ageing and pathology through the accumulation of mitochondrial DNA (mtDNA) mutations induced by mutagens such as ROS or NO. It is well established that mtDNA accumulates mutations with ageing, especially large-scale deletions and point mutations [49]. The accumulation of deletions and point mutations correlates with decline in mitochondrial function. In the mtDNA control region, point mutations at specific sites can accumulate to high levels in certain tissues: T414G in cultured fibroblasts, A189G and T408A in muscle and C150T in white blood cells [50]. However these control region “hot spots” have not been studied in chondrocytes. One hypothesis is that somatic mtDNA mutations acquired during ageing contribute to the mitochondrial activity decline that occurs in OA chondrocytes. Accumulation of mtDNA mutations may be due to increases production of ROS or to a defect in the mitochondrial anti-oxidant system [23]. One of the primary scavangers of ROS is superoxide dismutase, which catalyzes the dismutation of superoxide to hydrogen peroxide and then to water. Local deficiency of SOD can lead to the formation of peroxynitrite and other oxidizing species. Three distinct SODs are found in the human body: SOD1 (Cu/Zn SOD) which is found in the mitochondria, which localized primarily to the cytosol, SOD2 (MnSOD) and SOD3 or extracellular SOD (EC-SOD). SOD2 is the enzyme that plays an essential role in oxidative stress mitochondrial protection [23]. The role of SOD2 in OA chondroyctes is unknown, however the levels of SOD2 protein in human articular chondrocytes decreases with aging [51]. Interestingly, it has been reported other mechanisms to induce somatic mutations in mtDNA. Several groups have addressed the issue of causation using a clever approach to generate mtDNA mutations experimentally. mtDNA replication is carried out by mtDNA polymerase-γ (POLG), which has 3-to-5 exonuclease (proofreading) activity in addition to its 5-to-3 polymerase activity [52]. If the proofreading activity of POLG is eliminated and the polymerase activity preserved, mtDNA mutations accumulate because of uncorrected errors during replication. In mice with such proofreadingdeficient POLG (mtDNA-mutator mice), mtDNA mutations accumulate to high levels in all tissues. By 8 weeks of age, homozygous Polg–/– animals had 9 point mutations per 10 kb in cytochrome b. By contrast, normal mice had less than 1 mutation per 10 kb. This marked increase in mtDNA mutations resulted in decreased respiratory enzyme activity and ATP production. To begin with, the mice appeared normal, but by 25 weeks of age began to exhibit pathology frequently seen in human ageing, including weight loss, alopecia, osteoporosis, kyphosis, cardiomyopathy, anaemia, gonadal atrophy and sarcopaenia (presence of OA was not studied). Somatic mutations of mtDNA have been detected in synoviocytes from Rheumatoid Athritis and OA patients [53]. Authors described that RA synoviocyte mtDNA had about twice the number of mutations as the OA group. However they did not compare the number of mtDNA mutations with healthy group. There are not published studies describing the mutations in


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mtDNA from OA chondrocytes. In this sense, preliminary results obtained in our lab using Temporal Temperature Gradient Electrophoresis (TTGE) showed that mtDNA from OA chondrocytes has higher number of mutations than mtDNA from healthy chondrocytes [54].

4. Conclusions In summary, mitochondrial function (MRC activity and ATP synthesis) of OA chondrocytes is altered. Mitochondrial dysfunction may mediate several specific pathogenic pathways implicated in OA. These include oxidative stress, inadequacy of chondrocyte biosynthetic responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, increased chondrocyte death (e.g. apoptosis), and pathologic cartilage matrix calcification. Mitochondrial dysfunction in OA chondrocytes may be originated by somatic mutations in mtDNA. Another explanation is the direct effect of some pro-inflammatory mediators (cytokines, prostaglandin, ROS and NO) on CRM and ATP synthesis. The weight of evidence reviewed herein strongly supports chondrocyte mitochondrial impairment as a mediator of the establishment and progression of cartilage degradation during OA. Thus, therapies targeting basic mitochondrial processes, such as energy metabolism or free-radical generation, hold great promise to treat OA diseases.

Acknowledgements This study was supported by grants from Ministerio Educacion y Ciencia (SAF 2005-06211), Secretaria I+D+I Xunta Galicia (PGIDIT06PXIC916175PN and from Fondo Investigación Sanitaria (CIBER- CB06/01/0040)-Spain, with participation of fundus from FEDER (European Community). MJ López-Armada was supported by Contrato Investigadores SNS (Fondo Investigación Sanitaria, Spain, CP06/00292). Ignacio Rego was supported by Contrato de Apoyo a la Investigación-Fondo Investigación Sanitaria (CA06/01102).

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XIII Subchondral Bone and Osteoarthritis Progression: A Very Significant Role Johanne MARTEL-PELLETIER, PhD, Daniel LAJEUNESSE, PhD and Jean-Pierre PELLETIER, MD Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec, Canada H2L 4M1 Abstract. Osteoarthritis (OA) is considered a complex illness in which cross-talk between the different tissues of the joint plays a significant role in the evolution of the disease. Although we may not yet completely know all the initiating factors involved in the degeneration of the articular tissues, significant progress has been made with respect to the proposal of new concepts regarding the etiopathogenesis of this disease. For decades, the prevailing concept centered around the destruction of articular cartilage as the focal pathological feature of OA. Consequently, it is not surprising that investigators concentrated their efforts at identifying mechanisms involved in the destruction of this tissue. There is now substantial evidence, from preclinical and clinical studies, that changes in the subchondral bone metabolism comprise an integral component of the disease process, and its key role in the initiation and/or progression of cartilage degeneration may have been largely underestimated. This concept as well as the complex pathophysiological mechanisms taking place in this tissue during OA, are the focus of this chapter.

Introduction Osteoarthritis (OA) is the most common disabling chronic condition in the western world. It is not a single disease entity but, rather, involves several subgroups with different underlying pathophysiological mechanisms. OA is a disease of an entire organ system, in which both anatomical and metabolic changes act together to bring about the structural changes. The notion that the degeneration and erosion of cartilage is the primary pathophysiological mechanism of OA has recently been challenged, in view of the identification of prominent changes in the subchondral bone which provide evidence suggesting that this tissue may also play a key role in the etiology of the disease. In fact, data strongly suggest that the subchondral bone could be a driving force behind the cartilage degradation observed in OA. It is common knowledge that, in OA animal models, changes in bone density osteoid volume and subchondral bone thickness are often more severe than cartilage changes. The severity of cartilage fibrillation and loss generally exceeds bone changes only in advanced OA in primate animal models [1,2].

J. Martel-Pelletier et al. / Subchondral Bone and Osteoarthritis Progression

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Subchondral Bone as a Risk Factor in OA Magnetic resonance imaging is ideal for assessing the structural changes that take place in OA, particularly in longitudinal studies of cartilage and bone, and for identifying the very early changes often overlooked by other, less sensitive, imaging technologies. It is now possible to quantify the trabecular architecture, the volume of subchondral bone marrow oedema and cyst lesions, as well as bone attrition and volume of cartilage. At first, a number of studies focused on the exploration of structural changes in the subchondral trabecular bone in knee OA patients. In a cross-sectional study, Beuf et al. [3] found that, in OA, the loss of femoral trabecular bone was correlated to the severity of the disease. For their part, Blumenkrantz et al. [4] showed that the loss of cartilage volume and the deterioration of the subchondral bone structure were interdependent, and a positive correlation was established between the loss of cartilage and subchondral bone sclerosis and osteopenia of the underlying trabecular bone. Moreover, other studies have recently reported that bone marrow lesions, including oedema and cysts, have a high prevalence in human knee OA [5–7], and a statistically significant correlation was found for the medial compartment between the increase in oedema and cyst size over time (2 years) and the loss of cartilage volume juxtaposed to the location of the lesions [8]. These data underline the importance of the subchondral bone remodeling in OA pathophysiology, and that bone marrow lesions are markers and strong predictors of structural worsening of knee OA cartilage lesions. Although these data suggest that subchondral bone alterations may be more intimately related to the OA process than merely being a consequence of the disease, a question that still remains is whether changes in subchondral bone induce or participate in disease progression. Studies have shown that in knee OA, the subchondral bone is stiffer and can increase trabecular bone strain in the proximal tibial plateau and distal tibia [9–11]. Bone strain could then lead to subsequent cartilage lesions. Hence, a steep stiffness gradient in the underlying subchondral bone may be an initiating mechanism of OA, as the integrity of the overlying articular cartilage depends on the mechanical properties of its bony bed. Inhomogeneities in density or stiffness of the subchondral bone may thus be key factors that could modulate cartilage loss. Indeed, articular cartilage above a less dense and more compliant bone will deform more than that above a denser and stiffer bone. Such deformation can then stretch the articular cartilage at the edge of the joint contact area, generating tensile and shear stresses [12].

OA Subchondral Bone Material Versus Mineral Density It has been shown that in knee OA, the progressive joint space narrowing correlates positively with the bone density of the tibial subchondral bone [13]. However, further studies point to the fact that the higher density of this tissue in OA is due to an increase in material density, not an increase in mineral density [14–16]. The higher density observed in this diseased tissue appears to be due to an increased osteoid collagen matrix and an increase in trabecular number and volume. However, in vivo and in vitro studies have revealed the presence of an abnormal mineralization process in OA subchondral bone, resulting in a hypomineralization of this tissue [15,17,18]. Thus, subchondral bone sclerosis results from an increased stiffness, and not an actual increase in bone mineral density. Some clinical studies have shown that the indices of bone resorption

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Figure 1. Cartilage and subchondral bone. Osteoarthritic subchondral bone showed an increased resorption at the early stage of the disease and sclerosis at the late stage. From reference [19].

are increased early in the disease, while subchondral bone sclerosis is a relatively late occurrence (Fig. 1; [19]). Consequently, the acceleration of bone turnover in OA results in the deposition of a hypomineralized bone, which reduces its stiffness for a given apparent density but increases stiffness if this is offset by increased bone volume.

OA Subchondral Bone: Early Versus Late Stages Several reports have indicated that the subchondral bone remodeling that occurs in OA involves both bone resorption and bone formation. However, a major area understudied is the characterization of specific changes that can distinguish advanced from early disease. Studies from animal models that allow for a chronological evaluation of these changes suggest that in the more advanced stage of the disease, bone formation is predominant [1,2,20,22]. In contrast, in the early phase there is a remodeling process that primarily favors bone resorption. In experimental OA animal models representing an early stage of the disease, it has been shown that the subchondral plate and the underlying trabecular bone become thinner, indicating excess bone resorption (Fig. 2) [21,23,24]. This agrees with the study of Bettica et al. [25] who demonstrated that general bone resorption, as defined by the level of type I collagen N- and C-terminal telopeptides biomarkers, is increased in patients with progressive knee OA. Similarly, Messent et al. [26], with the use of fractal signature analysis, showed that bone loss occurred in patients with knee OA, specifically in the medial compartment, and that these changes were associated with an increase in the number and size of the remodeling units.

Abnormal Biochemical Pathways Many studies have demonstrated that the subchondral bone is the site of several dynamic morphological changes that seem to be part of the disease process. These changes are allied with many local abnormal biochemical pathways, including the increased synthesis of several bone markers, growth factors, cytokines, proteases and inflammatory mediators. The levels of alkaline phosphatase, osteocalcin, collagen type


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Figure 2. Morphometric analysis of subchondral bone in normal (n=6) and osteoarthritic (OA) (n=7) anterior cruciate ligament dog model. OA subchondral bone specimens were selected at the lesional (L) and nonlesional (NL) areas of the tibial plateaus. Right panel: Data are the bone surface at different depths starting from the calcified cartilage 0–500 µm or 500–1000 µm. Morphometric data are presented as box plot, where the boxes represent the first and third quartiles, i.e. line within the box represents the median and lines outside the box represent the spread of values. The statistical analysis data are the comparison made between the normal and the OA groups using the Mann-Whitney U test. Left panel: Representative morphological sections of subchondral bone. From reference [23] with modification.

I, IL-6, IGF-1, TGF-ß, PGE2, LTB4, proteases including urokinase, cathepsin K and metalloproteases (MMPs) have all been found elevated in OA subchondral bone osteoblasts [27–30]. An abnormal level of production of mature collagen type I [14,27,31] accompanied by a significantly elevated level of the collagen type I α1 chain has also been found in OA subchondral bone. Collagen type I is composed of heterotrimer α1 and α2 chains. In normal bone an average ratio of 2.4:1 is found. Yet, in OA this ratio varied from 4–17:1 [14,27,31]. The abnormal production of the α chains is of great importance and could provide an explanation for the reduction in bone mineralization, as it has been shown that the increase in α1 homotrimers in bone causes a 50% reduction in the strength of this tissue in α2 knock-out mice [32]. The higher levels of osteocalcin found in the subchondral bone of OA patients, even at non-weight-bearing sites, and the finding that in vitro subchondral human OA osteoblasts produce an increased amount of osteocalcin, could also explain the abnormally low bone mineralization in these individuals, as this factor has been suggested to retard normal mineralization [33]. Thus, an imbalance in collagen and non-collagen

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Figure 3. Immunohistochemical detection of osteoclasts staining positive for cathepsin K (arrows) in subchondral bone in normal (n=6) and osteoarthritic (OA) (n=7) anterior cruciate ligament dog model. OA subchondral bone specimens were selected at the lesional (L) and non-lesional (NL) areas of the tibial plateaus. Right panel: Data are presented as box plot, where the boxes represent the first and third quartiles, i.e. line within the box represents the median and lines outside the box represent the spread of values. The statistical analysis data presented are the comparison made between the normal and the OA groups using the MannWhitney U test. Left panel: Representative sections of the immunohistological detection. From reference [23] with modification.

protein production, such as osteocalcin, could contribute to an increase in bone volume without a concomitant increase in the bone mineralization pattern. Urokinase, through its wide proteolytic activity, seems to be a particularly important factor in OA subchondral bone remodeling, since this protease has been shown to be capable of activating factors involved in bone resorption and formation, such as TGF-ß and IGF-1, and can also directly degrade the bone matrix [34–37]. The higher levels of PGE2, IL-6 and LTB4, which occur in OA subchondral bone osteoblasts are capable of promoting bone formation and the deposition of a new matrix, yet this matrix may be undermineralized. Indeed, PGE2, at low concentrations, stimulates bone formation while it may have inhibitory activity at high concentrations [38–40]. In addition, PGE2 stimulates collagen synthesis and can promote the proliferation of osteoblasts. Conversely, IL-6 and PGE2 may also be responsible for the increased number of active osteoclasts found in OA subchondral bone. Lastly, LTB4 stimulates osteoclast differentiation and bone resorption [41]. Two other proteases, cathepsin K and MMP-13, known to be potent bone resorptive factors, are also present in increased amounts in OA subchondral bone and calcified cartilage [23,42]. Cathepsin K has been found to be present quite selectively in the subchondral bone osteoclasts, and preferentially located in the zone where there is a very active bone resorption (Fig. 3). MMP-13 has been found in chondrocytes from calcified cartilage, in subchondral bone osteoblasts and also in osteoclasts. As this enzyme is known to work in conjunction with cathepsin K in the induction of bone resorption, their combined effect is likely to be very potent in inducing the resorption of subchondral bone. The activity of two other MMPs, MMP-2 and MMP-9, is elevated in


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OA proximal cancellous bone tissue, a situation possibly linked to abnormal collagen matrix deposition [16]. Some of the factors belonging to the TNF family, namely OPG and RANKL, are of key importance in regulating bone metabolism [43]. RANKL is a factor synthesized by osteoblasts, and is essential for osteoclast differentiation and bone resorption [44–46]. It exists as a cell membrane or extracellularly in soluble form, both of which stimulate osteoclastogenesis and osteoclast action after binding to and activating the cell surface RANK, located on osteoclast precursors and osteoclasts. In addition, a soluble decoy receptor for RANKL, OPG, has been identified [47]. This decoy receptor blocks the binding of RANKL to RANK, thus blocking RANK activation and subsequent osteoclastogenesis and, as a result, inhibiting bone resorption. An abnormal production of these factors by human OA osteoblasts has recently been identified [48]. Interestingly, a recent study reported a differential level of these factors according to OA osteoblast metabolic state [49], suggesting that, in some cases, OA subchondral bone tissues are enriched in factors promoting bone resorption, while others, conversely, have reached a different metabolic state which favors a reduction in resorptive properties. Some factors produced in the subchondral bone by cells derived from the bone marrow were also suggested as being involved in the pathological process. Aspden et al. [50] put forth the concept that OA is a metabolic disease in which systemic and/or local factors induce changes in skeletal tissues by modifying the formation and activity of mesenchymal precursor cells, suggesting a possible link between abnormal lipid metabolism and OA subchondral bone structural changes. This hypothesis is supported by the observation that osteoblast maturation from bone marrow stromal cells in OA patients is enhanced while that of adipocytes and chondrocytes is blunted [51]. Moreover, OA patients have, in general, higher than average body weight, and obesity is a major risk factor for OA [52–54]. In this context, leptin, a protein involved in lipid metabolism, has been suggested as an important pathological factor in OA [55]. Leptin is the product of the obese gene. Its protein level is increased in human OA cartilage [56], however, its expression is greatest in OA subchondral osteoblasts [57]. A point of interest is the fact that within the bone marrow, leptin favors the differentiation of mesenchymal stromal cells into osteoblasts while impeding the maturation of adipocytes, a situation that is also observed in OA [51]. It is noteworthy that leptin injections in rats stimulate the expression of IGF-1 and TGF-ß, increase alkaline phosphatase activity, and the level of synthesis of osteocalcin and collagen type 1 α1 chain [58], factors found at higher levels in OA subchondral bone tissue. In addition, leptin was also found to be associated with inflammatory states and to stimulate PGE2 and leukotriene production [59–63]. Other studies have shown that leptin can enhance the synthesis of endothelin-1 (a factor also found to be involved in human OA cartilage), trigger nitric oxide production and activate the p38 MAP kinase, changes which are commonly found in OA tissues. A clear link between circulating leptin levels and OA remains to be established. To that effect, the role of serum leptin and obesity in the progression of knee OA has recently been investigated. Data showed [64] that weight loss in the obese OA patients resulted in a significant decrease in the level of serum leptin. Although speculative, these findings could imply that a decrease in serum leptin may be one of the mechanisms by which weight loss could possibly slow progression of the disease in OA patients. However, it would not preclude the involvement of other local factors, as leptin might be a contributing factor that alone may not be sufficient, although necessary, to

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promote joint damage in OA. On the other hand, and logically, the local levels of leptin in the joint may be more relevant than circulating leptin toward OA progression. On the subject of lipoprotein, recent genome-wide scans have revealed an OA susceptibility locus on chromosome 11q, in close proximity to the low-density lipoprotein receptor-related protein 5 (LRP5). An altered haplotype of LRP5 was observed in individuals conferring a 1.6-fold increased risk of OA [65]. This gene product controls bone mass, and therefore could explain the abnormal bone tissue mineralization and remodeling observed in OA patients.

Subchondral Bone and Cartilage Cross-Talk Although the initiating event responsible for the degradation of cartilage in OA patients remains unclear, the concept whereby subchondral bone and cartilage should be considered as an interdependent functional unit is gaining strong support. The idea of a biological link between bone and cartilage that was originally hypothesized a few decades ago, was halted for a while by the concept that the calcified cartilage was an impenetrable structure. This was challenged by independent groups that demonstrated the presence of channels between the subchondral region and the uncalcified cartilage, and the presence of microcracks in the articular cartilage [66–69]. The presence of both microcracks and vascularization in the subchondral bone plate could then facilitate the transfer of factors from the subchondral bone region to the cartilage by diffusion through the basal layer of OA cartilage. These findings could explain that cytokines, proteases, growth factors, and eicosanoids produced locally by subchondral bone tissue seep through the bone-cartilage interface and stimulate cartilage breakdown. In that respect, if the abnormal production of proinflammatory cytokines, PGE2 and LTB4 by OA osteoblasts under in vitro conditions reflects in situ conditions, it could very well be that these cells have a significant influence on the metabolism of the contiguous cartilage. Recent data have also shown that bone resorption pits in subchondral bone may be an important factor in cartilage destruction via the release of proteases, thereby establishing a clear link between subchondral bone activity and altered metabolism, and cartilage loss. Two of the enzymes that are produced in the resorption areas of the subchondral bone, cathepsin K and MMP-13, may also contribute to the degradation of the articular cartilage matrix macromolecules. The findings that, in situ in OA cartilage, MMP-13 synthesis is located preferentially at the lower intermediate and deep layers [70,71], support this hypothesis. Hepatocyte growth factor (HGF), has recently been proposed as another possible candidate for this cross-talk between these two tissues. First identified in the liver, this multifunctional factor can induce proliferation, motility, morphogenesis, and it has chemotactic properties. HGF was found to be preferentially located in the deep layers of OA cartilage [72]. However, data have shown that chondrocytes do not express HGF, but OA osteoblasts do and they produce higher levels when compared to normal [73]. From these findings, it would seem most likely that this factor originates from the subchondral bone. The report that HGF induces MMP-13 production by chondrocytes also adds credibility to the hypothesis [74]. In OA subchondral bone, HGF could have a dual role, sclerosis and resorption. Indeed, as a growth factor HGF was shown to increase bone mass, conversely in the presence of osteoblasts it stimulates osteoclast resorption [75] and elevated serum concentration of HGF correlates


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with markers of disease activity in multiple myeloma associated with bone destruction [76] while it directly increases osteoblastic function [77–79]. In addition, HGF could act on the neovascularization of the subchondral bone, as it is known to induce it in other tissues [77,80,81]. Another factor which is found in both cartilage and in subchondral bone, TGF-ß, could very well be an important factor contributing to OA cartilage degradation. The stimulation of normal cartilage explants with TGF-ß mimics the increased production of MMP-13 found in situ in the lower zones of OA cartilage [71]. The level of TGF-ß as well as its receptor level are increased in the low intermediate and deep zones of OA cartilage [82], and in OA subchondral bone osteoblasts [28]. This particular distribution of TGF-ß and its receptors in cartilage therefore suggests that bone-derived TGF-ß could be responsible, to a certain extent, for the upregulation of MMP-13 in OA cartilage. Vitamin A derivatives can promote subchondral bone proliferation whereas they lead to progressive atrophy of articular cartilage at the same time that they initiate ectopic collagen type I production in cartilage, a feature reminiscent of OA [83]. It was also shown that subchondral bone proliferates into the cartilage, and the affected cartilage produces a developmentally regulated matrix molecule, osteoblast-stimulating factor-1, normally expressed by fetal and epiphyseal growth plate cartilage but not by articular cartilage, indicating that vitamin A derivatives may promote de-differentiation of chondrocytes as observed in OA. The intimate link between the articular cartilage and subchondral bone is also reflected in the observation that autologous chondrocyte implantation for cartilage repair is less effective than osteochondral cylinder transplantation [84].

Therapy Directed Against Subchondral Bone Remodeling Joints affected by OA have an increased bone turnover, which consequently increases the possibility of benefiting from drugs that alter bone metabolism, particularly the antiresorptive agents such as bisphosphonates. There is a good rationale to use such agents since they are safe for long-term administration (as they are used for osteoporosis) and easy to administer. Carbone et al. [85] conducted a study in which they examined the association between use of medications that have a bone antiresorptive effect, including estrogen, raloxifene, and alendronate, and the structural features of knee OA. Although the women treated with both alendronate and estrogen showed significantly less subchondral bone attrition and bone marrow oedema-like abnormalities than those who had not received these medications, no significant effect was found on the progression of cartilage damage. Others [86] looked at the effect of risedronate, another bisphosphonate, on joint structure and symptoms of OA patients. A definite trend toward improvement was observed in a Phase II study in both joint structure and symptoms in patients treated with risedronate [85]. The results of this study, however, could not be confirmed in a Phase III study [87]. Explanations for these disappointing data are that patients selected for these studies had long disease duration upon study entry and that the technologies used (X-rays) were not sensitive enough for the cartilage structure. Before definitely eliminating this line of drugs from OA treatment, patients with less advanced disease should be tested and a more reliable and sensitive imaging technology, such as magnetic resonance imaging, should be used.

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The most recent knowledge of the underlying molecular pathological mechanisms leading to bone remodeling/resorption in OA such as the RANK, RANKL and OPG will help to bring new therapeutic strategies to clinical practice. Clinical trials are already underway using OPG and RANKL antibodies to test their efficacy in the treatment of osteoporosis and of bone erosions in rheumatoid arthritis patients [88]. The potential of these agents in OA is also very appealing.

Conclusion Several processes are altered in cartilage during the OA process. Recent data, however, indicate that the subchondral bone plays a major role in this process, and is not merely a secondary manifestation of the disease. This tissue is now considered part of a more active component of the initiation and/or progression of OA. Some bone parameters, including abnormal bone mineral density, osteoid volume, bone mechanical parameters or indicators of bone turnover, are altered in OA patients. Similarly, several biochemical factors are also found elevated in the subchondral bone tissue in OA, and these may seep through clefts or channels in the tidemark to invade the overlying cartilage and promote its degradation. Therefore, even though the initiating event or effector in OA has yet to be identified, subchondral bone can now be focused upon as a potential candidate.

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XIV Osteoarthritis and Inflammation – Inflammatory Changes in Osteoarthritic Synoviopathy Thomas AIGNER a,1, Peter VAN DER KRAAN b and Wim VAN DEN BERG b a Osteoarticular and Molecular Pathology, Institute of Pathology, University of Leipzig, Germany b Experimental Rheumatology & Advanced Therapeutics, Nijmegen Centre For Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands

Abstract. OA research traditionally focuses on understanding events that occur within the degenerated articular cartilage whereas changes in the synovial membrane are largely neglected. However, inflammatory changes do occur in the synovium that may contribute to the overall effects observed in at least a subsets of OA patients. This implicates that the inflammatory and degradative activities of synoviocytes represents an interesting (therapeutic) target in OA research. Keywords. Osteoarthritis, Cartilage, Synovial membrane, Synovitis, Inflammation, Interleukin

1. Osteoarthritis and Inflammation Osteoarthritis (OA) is a multifactorial disease, which results primarily in degeneration of articular cartilage tissue. Disruption of the homeostatic anabolic and catabolic events in articular cartilage is thought be the initiation point that causes this condition [1]. However, other joint structures such as the subchondral bone plate and the synovium play their own roles within the OA disease process [2]. Thus, the importance of the synovial membrane and joint capsule in terms of causing disease symptoms is apparent and worthy of further investigation. The two main clinical symptoms of OA, pain and joint stiffness, are both significantly related to synovial inflammation and capsular fibrosis. However, the role of synovial inflammation in the pathogenetic process of cartilage destruction is largely unknown. The synovial (inflammatory) reaction observed in OA joint disease is primarily considered to be a secondary effect resulting from the release of cartilage debris from the damaged articular cartilage [3–7]. This is in contrast to the situation found in rheu1 Osteoarticular and Molecular Pathology, Institute of Pathology, University of Leipzig, Liebigstr. 26, D-04103 Leipzig, Germany, E-mail: [email protected].

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matoid arthritis, which is considered to originate from a synovial inflammatory autoimmune reaction with secondary cartilage destruction. However, inflammatory reactions in the synovial membrane do occur to some degree in all OA joints as discussed below. Also, the fact that most OA patients display a minor elevation of C-reactive protein within the serum [8,9] suggests that the inflammatory component plays some role within the disease process. In addition to local and/or general inflammatory responses within the synovial membrane, the activation of inflammatory pathways within the chondrocytes themselves may also play a crucial role in disease progression. Activation of such processes would be independent of direct inflammatory cell infiltrates, which are not present in OA articular cartilage. In fact, inflammatory signaling pathways have been shown to induce catabolic responses in chondrocytes, namely matrix degrading proteases such as MMP-13, MMP-1 and others. One of the most prominent catabolic cytokines in OA is the pro-inflammatory cytokine interleukin 1 (IL-1) [10,11]. Elevated levels of IL-1 are found in synovial fluids of patients suffering from rheumatoid arthritis and, to a lesser extent, in synovial fluid from OA patients [12]. An increase in IL-1ß levels in OA cartilage has been reported using immunolocalization technology [13]. Although own studies could not confirm an increased expression of IL-1 mRNA in OA chondrocytes by sensitive PCR technology [14], this may still reflect increased levels of IL-1 protein diffused into cartilage from the synovial space. IL-1 significantly affects gene expression patterns within articular chondrocytes [15] via multiple intracellular pathways, particularly the MAPkinases and NFkB-pathways (Fig. 1) (for review see Saklatvala 2007 [16]). IL-1 down-regulates the expression of the major cartilage matrix components, aggrecan and collagen type II [17–19] and, thus, counteracts the effects of anabolic factors on matrix synthesis. Additionally, IL-1 induces the expression of matrix degrading enzymes such as MMP-1, MMP-3, MMP-13 or ADAMTS-4, which are all potential major players in the destruction of cartilage matrix components [18–21]. Besides these direct effects, IL-1 also induces other cytokines with synergistic (catabolic) effects such as IL-6 and LIF (leukaemia inducing factor) [14,22,23].

2. Physiology of the Synovio-Cartilage-Interaction Joints are highly specialized organs that allow repetitive pain-free, frictionless movements. These properties are provided by the articular cartilage and its extracellular matrix which, under physiological conditions, is capable of sustaining high cyclic loading. Joints are, however, complex composites of different types of connective tissue including subchondral bone, cartilage surfaces, ligaments and the joint capsule. All the different joint tissues together provide their own specific roles to permit correct functioning of the joint. The synovial capsule and, in particular, the synovial membrane (i.e. the synovial lining cell layer (Fig. 2 a,b)) vastly contributes to the physiological functioning of the articulating joints. It is the synovial capsule together with the ligaments that provide the mechanical stability of the joints and ultimately determines the flexibility or range of motion of the joint. The synovial membrane, containing high metabolically active surface cells (synoviocytes) plays a crucial role in nourishing the chondrocytes as well as removing metabolites and matrix degradation products from the synovial space. Therefore, the synoviocytes maintain the normal metabolic milieu within the joints. Furthermore, the synoviocytes produce large amounts of hyaluronic acid and other factors


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IL1

IL1-R MAPKKKK NIK

MAPKKK / TAK1 / TAB1

MAPKK NFΚB

JNK

p38

ERK

c-jun, ATF, SAP-1, ELK,…

catabolic genes

catabolism Figure 1. Schematic representation of the IL-1 signaling pathway.

such as lubricin/superficial zone protein [24], which provide the joint surfaces with its lubrication capacity. In addition, the synovial fluid is composed of other substances that diffuse between the articular cartilage and the synoviocytes including chondrocytederived nutrients and metabolites as well as oxygen molecules.

3. Synovial Changes in OA – Histologic Reaction Pattern We and others have shown that all cases of clinically relevant OA joint disease are associated with some sort of synovial pathology [25,26]. This reflects the notion that there is a direct relation between clinical symptoms and the synovial reaction in OA. This suggests also that changes in the synovial membrane are partly involved in the progression of the disease [27]. In OA synovial specimens, in principle, four different types of OA synoviopathies are found: hyperplastic, inflammatory, fibrotic, and detritus-rich synoviopathy (Table 1) [26].

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Figure 2. a,b: Histological appearance of normal synovial membrane with flat, non-activated synovial lining cells at the surface (b: detail). c: Typical picture of hyperplastic synoviopathy with numerous synovial villi. d,e: Typical picture of fibrotic synoviopathy with a very much thickened and fibrotic capsule (note at the surface also some hyperplastic synovial villi; e: collagen stain (van Gieson´s stain)) f,g: Inflammatory synovitis in osteoarthritic patients with a minor to moderate lymphocytic infiltrate, partly organized in lymph follicles (g). h,i: detritus-rich synovitis, which is a typical feature of rapid-progressive end-stage disease with numerous bone and cartilage particles intermixed with fibrin and partly incorporated into the synovial stroma. Large fragments get successively degraded by osteoclast-type multinuclear giant cells (i: here labeled with CD68, a marker of phagocyting cells).

Detritus-rich synovitis, which is found in end-stage OA disease [28,29], is due to abundant macromolecular cartilage and bone detritus (i.e. bone and cartilage fragments attached to or incorporated into the synovial membrane; Fig. 2h) in addition to abundant molecular debris, not visible microscopically. Besides the debris, a significant

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Table 1. Table listing the major histopathological features of the four pattern of osteoarthritis associated synoviopathy in comparison to each other and to normal synovial membrane. Bold letters indicate key diagnostic criteria normal hyperplastic inflammatory fibrotic detritus-rich villous hyperplasia

–

++(+)

++(+)

++(+)

++(+)

synovial lining – proliferation

–

+

++

++

++(+)

synovial lining – activation

–

+

++

+

+

fibrinous exsudate

–

–

(+)

+

++(+)

capsular fibrosis

–

–

(+)

+++

+++

(macromolecular) cartilage and bone debris

–

–

(+)

–

+++

granulocytic infiltrate

–

–

–

–

+

lymphoplasmacellular infiltrate – diffuse

–

–

++

(+)

+(+)

lymphoplasmacellular infiltrate – aggregates/follicles

–

–

++

(+)

(+)

amount of fibrinous exudate is found either at the surface of the synovial membrane. This exudate may be combined with incorporated fibrin reflecting longer ongoing fibrinous exudation already being organized (i.e. resorbed). Detritus-rich synoviopathy usually contains a minor inflammatory cell infiltrate consisting of lymphocytes and granulocytes as well as some foreign body giant cells (Fig. 2i). Another form of OA synoviopathy found in late stage disease, fibrotic OA synoviopathy (capsular fibrosis) [26,30] (Fig. 2d,e), is mainly characterized by the shortening and thickening of the joint capsule, which is partly responsible for some symptoms, in particular joint stiffness, seen in OA patients. The most interesting of the OA synoviopathies in terms of pathogenesis is the inflammatory OA synoviopathy, which displays moderately extensive lymphocytic infiltrates [26,31,32] (Fig. 2f,g). Histologically, this condition resembles a less severe case of rheumatoid synovitis where dense infiltrates of B-lymphocytes, plasma cells and T-lymphocytes (CD4- and/or CD8-positive) are detected. It is intriguing to speculate whether this condition reflects some kind of autoimmune aspect that may be occurring, at least in this subset of OA patients. In fact, this opens up the possibility that “overlapping forms” of rheumatoid arthritis and OA might exist, but this has not been investigated in any detail yet. Interestingly, the lymphocytic infiltrate in the subsynovial stroma appears to correlate directly with Il-1ß in the synovial fluid as well as MMP-1 expression by synoviocytes [33,34] suggesting a direct stimulatory role of the inflammatory cells on the activity of the synovial lining cells. In any case, the presence of inflammation in a significant portion of OA patients clearly points to the option of antiinflammatory therapy at least for some subsets of OA patients. In early OA, mostly hyperplastic OA synoviopathy is found (Fig. 2c,d). This pattern shows only moderate synovial hyperplasia with or without cellular activation, but without significant capsular fibrosis and thickening and without significant inflammatory infiltrates or macromolecular detritus [26,35]. Overall, three forms of alterations of the synovial surface can be observed: 1) increased cytoplasmic volume of the usually flat synovial lining cells. These cells may even become cuboidal or even cylindrical in

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shape suggesting that they have been activated in some way; 2) the in normal conditions single cell layer of synovial lining cells can proliferate to form as many as five cell layers; 3) the whole synovial surface, including the underlying stroma, can become hyperplastic and form the classical synovial villi. Synovial hyperplasia per se can be found in all forms of OA synoviopathy and in chronic synovitis. Thus, villous hyperplasia is largely a non-specific feature of chronic synovial alteration and activation. Synoviocyte activation and proliferation as well as synovial hyperplasia presumably all represent reactive changes responding to increased demands for clearance of molecular debris in the synovial fluid of the joint [3,7,36]. This also explains the increase in the amount of CD68-positive type A synoviocytes, which have phagocytic capacity, in the synovial lining layer [5,7,26,37–40]. Of note, the highest percentage (up to 60%) of type A synoviocytes is found in the inflammatory OA synoviopathy suggesting that this subform is associated with a very significant matrix catabolic activity. Although a cellular inflammatory component is missing in synovial hyperplasia, the proliferation and activation of the synovial lining cells might generate significant problems for the articular cartilage as these cells are able to secrete matrix-degrading proteases (MMPs) and catabolic cytokines (IL-1, TNF-alpha) [34,41]. It is therefore intriguing to speculate that the cartilage matrix catabolism may be partly induced by catabolic mediators (e.g. Il-1ß and TNF-a) secreted by the activated synoviocytes. This leads back to the notion discussed above where, in addition to the direct involvement of inflammatory cell infiltrates, which do not occur in OA cartilage, inflammatory pathways induced in the articular chondrocytes themselves may have the potential to play a critical role within OA cartilage destruction. Altogether, it appears that the production of inflammatory mediators by the synoviocytes, in particular if they are activated, can play a very important role in the OA disease process (Fig. 3).

4. Synoviopathy and Cartilage Matrix Degradation The common perception of OA is a disease in which deranged processes in cartilage and possibly bone are responsible for the destruction of the articular cartilage. Synovium has been considered of minor importance and to contribute little to OA pathogenesis. However, in addition to OA patients without obvious synovitis there has been a subgroup of cases identified in which joint inflammation and synovial activation is a major hallmark. It is apparent that OA synovial tissue shows typical activation markers such as expression of transcription factor nuclear factor kappaB (NF-kappaB) [42]. Adenoviral gene transfer into osteoarthritis synovial cells of the endogenous inhibitor IkappaBalpha showed that the synthesis of inflammatory and destructive mediators from OA synovial tissue was NF-kappaB dependent [43]. The activated synovial tissue is proposed to contribute to degradation of the articular cartilage matrix. Synovitis has been demonstrated to be correlated with greater severity and accelerated progression of structural damage in OA. Ayral et al found in patients with knee osteoarthritis that activation of the medial perimeniscal synovium was associated with more severe medial chondropathy [27]. Inflammation of the medial perimeniscal synovium could be regarded as a predictive factor of succeeding increased degradation of medial cartilage damage. In a study of Loeuille in patients with knee OA, histologically scored synovitis was associated with chondral lesions in the medial femorotibial compartment but not at other locations [44]. Synovitis scored with


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Figure 3. Interaction action between synovium and cartilage in osteoarthritis. Molecular detritus from the cartilage activates the synovial lining cells. The synovial lining cells produce cytokines, growth factors and (latent) enzymes. Synoviocyte-derived cytokines and growth factors further activate the chondrocytes. Enzymes produced by the synovial lining cells can directly degrade matrix molecules if not inactivated by inhibitors in the synovial fluid. Latent enzymes can be activated in the milieu of the osteoarthritic cartilage.

MRI could not discriminate patients with moderate cartilage damage from patients with severe cartilage lesions demonstrating the lower sensitivity of MRI compared to histology of tissue biopsies to detect inflammation [44]. Synovitis appears to play a role in the destruction of cartilage in part of the OA patients. This can be either a direct effect of factors produced by the synovial tissue or an effect of activation of chondrocytes by mediators released by the activated synovium. The cells in the synovium can function as a source of cytokines, growth factors, metalloproteinases (MMPs), reactive oxygen species and various other mediators. The role of cytokines and growth factors in synoviopathy in OA will be discussed in the section below. Many studies have evaluated MMP levels in synovial fluid of OA patients but a smaller number of studies have analyzed MMP expression in OA synovium. However, already in the early 1980’s elevated synthesis of MMPs in OA synovium has been reported [45,46]. Increased levels of collagenase and stromelysin were detected in synovial tissue of OA patients [34,47,48]. Davidson et al. compared expression of MMP and ADAMTS genes in synovium from patients with either hip OA or femoral neck fracture [49]. Genes upregulated in OA synovium compared to synovium from the femoral neck fracture patients were MMP-9, -11, -13, -16 and -28 and ADAMTS-2, -10, and –16. For MMP-9, -10, -12, -17, -23, -28, ADAMTS-4, and -9, there was a significant correlation between expression levels in the synovium and cartilage, suggesting similar mechanisms of regulation [49]. In a zymography study of MMPs in knee OA, latent and activated forms of MMP-2 and MMP-9 were found to be produced by

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cultures of synovial tissue. Moreover, it was found that both protein and mRNA levels in lesional cultures were significantly higher than those in paralesional ones [50]. Kanbe et al. performed a very interesting study on the effect of synovectomy on serum MMP levels in OA patients. Levels of MMP-9 and MMP-13 decreased 7 to 9 fold in serum after arthroscopic knee synovectomy [51]. This study indicates that inflamed synovial tissue contributes significantly to systemic MMP levels and it can be expected that it will have similar or even greater effects on local MMP levels in the knee joint. In experimental OA we have detected elevated expression of MMP-13 by quantitative RT-PCR in synovial tissue of OA prone STR/ort mice and C57Bl/6 mice with collagenase-induced OA. These results indicate that in OA synovium the synthesis of MMPs is turned on. Before becoming biologically active MMPs have to be activated. Plasminogen activator and plasmin have been found elevated in OA synovium and are thought to play a significant role in the activation of MMPs [52]. Proteases (MMP and ADAMTS) produced in OA synovial tissue might contribute to the progression of OA by damaging joint structures such as cartilage and ligaments. However, MMPs produced by the activated synovium have to travel through the synovial fluid to reach articular cartilage unless synovium and cartilage are in close contact. In the synovial fluid MMP inhibitors such as Tissue Inhibitors of Metalloproteinases (TIMPs) and alpha1-antitrypsine and alpha2-macroglobuline are present [53–55]. These factors limit the action of MMPs on articular cartilage. On the other hand, in general only active forms of MMPs are inhibited by protease inhibitors and one can envision that inactive MMP reach the articular cartilage and these are locally activated leading to degradation of cartilage matrix components. Not only proteases but also other factors are expressed by inflamed OA synovium. OA synovium produces increased amounts of reactive oxygen species (ROS), such as nitric oxide, peroxynitrite and superoxide anion [56–58]. Immunohistochemistry showed that not only iNOS but also cNOS was expressed by cells in the synovial lining and subsynovium of patients with OA [59]. These data indicate that NO is produced by the activated synovial lining not only by iNOS but also by cNOS. The presence of functional NO synthetases is confirmed by studies that demonstrate that cultured synovial tissue of OA patients spontaneously produce nitric oxide [60]. Both fibroblast and macrophages contributed to this production [60]. The presence of enzymes involved in the synthesis of lipoxygenase products, prostaglandins and leukotrienes, has also been demonstrated in OA synovium [61–63]. Reactive oxygen species can either directly damage matrix components or indirectly by inhibiting matrix synthesis, inducing apoptosis or by activating latent metalloproteinases. Lipoxygenase products are known to activate chondrocytes and to stimulate the synthesis of catabolic factors by chondrocytes [64,65]. Inhibition of cyclooxygenases and lipoxygenase pathways in chondrocytes inhibit MMP-13 production in human osteoarthritic chondrocytes [64,65]. These data show that ROS and lipoxygenase products synthesized in inflamed OA synovium could be harmful in OA cartilage and contribute to the process of cartilage destruction in this disease. Synoviopathy and Cytokine/Growth Factor Balance In general, cytokines are defined as peptide factors that are produced by, and act on cells habitually in close vicinity. In that respect, the definition includes the various growth factors. To call a factor a cytokine or a growth factors is in most cases based on historical grounds and not directly related the mode of action and activity of a particu-


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lar factor. Various cytokines and growth factors are found in augmented quantities in activated OA synovium and can have a direct impact on chondrocyte function. Even though absolute levels of specific mediators may be indicative of their importance, the net effect is for the most part determined by the balance of synergizing, counteracting, and regulating mediators. Increased production of various mediators is found in synovial tissues of OA and RA patients and differences are mostly quantitative and not qualitative. In terms of their most distinctive effect on chondrocytes, cytokines and growth factors can be broadly categorized in three classes: catabolic, anabolic and regulatory. However, a strict distinction is not always possible and several growth factors and cytokines show so-called contextual actions on cellular behaviour [66]. Several studies have identified cytokines and growth factors presence in both OA and RA synovial tissue using immunohistochemistry. Most studies were focused on RA synovial tissue, and OA synovium was in general used as a control. Most cytokines and growth factors were found in both OA and RA synovium. The differences found were mainly quantitative and not qualitative. A study in patients with a range of disease stages of osteoarthritis demonstrated that highest IL-1 and TNF-α expression was related with the most severe cases of inflammation, the latter resembling RA synovial tissue [35]. It is generally accepted that both TNFα and IL-1 are the prevailing cytokines in RA synovium. IL-1 is also synthesized in substantial quantities in OA synovial tissues and this may be a major source of the increased IL-1 levels in OA synovial fluid [67–69]. TNFα is less abundant and this is in line with the observation that TNFα can only be found in a limited number of OA cases [67–69]. Of importance, IL-1 is regarded to be the driving force for the production of MMPs and ADAMTS in OA synovial tissue. This is demonstrated by the striking inhibition of enzyme production when culturing OA synovium in the presence of IL-1 receptor antagonist (IL-lra) [70]. A cytokine resembling IL-1, IL-18, is found in low levels in OA synovial tissue and the enzyme responsible for the activation of IL-1β and IL-18, ICE (Interleukin1beta-converting enzyme), has been identified in OA synovial tissue [71–75]. The expression of the proinflammatory cytokine IL-12 was found to be similar, both on the mRNA and protein level, in RA and OA patients [76]. Remarkably, expression of both Th1 and Th2 cytokines have been detected in OA synovium [77]. The number of interferon-gamma positive cells was higher than the number of IL-4 positive cells, indicating that Th1 cells dominate in the synovium of OA patients. Members of the TGF beta superfamily are found in OA synovium. Synovial tissues from patients with osteoarthritis express and secret TGF beta, mainly TGF beta1 [78]. Expression of BMP-2 and –4 was reduced in OA synovial tissue compared to controls [79]. The latter suggests that diminished BMP levels in OA synovium can contribute to a loss of joint homeostasis in this disease. The BMP antagonists follistatin, gremlin, chordin are expressed by synovial fibroblast from OA patients at similar levels as in fibroblast from controls while expression was reduced in OA cartilage [80]. In experimental (collagenase-induced) OA in mice we have found an increased expression of BMP-2 and –4 in synovium using Immunohistochemistry [81,82]. Depletion of macrophages from the synovial lining using chlodronate-loaden liposomes resulted in diminished expression of BMP-2 and –4 indicating that these cells are responsible for a major part of BMP-2 and -4 production in activated synovial lining cells. Vascular Endothelial Growth Factor has been detected in OA synovium and immunoreactivity increased with increasing histological inflammation grade. In the synovial lining, VEGF immunoreactivity was localized to macrophages [83]. Synovial tissue from patients with OA expressed basic Fibroblast Growth Factor (bFGF) mainly in

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hyperplastic lining synoviocytes [84]. Arthroscopic synovectomy resulted in a five-fold reduction in the serum levels of the chemokine stromal cell derived factor 1 (CXCL12) in OA patients, indicating that synovium contributes significantly to the production of this factor in these patients [43,85]. The parathyroid hormone-related peptide (PTHrP) is more strongly expressed by RA synovium than by OA synovial cells [86,87]. However, synovial fibroblasts from OA patients have been shown to produce PTHrP after incubation with inflammatory cytokines such as IL-1 [88]. The adipokines, such as leptin, adiponectin and resistin, could give a clue for the relationship between obesity and OA development [89,90]. Leptin levels correlate with body mass index [89,90]. Synovia from OA patients have been shown to be a major source of leptin and adiponectin [91]. The cytokines and growth factors described in the previous section are produced in the synovial tissue, the articular cartilage, or in both tissues. The factors produced in the synovium are thought to be able to reach the articular cartilage and modulate in this tissue the cytokine/growth factor balance. Changes in the availability of cytokines and growth factors will alter the behaviour of the chondrocytes and his can lead to loss of cartilage homeostasis and OA. Catabolic Factors A key example of a destructive cytokine is IL-1. A critical role of IL-1 in early stages of OA seems unlikely. Inflammatory models demonstrate that chondrocyte proteoglycan synthesis is strongly reduced shortly after induction of inflammation and that synthesis remains being suppressed during ongoing inflammation [92,93]. Blocking IL1 with neutralising antibodies and IL-lra provided persuasive evidence that IL-1 is the key mediator of the inhibition of proteoglycan synthesis [94–96]. In marked contrast, chondrocyte proteoglycan synthesis, and collagen synthesis, has been reported to be enhanced in early stages of OA [97–99]. This appears to exclude an essential role for IL-1, or at least suggests an overkill by anabolic factors. An OA phenomenon that may be attributable to IL-1 is the alleged shift in chondrocyte phenotype during OA. It has been shown that after prolonged exposure to IL-1, synthesis of cartilage specific collagen types such as type II and type IX is reduced while synthesis of types I and III collagen is increased [100,101]. This shift could account for the unsuccessful matrix repair in OA. Moreover, IL-1 can play a major destructive role in later stages of OA, which is characterized by reduced matrix synthesis and overt cartilage destruction. The functioning of cytokines, such as IL-1, is regulated by cytokine-specific soluble receptors. Additionally, IL-1 is counteracted by the IL-1 receptor antagonist (IL-lra). Synovia from OA patients abundantly produce IL-lra but it must be kept in mind that a 1000-fold excess of antagonist over IL-1 is needed to entirely block the IL-1 activity. This makes it unlikely that the levels of IL-lra produced in the synovial lining are sufficient to fully counteract IL-1 effects on chondrocytes. Anabolic Factors In addition to overproduction of catabolic factors, OA pathology may be linked to lack of anabolic growth factors. IGF-I is one of the most potent anabolic factors for normal cartilage. There is no proof that IGF levels are limited in synovial fluid of OA patients. However, as a result of joint inflammation chondrocytes become non-responsive to IGF-I. Chondrocytes in OA cartilage are reported to be less stimulated by IGF-I due to enhanced


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levels of IGF binding proteins, limiting the anabolic action of IGF [102]. This lack of response may be overcome with high levels of IGF and could justify therapeutic approaches with high doses of IGF-I [103]. Other anabolic factors are FGF and PDGF, which demonstrate stimulation of proteoglycan synthesis above the IGF-I effect and may contribute to cartilage repair by stimulation of chondrocyte proliferation. However, it was demonstrated that previous incubation of chondrocytes to FGF stimulates the protease release after IL-1 exposure [104]. PDGF also stimulated IL-1 dependent protease release, but inhibited IL-1 mediated reduction of proteoglycan synthesis [105,106]. These data indicate that a number of factors not only stimulate repair but enhance breakdown as well. The members of the TGF beta growth factor superfamily are considered as anabolic factors due to their role in cartilage formation during embryogenesis. We have shown that repeated local injection of TGFβ markedly upregulated chondrocyte proteoglycan synthesis and induced osteophytes in murine knee joints [107,108]. Both features could be an indication of a role of TGFβ in early OA. Osteophytes are characteristic features in OA and it was observed that repeated local injection with another growth factor, IGF-I, does not induce these hallmarks. Moreover, our group recently showed that blocking of TGF beta activity in the synovial lining by adenoviral overexpression of the TGF beta inhibitor SMAD7 significantly inhibited osteophyte formation and synovial fibrosis [109]. Finally, prolonged exposure to TGF beta results in proteoglycans loss close to the tidemark in femoral cartilage of the murine knee joints. Both suboptimal and supraoptimal level of TGF β appear to result in cartilage pathology and ultimately osteoarthritis. We have found profound synovial expression of Connective Tissue Growth Factors (CTGF/CCN2) in murine experimental models of osteoarthritis. A role for CTGF in the repair of cartilage damage in a full thickness defect model and an osteoarthritis model have been reported, suggesting that CTGF functions in regeneration of articular cartilage [110]. In contrast, we have found that in normal murine knee joints overexpression of CTGF induced transient synovial fibrosis, as shown by extracellular matrix accumulation and an increase in the number of procollagen type I-expressing cells [111]. The fibrotic tissue showed elevated mRNA levels of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5 and TIMP-1. CTGF overexpression led to proteoglycan depletion in the articular cartilage. The observed damage is either a direct effect of CTGF on the articular chondrocytes or mediated by factors released by the CTGF-induced fibrotic tissue. The discrepancy between our study and the study of Nakao et al could be based on the recent observation that CTGF can modulate TGF beta action [90]. Wahab et al showed that CTGF enhances TGF beta action by suppression of SMAD7 transcription and induction of the transcription factor TIEG11. SMAD7 is a known specific inhibitor of TGF beta signaling while TIEG11 is a known repressor of SMAD7 transcription. It can be expected that in the defect model and the OA model used by Nakao et al TGF beta was present and that CTGF has amplified the anabolic effects of TGF beta on cartilage repair. Bone Morphogenetic proteins have been shown to stimulate matrix production in normal and OA chondrocytes and are considered to be suitable activators of chondrocytes anabolism [112–117]. It has been shown that BMP-2 expression in OA cartilage co-localizes with newly synthesized type-II procollagen [117]. Moreover, expression of BMP-2 favored the expression of the differentiated-chondrocyte specific type II collagen isoform IIB [113]. This indicates that BMPs derived from OA synovial tissue can

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contribute to stimulation of matrix synthesis by chondrocytes and this might contribute to delayed cartilage degradation.

5. Final Remarks OA research traditionally focuses on understanding the events within the degenerated articular cartilage, the major tissue where OA is presumed to commence. However, changes occurring in the synovial membrane are largely neglected. The synovial capsule and, in particular, the synovial lining cells represent an important portion of the joint as an organ and must also play an important role in its normal physiology. Of importance are the inflammatory changes observed in the synovial membrane, which occur to some extent in all OA joints. This supports a pathogenetic role of OA synoviopathy in OA cartilage degeneration and implies that targeting the inflammatory and degradative activities of synoviocytes may be an interesting target for research and therapy.

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Part IV Imaging and Clinical Applications



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XV Magnetic Resonance Imaging of Cartilage: New Imaging and Clinical Approaches Daniel R. THEDENS a,∗ , James A. MARTIN b and Douglas R. PEDERSEN b a Department of Radiology, The University of Iowa, Iowa City, IA b Department of Orthopaedics and Rehabilitation, The University of Iowa Abstract. Magnetic resonance imaging (MRI) remains the imaging method of choice for depicting the morphological changes associated with osteoarthritis (OA) and other diseases of cartilage, but the early stages of OA are characterized by tissue level changes which are not evident with standard MRI protocols. Several emerging MR-based techniques show promise for detecting changes in water, collagen, and proteoglycans that are the hallmarks of cartilage degeneration. In this review, the principles and application of several of these techniques, including T2 mapping, T1ρ imaging, delayed gadolinium MRI of cartilage (dGEMRIC), and sodium MRI are outlined and compared. Keywords. Cartilage, osteoarthritis, magnetic resonance imaging, T1ρ imaging, dGEMRIC, T2 mapping, sodium imaging

Introduction As one of the most widespread causes of disability among adults, degenerative diseases of articular cartilage such as osteoarthritis (OA) remain a significant public health concern. OA can arise as a result of traumatic joint injury or may result from a general degeneration of cartilage over time. The consequence of this cartilage breakdown is pain and loss of motion in the affected joints. OA is generally a progressive disease. By the time painful symptoms appear, the degenerative processes causing cartilage damage have been long underway. Currently available clinical imaging techniques such as magnetic resonance imaging (MRI) provide superb depiction of cartilage anatomy and morphology noninvasively and can readily show cartilage surface defects and thinning. But these imaging markers of OA are also not exhibited until long after the onset of the disease process. The initial steps in the disease process occur at the tissue level, and thus the lack of noninvasive ∗ Corresponding Author: Daniel R. Thedens, Department of Radiology, The University of Iowa, Iowa City, IA 52242, E-mail: [email protected].

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imaging methods that can detect such changes hampers effective diagnosis and treatment of these conditions. Among imaging modalities, magnetic resonance imaging (MRI) is one of the most versatile as it can generate images sensitive to a wide range of intrinsic tissue parameters. MRI is thus not limited to depictions of anatomical features, but can show changes in functional and chemical characteristics as well. In the field of cartilage imaging specifically, there are several emerging MRI-based methods that are showing promise for detecting the tissue changes associated with the early stages of OA. The successful development and clinical validation of such methods for assessing cartilage health would create a valuable clinical tool for more effective diagnosis and treatment of progressive cartilage diseases such as OA.

1. Cartilage Structure and Composition Articular cartilage is a complex connective tissue consisting of a relatively small number of cells (chondrocytes) that synthesize and maintain the extracellular matrix. The extracellular matrix is composed primarily of water and macromolecules, including collagen and proteoglycans. The collagen (type II being the most abundant component) acts as the “scaffold” of the tissue, providing its shape and mechanical stability. The highly-charged proteoglycans provide the ability of the tissue to take up water and swell, yielding its cushioning and shock-absorbing properties. Cartilage is an avascular tissue, relying on diffusion of required nutrients, which accounts for its poor ability to repair itself. The primary purposes of articular cartilage are to provide a low friction surface over which articulating bones can smoothly move and to act as a shock-absorbing cushion for the joints. These functions depend on the tensile restraint provided by the collagen fiber network and the osmotic pressure provided by proteoglycans. The precise composition of this extracellular matrix varies with depth [1,2] in a manner which affects the distribution of stresses and strains in the tissue during loading. In the body the superficial zone experiences hydrostatic pressure, fluid flow, and tensile stress along with high compressive strains. In response to this environment, chondrocytes appear to be flattened and the region is relatively richer in collagen (type II) and poorer in proteoglycans [3,4]. Cells in the middle and deep zones experience more hydrostatic pressure but very minimal strain and fluid flow, resulting in synthesis and maintenance of large amounts of glycosaminoglycan (GAG), uronic acid and type II collagen [5,6]. The superficial zone has higher tensile modulus by a factor of 2, but lower compressive modulus by a factor of 3 compared to the deep zone [2]. Healthy cartilage must be able to retain its mechanical stiffness and cushioning abilities, which are in turn preserved by the integrity of the collagen matrix and proteoglycan networks and maintained by functioning chondrocytes. A disruption in any of these components can begin a cascade of degenerative processes. Deterioration of the articular cartilage extracellular matrix results in progressive changes in cellular response and composition of the tissue. The onset of degeneration charts a path from microscopic biochemical events (proteoglycan loss, cell senescence, cell apoptosis and collagen degradation) to irreversible morphological changes (cartilage thinning, clefts, lesions and fibrillations) resulting in total disruption of the cartilage.


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OA is one of the most common degenerative diseases of cartilage. OA may manifest as a secondary effect of traumatic joint injury or other abnormal joint loading condition, or it may arise from normal “wear and tear” on joints with no other primary cause. In either case, the general progression of the disease is broadly similar as outlined above. The initial changes in OA occur at the microscopic level with changes in collagen and macromolecular components of the extracellular matrix. These changes compromise the cartilage mechanical properties, leading to further disruption until the characteristic symptoms of pain and stiffness are experienced, and radiologic evidence such as joint space narrowing and cartilage thinning appear. While these significant effects of cartilage degeneration are readily appreciated, they are experienced in the later disease stages after the responsible microscopic processes have long been underway. For effective diagnosis and treatment, it is imperative to intervene at the onset of the disease to restrict if not reverse its progression. Noninvasive diagnostic procedures sensitive to these early changes in tissue microstructure are needed if such early identification and treatment are to be made possible.

2. MRI of Cartilage: Morphology The exquisite soft-tissue contrast of MRI and the multiplicity of contrast mechanisms available in a single exam have established MRI as the method of choice for clinical imaging of cartilage and joint anatomy. A typical MRI protocol will acquire proton density, T1, T2, and fat-suppressed images with rapid spin-echo-based techniques such as fast spin-echo (FSE), which can give a comprehensive picture of the morphologic changes associated with injury and subsequent degenerative processes [7]. T1-weighted imaging provides visible distinction between cartilage and subchondral bone, although contrast between cartilage and fluid is poor. For cartilage, fat suppressed T2-weighted imaging is particularly valuable and routinely acquired as it yields good contrast with synovial fluid at the cartilage surface, permitting identification of cartilage surface lesions. FSE acquisitions primarily produce two-dimensional (2D) data sets, with relatively coarse resolution in the slice direction, limiting the possibilities for multiplanar reformatting of the curved surfaces. Three-dimensional (3D) imaging with gradient echo (GRE) acquisitions, particularly with fat suppression via water-selective excitation, can generate isotropic image volumes with high cartilage signal relative to surrounding tissues. The thinner slices and reformatting capabilities result in high sensitivity and specificity for identification of cartilage defects and the potential for accurate quantitative volume measurements [8]. The cost is a relatively lengthy scan time (10 or more minutes) and the possibility of motion artifacts during that time. At present, clinical practice for noninvasively assessing cartilage condition with MRI in OA is primarily based on morphological characteristics and parameters such as cartilage thickness and the appearance and identification of significant defects. Quantitative measures of cartilage volume are also possible with MRI. As noted previously, however, these gross changes in cartilage appearance are seen long after the degenerative processes have begun. Current standard imaging methods are thus not well suited to identification of the early markers of OA. The lack of quantitative measures of cartilage

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condition hampers treatment planning and meaningful and timely assessment of joint injuries.

3. Functional MRI of Cartilage Ultimately, it is biomechanical properties of cartilage that determine its functional state. Direct assessment of biomechanical parameters is not presently possible with a noninvasive exam, and so there is no effective way to objectively assess this element of cartilage health noninvasively. The next best alternative to determining biomechanical characteristics of cartilage is to base assessments on the related biochemical composition of the extracellular matrix, especially in terms of proteoglycan and collagen content, the primary components responsible for the mechanical strength of healthy cartilage. The early stages of OA are characterized by numerous changes in the extracellular matrix at the microscopic level, some of which may be detectable by imaging. As outlined previously, disruption of the organization of the collagen fiber network is initially seen along with an increase in water content, causing the cartilage to swell. Proteoglycans are subsequently lost from the matrix as the disease progresses, increasing susceptibility to mechanical stresses and continuing degradation. The magnetic resonance (MR) signal characteristics of cartilage are affected by all three of the main components of the extracellular matrix (water, proteoglycans, and collagen) that undergo changes in early OA. The water content of tissue has a direct effect on T2 relaxation parameters. The structure and orientation of collagen fibers further influences T2 relaxation in cartilage. In particular, the regular structure of collagen restricts the motion of water molecules and increases the interactions that are the basis of T2 relaxation. Molecular interactions between proteoglycans (being highly charged macromolecules) and water or other molecules also influence the MR behavior and thus the qualitative appearance and quantitative relaxation characteristics of the magnetic resonance signal. Given the ability to design MRI acquisition techniques that are sensitive to and quantitative in these tissue relaxation changes, MRI-based methods show considerable promise for early detection of these characteristic biochemical markers of OA. Numerous MR-based methods have been proposed to noninvasively display and quantitate changes in the composition and integrity of cartilage extracellular matrix in vivo, including T2 MRI, T1ρ MRI, delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC), and sodium MRI. In brief, quantitative mapping of T2 relaxation times is sensitive to several processes involved in cartilage degeneration, including water content and derangements in collagen fibril orientation. T1ρ MRI is most sensitive to interactions between tissue water and macromolecules and thus correlates strongly with proteoglycan content. dGEMRIC introduces an exogenous charged contrast agent that alters T1 relaxation properties and whose distribution depends on the local concentration of charged GAG molecules. Sodium MRI similarly probes the charge density (and thus concentration) of GAG by directly measuring the presence of the balancing concentration of sodium in the tissue. Each of these methods has significant advantages and disadvantages in terms of their sensitivity and specificity to relevant biochemical properties and their implementation and practicality for in vivo and clinical work. Broadly, the methods are ordered above by their specificity to relevant biochemical changes in cartilage (from least to most specific) and their ease of translation to a clinical setting (from easiest to most difficult).


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3.1. T2 Mapping Qualitative and quantitative T2-based MRI acquisitions are universally available and mature techniques in diverse areas of clinical MRI. The T2 relaxation times of many normal and diseased tissues are widely studied and T2-weighted images are routinely acquired as part of virtually all musculoskeletal MRI protocols. The quantification of T2 relaxation times is also a readily available technique on most clinical scanners, producing pixel-by-pixel maps of T2 relaxation times and color maps that can highlight areas of abnormality. Generation of this information requires only a standard spin-echo-based acquisition protocol and fundamental data analysis tools for nonlinear curve fitting of the variation of local signal intensity with the change in acquisition parameters (the echo time or TE). As noted previously, the T2 relaxation time can be altered by changes in water content and collagen fiber concentration, orientation, and condition. Specifically, degradation of the collagen matrix permits greater mobility of the water component of the extracellular matrix, which results in longer T2 relaxation times. Several in vitro studies have confirmed this effect in animal and cadaveric models, demonstrating a strong correlation between T2 changes and histologic changes [9]. In contrast, proteoglycan content does not appear to have a strong direct influence on T2 relaxation time [10,11]. Clinical studies have also begun to appear that show significant changes in T2 relaxation times in areas of cartilage degeneration. These changes may be seen in subjects with OA in the absence of volume and thickness changes, indicating sensitivity to the biochemical changes that are hallmarks of early OA [12]. The changes were significant between subjects with and without OA, but did not demonstrate graded differences with the severity of OA. Dependencies on the age of subject have also been observed [13] suggesting sensitivity to the general deterioration of the cartilage over time. The observed changes in T2 relaxation time in these studies were significant but relatively small, and may result from multiple mechanisms including water content changes and collagen concentration and orientation, with the latter having the greatest influence. In particular, the dependence on collagen fiber orientation gives rise to spatial variations in T2 relaxation within healthy cartilage which can confound the detection of abnormalities. Fiber orientation effects can be diminished in in vitro studies, but may be problematic when interpreting results from clinical exams. Thus, while T2 changes in cartilage are seen in degenerative processes and may often be diagnostically valuable prior to visible morphologic changes, it can be unclear which of these mechanisms is the predominant cause, potentially limiting its sensitivity and specificity. Nevertheless, T2 mapping may provide valuable information on the biochemical and biomechanical properties of cartilage and is deserving of continued study. Its primary (though complex) dependence on collagen condition and water content rather than proteoglycans means it may also serve as a useful adjunct to one or more of the imaging techniques described below, which are predominantly sensitive to proteoglycan content. 3.2. T1ρ Imaging T1ρ describes an alternative relaxation characteristic in MR experiments, “spin-lattice relaxation in the rotating frame.” As the name implies, this relaxation mechanism is related to T1 relaxation, but is measured in the presence of an externally applied radiofrequency

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Figure 1. T1ρ pulse sequence. The T1ρ magnetization preparation step consists of a 90◦ tip-down, spin-lock RF for variable T1ρ weighting, a 90◦ tip-up, and crusher gradient to eliminate any residual transverse magnetization. A standard fast spin echo pulse sequence follows for image acquisition.

(RF) magnetic field (the B1 field) applied in a direction perpendicular to the large main magnetic field (the B0 field). This is the same type of magnetic field used to perform the excitation that generates the MR signal. T1ρ relaxation has been experimentally shown to be sensitive to changes in proteoglycan content both in tissue and in vivo studies, and thus shows considerable promise for detecting some of the early biochemical changes associated with OA both in tissue and in vivo. In a common type of T1ρ acquisition, a standard 90◦ pulse is applied to tip the magnetization into the transverse plane, as shown in Fig. 1. Next, a constant low-power RF pulse (the spin-lock pulse) is applied for some relatively long duration (several milliseconds). This pulse counteracts the usual effects of the interactions that cause T2 signal decay, but other relaxation mechanisms similar to T1 relaxation still yield some characteristic signal loss. Hence, the T1ρ relaxation time is necessarily longer than the T2 relaxation time (the signal loss that would occur in the absence of the spin-lock pulse). At the completion of the spin-lock RF pulse, a –90◦ pulse restores the remaining magnetization back to the longitudinal (B0 ) axis. The signal available for a subsequent image acquisition will be modulated by the signal loss during the spin-lock pulse due to T1ρ relaxation. The methodology for generating a quantitative measure of the T1ρ relaxation time for a given tissue is then conceptually similar to that for quantitation of T2 relaxation time. A series of images are acquired, each utilizing a spin-lock pulse of identical amplitude but different duration, producing a sequence of signal intensities dependent on the pulse duration. A nonlinear (decaying exponential) curve fit of the resulting signals to the applied pulse durations provides a best-fit estimate of the relaxation parameter. Such an analysis can be carried out on a pixel-by-pixel basis to generate a T1ρ map. The T1ρ sensitizing step acts as a magnetization preparation step which can be applied to most any type of subsequent imaging acquisition, such as FSE [14] or 3D GRE [15]. The application of T1ρ imaging to cartilage is predicated on its sensitivity to the low-frequency interactions among water and macromolecular protons that contribute to the relaxation (i.e. signal decay). In the case of cartilage, the macromolecules of interest are proteoglycans. A depletion of proteoglycan content in cartilage will reduce these interactions and signal decay and yield a corresponding increase in the measured T1ρ


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relaxation time. While the complete set of dependencies of T1ρ relaxation in cartilage is not fully known, experimental models with enzymatically degraded cartilage have shown strong correlation of T1ρ with proteoglycan content and minimal dependency of T1ρ on collagen content [16], though some experiments have noted an influence from interactions due to collagen fibril orientation [17]. As it relies only on a set of magnetization preparation pulses as described above, T1ρ MRI is a completely noninvasive method for generating quantitative information about cartilage proteoglycan content without the need for special hardware or exogenous agents [18,19], and can therefore be implemented on any clinical scanner with a variety of imaging pulse sequences [20,21]. The primary factor limiting resolution and coverage at high field strength is RF energy deposition, arising from the low-power but relatively lengthy spin-lock pulse. These energy deposition factors scale as the square of the field strength, so much of the initial clinically-oriented work has been done at a field strength of 1.5 T. However, advances in parallel imaging capabilities are rapidly eliminating these limitations [22], along with the use of extended readout techniques such as spiral and echo-planar imaging [20]. As described, the image analysis techniques required to quantify T1ρ are very similar to those required for T2 measurements and are straightforward to implement at the scanner console. The close ties between proteoglycan content and cartilage function make T1ρ MRI a much more direct and valuable indicator of cartilage health and treatment efficacy than purely anatomic imaging. Because of the dependence on proton exchange between water and proteoglycans, quantifying proteoglycan depletion associated with early OA development with T1ρ is a more discriminatory cartilage assessment than T2 mapping [23]. The changes in T1ρ parameters are of a considerably greater magnitude, offering the likelihood of earlier and greater sensitivity to degenerative processes [24], though both may be valuable as sources of complementary information. Thus, T1ρ MRI pulse sequences may be able to detect cartilage compromise occurring both shortly after acute injury such as anterior cruciate ligament (ACL) rupture as well as early stages of degenerative changes arising from OA. Both in vitro and in vivo studies suggest that the relationship between T1ρ and proteoglycan content is sufficiently strong and sensitive to detect harbingers of OA while being simple to implement in clinical settings. Sample images from a recent in vitro study of the ability of T1ρ to assess changes in proteoglycan content are shown in Fig. 2. A set of fresh cartilage cylindrical explants taken from the tibial plateau of amputees with no known history of OA underwent three one-hour episodes of cyclic mechanical loading over a period of 12 days in order to induce proteoglycan depletion. The corresponding T1ρ maps derived from the subsequent imaging experiments showed increased T1ρ relaxation time in explants with the highest loading and greatest loss of proteoglycans, as confirmed by biochemical assay [25]. Figure 3 shows the relationship between T1ρ relaxation and proteoglycan content for all ten samples used in the study, demonstrating a highly significant correlation between the imaging and biochemical assay, indicating the sensitivity of the T1ρ technique to this marker of cartilage condition. T1ρ imaging has seen initial clinical use for early OA cartilage degeneration [19,26], and its ability to detect arthroscopically confirmed cartilage abnormalities not otherwise seen on morphologic imaging has been recently demonstrated [27]. Figure 4 shows a T1ρ map of cartilage along with T2-weighted imagery from a patient with an acute ACL injury. The continued development and validation of T1ρ MRI as an objective and quan-

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Figure 2. Cartilage samples and corresponding T1ρ maps for cartilage explants from tibial plateau of a 51-year-old male. Images were acquired at a magnetic field strength of 4.7 T. An increase in T1ρ corresponds to a decrease proteoglycan concentration ([PG]).

Figure 3. Comparison of computed T1ρ relaxation time and GAG content from biochemical assay in ten cartilage sample explants. Proteoglycan depletion was accomplished by a multiday schedule of mechanical loading of the explants.

titative measurement standard of cartilage condition may provide a new and clinically viable tool to improve the assessment and understanding of acute injury, diagnosis, and treatment. 3.3. dGEMRIC Both T2 and T1ρ mapping depend only on intrinsic tissue relaxation characteristics to derive information as to water and collagen (T2) or proteoglycan content (T1ρ) in cartilage. An alternative method to improve the specificity of measurements of biochemical composition is to introduce and detect the distribution of an exogenous contrast agent with a dependency on tissue characteristics. In cartilage, GAG (components of proteoglycans) acts as a source of fixed charge density in the tissue. Delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) is an imaging technique that utilizes a widely available anionic MRI contrast agent, gadopentate dimeglumine (Gd-DTPA2− , commercially available as Magnevist, Berlex Laboratories, Wayne, NJ). Gadolinium-based contrast agents reduce


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Figure 4. In vivo T1ρ acquisition from a patient with acute ACL injury. A color-coded map of T1ρ relaxation time is overlaid on a fat-suppressed T2-weighted image in the sagittal plane. The T2-weighted image shows evidence of bone bruise, while the lengthened T1ρ relaxation of the underlying cartilage is suggestive of local proteoglycan loss (decreased [PG]).

the T1 relaxation time of the tissues that they penetrate in proportion to their concentration in the tissue. This contrast agent is introduced intravenously and allowed to penetrate the cartilage (usually helped along by a period of light exercise after administration). Because of the negative fixed charge density of the GAG (which is linked to core proteins to form proteoglycans) and the like charge of the contrast agent, the concentration of the contrast agent that equilibrates in the cartilage will be inversely related to the GAG concentration of the tissue [28]. As noted, the T1 relaxation time measured in the tissue will be dependent on the normal (without contrast) T1 relaxation time of the cartilage and the concentration of the Gd-DTPA2− agent that penetrated the joint. The Gd-DTPA2− concentration can be quantified through measurement of T1 relaxation time with standard imaging and processing techniques and application of a physical model (such as an electrochemical equilibrium model) to quantify the fixed charged density to yield a marker of GAG concentration and proteoglycan loss. Since the T1 relaxation time of cartilage does not show a dependency on proteoglycan content in the absence of contrast agent, the post-contrast T1 measurements provide a direct marker for the in vivo GAG concentration. As with T2 and T1ρ mapping, the results can be presented both qualitatively as a color-coded image overlay as well as quantitatively in terms of local and regional relaxation parameters implicitly or explicitly linked to tissue composition. The dGEMRIC protocol is currently the most frequently used and widely validated of the emerging methods for in vivo characterization of proteoglycan loss. Numerous in vivo and in vitro studies suggest dGEMRIC is the most specific for GAG concentrations among the described methods, demonstrating very direct correlations between dGEMRIC measurements and GAG concentration and even mechanical properties [29,30]. Figure 5 shows two samples of human cartilage comparing GAG concentration as derived from T1 measurements of the contrast-infused cartilage with histological preparation [31]. When logistically feasible, dGEMRIC can be expected to yield more specific proteoglycan loss information than T1ρ imaging. This specificity comes at a cost of considerable complexity in implementing the exam in a clinical setting. The usual imaging

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Figure 5. Comparison of quantitative GAG concentration maps with histological analysis (toluidine blue staining) in human cartilage samples. The image-derived GAG concentrations show excellent correspondence with the histological standard. Courtesy of Martha Gray, Ph.D. (MIT/Harvard Division of Health Sciences and Technology).

protocol begins with subjects receiving an intravenous injection of contrast agent, followed by a period of mild exercise to distribute the agent. Image acquisition then takes place 1–2 hours after injection. In some cases a pre-contrast exam is also desirable to determine subject-specific baseline T1 measurements for robust quantitative measurement of relaxation changes. Thus, while dGEMRIC shows strong abilities for accurate measurements of proteoglycan depletion, the rigors of the examination require considerable planning and logistical coordination (these issues have been considered and discussed in Burstein et al. [32]). It requires some minimal invasiveness and is thus somewhat more cumbersome to implement clinically than the previously described techniques, but it yields results that are most directly correlated to proteoglycan content. Motivated by the unique quantitative information that can be derived, dGEMRIC has been successfully applied in several subject populations. Figure 6 shows an in vivo example of a GAG concentration map generated for assessment of cartilage in the knee joint [31]. Examples of its application include assessing GAG content in subjects with differing levels of physical activity [33], subjects with cartilage changes confirmed on arthroscopy [34], and correlation with pain and severity in early OA subjects with hip dysplasia [35]. Comparisons of dGEMRIC quantification and a variety of disease state parameters as determined radiographically have also been carried out [36]. In injury models, dGEMRIC has been applied to assessment of cartilage and synovial fluid GAG content in subjects with acute ACL injury [37] and in a case report of GAG changes in posterior cruciate injury [38]. 3.4. Sodium MRI Sodium (23 Na) NMR spectroscopy has long been applied to investigate proteoglycan depletion in cartilage [39]. The principle for this method is that loss of proteoglycans with their negatively charged GAG causes a reduction of fixed charge density (FCD) and a loss of charge-balancing sodium ions, as well as a change in sodium MR relaxation parameters. While initially applied to (non-imaging) spectroscopic measurements, these changes can also be seen and quantified from sodium-based MRI of tissue samples as


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Figure 6. Example of in vivo mapping of GAG distribution in the human knee joint. The image-derived GAG concentration is overlaid on the standard T1-weighted acquisition in sagittal and coronal planes, permitting both qualitative and quantitative assessment of cartilage condition. Courtesy of Martha Gray, Ph.D. (MIT/Harvard Division of Health Sciences and Technology). Reprinted from: Burstein D Bashir A, and Gray ML: MRI techniques in early stages of cartilage disease. Invest Radiol, 35:634, 2000.

well [40]. The referenced study demonstrated changes in image characteristics of 23 Na in cartilage with enzymatically depleted proteoglycan content. Because the signal used for image formation arises solely from the presence of sodium, 23 Na imaging presents a very direct correlation to proteoglycan content and changes. Feasibility studies have also demonstrated that this technique can be successfully applied in vivo [41,42]. However, as sodium MRI is not proton based, special imaging coils and signal processing hardware is required, both of which are rare in clinical environments. The signal strength of 23 Na is much smaller than that in standard proton MRI, and the T2 relaxation time is short, limiting the resolution, signal-to-noise ratio (SNR), and image quality that can be achieved. Sodium MRI must thus be regarded as an esoteric research-oriented application at present and is unlikely to translate to standard clinical scanners in the near future. Nevertheless, its unique ability to directly measure fixed charge density may be valuable for nondestructive evaluations in benchtop studies and as a validation technique at well-equipped research sites. 3.5. Diffusion Imaging Diffusion-weighted imaging (DWI) and diffusion-tensor imaging (DTI) apply extra pulses that create signal differences based on the rate of diffusion in the tissue of interest. The acquisition may be made sensitive to the local diffusion coefficient or to diffusion anisotropy, which in turn reflects variations in tissue microstructure and potentially early changes in OA. While diffusion-based techniques have been successfully applied in tissue samples [43–45], the relatively short T2 relaxation time of cartilage necessitates lengthy exam times for the needed resolution in vivo. This in turn exacerbates the already high sensitivity to motion inherent in diffusion weighted imaging. Feasibility of the technical aspects has been demonstrated in vivo [46], but not widely applied.

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4. Discussion As seen in the foregoing description, MRI is a flexible imaging modality capable of providing numerous mechanisms to characterize cartilage tissue in vivo. T2 mapping, T1ρ imaging, dGEMRIC, and 23 Na sodium imaging have all demonstrated sensitivity to important biomarkers and feasibility in in vivo applications. At present, however, these tools still need more widespread dissemination and more extensive validation in clinical populations before they can become routinely available as part of the clinical MRI toolbox. Nonetheless, the initial studies with all the methods suggest several observations regarding their future use. As a mature and already widely available tool in other application areas, T2 mapping has the most immediate potential to have a clinical impact. The majority of high field scanners already have the necessary pulse sequences and analysis tools to generate these maps directly on the scanner. However, initial studies with T2 mapping of cartilage have shown it to depend on numerous tissue factors (including collagen content and orientation as well as water content) that may be difficult to isolate into a single measure of cartilage condition. Its higher sensitivity to these factors compared to T1ρ and dGEMRIC may merit a place for T2 mapping as an adjunct to one of these other techniques, however. The dGEMRIC technique is highly attractive due to its specific sensitivity to GAG via the use of a exogenous charged contrast agent. The image acquisition protocol (quantitative T1 measurement using inversion recovery pulse sequences) is also one that is universally available at the present time. While the use of contrast material may constitute a (minimally) invasive step, the widespread use of MR contrast media in other applications suggests this is a relatively minor burden. The exercise and time delay requirements between contrast administration and image acquisition is the most challenging aspect in translating this technique to the clinical realm. T1ρ MRI represents a promising “middle ground” between the ease of application of T2 mapping and the specificity to GAG content of dGEMRIC. If properly validated, T1ρ MRI may become the most preferred of the methods as it can generate much of the same information on proteoglycan content as dGEMRIC and sodium MRI, but can be applied on standard MRI hardware and has less complex protocol requirements. It does require specialized software presently only available at research sites, though it has been implemented on systems from all major vendors. Sodium MRI may have a niche in tissue and research studies as a validating tool but is unlikely to achieve a place in the clinical imaging toolbox because of its demanding hardware requirements. Although diffusion imaging is widespread in other MRI applications, the obstacles to clinical imaging of cartilage remain to be surmounted, and it is too early to determine if it will have a place for cartilage imaging and tissue characterization.

5. Conclusion The appearance of techniques for robust and noninvasive assessments of cartilage composition, structure, and function would be invaluable for all phases of the disease process in OA. Such information may yield detection of early OA markers before morphologic changes or physical symptoms occur. It could also direct treatment options and assess


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recovery in later stages of OA development. There is therefore a great need to study and validate these noninvasive techniques in order to bring such tools toward fruition as clinical tools. Acknowledgments Thanks to Martha Gray, Ph.D. (Edward Hood Taplin Professor of Electrical and Medical Engineering and Director, MIT/Harvard Division of Health Sciences and Technology) and collaborators for images taken from her Elizabeth Winston Lanier Award lecture delivered at the 53rd annual meeting of the Orthopaedic Research Society. Supported by NIH grant P50AR048939. References [1]

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XVI Multimodality of Microscopy Imaging Applied to Cartilage Tissue Engineering D. DUMAS a, B. RIQUELME b, E. WERKMEISTER a, N.D. ISLA a and J.F. STOLTZ a a Groupe de Mécanique et Ingénierie Cellulaire et Tissulaire. – UMR CNRS 7563 LEMTA et IFR 111 CNRS –UHP-INPL-CHU b Faculté de Médecine, Nancy-Université. 54505 Vandoeuvre-lès-Nancy, France Facultad. De Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina [email protected]

Abstract. As a comparatively non-destructive imaging technique into living specimens, fluorescence microscopy has a number of strong advantages over alternative imaging modalities (X-ray, MRI, CT-scan, arthro-scan, etc.). The limited analysis in thick tissue has given rise to the development of other techniques, multiphoton excitation microscopy in particular. A need for increased sensitivity and resolution has been driving the development of new sophisticated fluorescence techniques based on microscopies to study: the tissue microstructure in situ (CLSM, SHG) on deeper thick sections of tissue (Multiphoton), molecular diffusion (FRAP, FCS) with fluorescent protein variants and molecular interaction (spectral, FRET, FLIM). In this paper, we have considered developments based on near infrared (NIR) femtosecond excitation in the imaging of articular tissue and discussed the technical limitations and perspectives. Keywords. Multiphoton Microscopy, Second-harmonic Generation, Fluorescence Correlation Spectroscopy, Fluorescence Lifetime Imaging, Spectral Imaging

Introduction Osteoarthritis (OA) is a degenerative disease of the joint characterized by fibrillation and erosion in cartilage, chondrocyte proliferation and osteophyte formation at the joint margins and sclerosis of subchondral bone. Articular cartilage damage and eventual loss is the primary pathological change and the capacity of articular cartilage to regenerate is very limited. Numerous studies have shown that mechanical load can affect the biosynthesis, turnover and structure of the macromolecules produced by the chondrocytes [1]. Tissue engineering of articular cartilage could represent an interesting pathway to solve the complex problem of cartilage regeneration. Several ways of biotherapies have been developed to induce regeneration, either by mobilization of native cells under chemical and biomechanical conditions for the neosynthesis of a replacement matrix (growth factors, cytokines, gene); either by delivery of viable cells (chondrocytes or mesenchymal stem cells). There is a growing interest for optical imaging

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methods in challenge due to the high level of autofluorescence on native unstained samples for cartilage tissue engineering [2]. All current imaging modalities derive information using ultrasound methods or electromagnetic radiation (nuclear, optical or magnetic resonance). The resolution and sensitivity of classical imaging techniques based on conventional radiography (X-ray, MRI, CT-scan, arthro-scan, etc…) are too low to collect any information about a specific cellular structure into a cellular scale (micrometer). Actually, clinical non-invasive techniques such as magnetic resonance imaging [3] and ultrasound lack resolution needed to distinguish complex structures of articular cartilage [4]. The minimally destructive nature of arthroscopy is very useful but this technique which requires anesthesia and surgery only provides information about cartilage structure at articular surfaces [5]. With a low ability to distinguish among biological constituents, Optical Coherence Tomography (OCT) provides full thickness, high resolution (around 10 µm), cross-sectional images of cartilage [6]. Even if these clinical non-invasive (optical biopsy) methods offer promise in the early detection of cartilage degeneration, they are mainly used to make a precise inventory of articular lesions in osteoarthritis (slow degradation of cartilage) and to investigate the superficial and deep layers of cartilage. Despite a considerable level of development, the major limitation of clinical techniques remains that the field of view is strictly limited to the surface and restrict their applicability to the study of large articular disease. By using visible and near-infrared windows of the electromagnetic spectrum, the increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopy applied to living tissue. But it was mainly in the past decade that it became possible to shift from whole-specimen analysis to smaller volumes, through microscopy techniques (far and near-field microscopies, fluorescence correlation FCS and FRAP, etc.), down to almost single-molecule exploration or interaction (FRET) [7]. In this situation, conventional light and fluorescence microscopies have shown in situ particular application for improving the ability for the characterization of organization within the structure of the extracellular matrix of cartilage. Among the physical methods available to investigate biological media, fluorescence, with its high analytical sensitivity and resolutions (spectral, spatial, temporal, order) offers interesting possibilities for cell or tissue biological analysis [8]. At this time, fluorescence microscopy is the most rapidly expanding microscopy method in both the biological and medical sciences. Molecular emission spectroscopic techniques (fluorescence, phosphorescence, chemo- and bio-luminescence), which correspond to excitation deactivation processes, are now widely used to study variably complex media such cells or tissues. Fluorescence microscopy is exempt from a number of constraints normally attached to standard techniques (probe concentration, low cellular density, exploration and display at the cellular scale). Furthermore, it is the only methods of investigation currently available with a high enough resolution to specify the distribution of the fluorescent probe, because it collects fluorescence signals emitted at the probe incorporation site. After staining, the ability to image a specific biological target or predictive molecular markers that could be associated with cartilage disease will represent a complementary approach to the anatomical imaging performed in clinical practice. There is a need for sensitive molecular imaging techniques dedicated to articular tissue autofluorescence which can be resolved by using new organic or inorganic fluorescent probes. Quantum Dots (QDots) are very small inorganic fluorophores (< 10 nm) based on a cadmium selenide semi-conductor core surrounded by a zinc sulphide shell. This core-shell complex can be finally used for immunohisto-

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chemistry (after antibodies or streptavidin coupling) with a great interest because they have several unique optical properties (single excitation wavelength for multiplexing, high level of brightness, photobleaching reduced, multilabeling available due to their narrow and symmetric emission). In the same way, multicolor imaging of proteins to study their function in the context of the living cells has become a fundamental technique in cell biology during the last decade. Natural fluorescent proteins (GFP, CFP, YFP, DsRed and variants) are also widely used as probes thanks to their brightness, resistance to photobleaching, and potential to be fused with virtually any gene product. For instance, these chimeric proteins have been used to determine a novel chondroprotective modality by overexpressing HSP70 in chondrocytes by using a vector carrying HSP70/GFP. To distinguish between implanted and host-derivate cells, in diseased states such as osteoarthritis cartilage matrix, Grossin at al developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death mainly due to an increase in apoptosis [9]. Scanning confocal microscopy has been confronted with the problem of using intact living cells and phototoxicity for imaging hard tissues. Most tissues have reduced absorption in the near-infrared part of the spectrum and nonlinear infra-red optical microscopy can exploit the “optical window” at 700–1000 nm. Multiphoton excitation is mainly characterized by less light scattering and deeper penetration of the light into the sample and usually entails less damage from photobleaching. This nonlinear optical microscopy allows living cells to be probed in real time under physiological conditions within the intact microenvironment and is beginning to emerge as a powerful contrast mechanism in combination with second and third harmonic generation imaging (SHG, THG) [10]. In this part, we have considered developments based on near infrared (NIR) femtosecond excitation in the imaging of articular tissue and discussed the technical limitations and perspectives.

1. Optical Scanning Microscopy/Deconvolution Conventional fluorescence microscopy was primarily used to determine the spatial distribution of fluorescent probes. One of the major limitations of fluorescence microscopy is the progressive bleaching of the fluorochrome during prolonged exposure. Unfortunately, when focusing at a particular depth within a transparent fluorescently labelled 3D specimens, fluorochromes molecules throughout the whole of its thickness are excited. The diffraction light disrupts the image which reduces contrast and spatial resolution, allowing few strategies to overcome these limitations. Contrary to confocal microscopy, each acquired 2D image (X, Y) contains data of its focal plane (clear area) and of all other planes (blurred area). The clear area of the image corresponds to data pertaining to the focal plane, the blurred part of the image originating from data pertaining to all other planes (Fig. 1). These interference data can be removed and images run through a computationally intensive set of algorithms that permit reassigning the photons from adjacent planes to the focal plane. These techniques are oriented towards modeling degradation phenomena (defocusing, noise) and to the application of reverse procedures (mathematical reversion or iterative deconvolution so as to obtain an approximation of the original scene (an estimate of the subject by using the transfer function image). The optical transfer function reflects the way in which a punctual source is deformed when displayed via an optical system and determines the impulse response of


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Figure 1. Before (A) and after (B) deconvolution process of isolated chondrocyte imaging with the appropriate Point Spread Function (Green Fluorescent Protein, GFP). CellScan EPR TM optical scanning acquisition system (IPLab-Scanalytics, Billerica, USA) equipped with a 12 bit CCD camera (Princeton Instruments). Z spacing: 0.25 µm. 60X/w1.2 NA PSF.

the optical system or point-spread function [11]. Then using a detailed knowledge of the degradation introduced by the optical system (convolution), images are corrected by computer deconvolution image processing (Fig. 1).

2. Confocal Laser Scanning Microscope The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. One of the first review in cartilage research using digital imaging CLSM was published in 1997 [12]. Confocal fluorescence microscopy has the ability to control the depth of focus by spatial filtering and as a result, it allows for collection of a series of optical sections throughout the sample, with reducing background fluorescence originating from sections that are away from the focal plane. The benefits of confocal microscopy are 1) Increased effective resolution 2) Improved signal to noise ratio 3) Clear examination of thick specimens 4) Depth perception in Z-sectioned images 5) Magnification can be adjusted electronically 6) Reduced blurring of the image from light scattering. The improvements were essentially aimed at offering solutions to the problems posed by CLSM 1) by multiple marking in fluorescence (cross-talking) 2) the too low scanning rate to catch rapid biological events, 3) the fast photodegradation of the fluorescent probe as well as cytotoxicity (under UV light mainly) 4) the low quantum efficiency of detector 5) the fluorescence emission restricted by optical configuration (pinhole). In contrast to cartilage which is highly scattering, a culture transparent medium such as alginate matrix, permits reflectance or transmitted mode imaging without the need for tissue fixation or exogenous dyes (Fig. 2). By this way, Yansen and al have followed the time-dependent formation of the cartilage matrix from 2 to 15 days [13]. Fluorescence confocal microscopy has showed significant variations in the shape, size and orientation of chondrocytes an chondrons revealing flattened discoidal chondrons in the superficial zone, rounded chondrons in the middle zone, and elongated chondron, multicellular chondrons in the deep zone [14]. The three-dimensional chondrocyte cytoskeleton is involved in mechanotransduction in the response to mechanical loads. The optimal processing method of fixation and permeabilization on the preservation of cytoskeletal structure has been

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Figure 2. Confocal images of fluorescence and light transmitted detection from articular cartilage chondrocytes. A-B; C: Autofluorescence of chondrocyte clusters of knee human osteoarthritis cartilage.

recently described for confocal imaging [15]. However, CLSM posed certain problems linked to the use of living cells due to the high density of incident light (laser source) focussed on a small volume (femtoliter) and very small illuminated diameter of the specimen (about 0.3 µm). To overcome these limitations, other 3D fluorescence techniques have been developed, such as multiphoton microscopy.

3. Multiphoton Microscopy Advances in multiphoton fluorescence microscopy continue to appear. Multiphoton excitation occurs during the simultaneous absorption of two or more photons by a fluorophore. This multiphoton process (simultaneous absorption of 2 photons) is made possible by a very high intensity combined with time-related concentration of a very brief (pico or femtosecond) and very high-frequency (about 80 MHz) laser flash [16]. Because in multi-photon mode spatial resolution is the result of absorption (and excitation) confinement to the focal event (smaller of one femtoliter) [17], the photobleaching patterns and photodegradation outside the focal plane are reduced [18]. According to the pulse duration/peak power ratio, two red photons (700 nm) can excite a fluorophore whose excitation spectrum is in UV (350 nm) for an emission spectrum in the blue (420 nm). Since the red or near infrared illuminating light used for 2-P excitation has approximatively twice the wavelength of that employed for 1-P excitation, scattering effects at the excitation wavelengths are greatly reduced allowing deeper penetration into tissues than with visible or UV excitation [19]. Fluorescence generated by two-photon absorption corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins as a redox imaging without the need for exogenous dyes [20]. The effects of the heat produced after high-frequency pulse illumination in a restricted volume (approx. 1 µm3) and the optical aberrations are thought to be negligible and similar to those observed with single-photon confocal microscopy [21]. To study how chondrocytes respond to alterations in their mechanical environment, numerous in vitro studies have been performed using 2P-excitation either viable cartilage explants or chondrocytes embedded in a gel matrix [22]. Figure 3 shows nucleus of chondrocytes excited by 2P-excitation pulsed laser light according to transparence and nature of culture material (500 µm native matrix or ∅ 2mm alginate bead). As shown, depth penetration in thick specimen is greatly enhanced (from 219 µm to 1.6 mm) for sodium alginate gel in the case of transparent medium.


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Figure 3. Multiphoton microscopy images of nucleus stained with Hoescht and illuminated by 2-Photon absorption light at 780 nm (Laser femtoseconde MIRA 900). A: Chondrocyte in rat cartilage head cap (depth illumination: 219 µm). B: Chondrocyte encapsulated in alginate bead (depth illumination: 1.6 mm).

4. FRAP and FCS Fluorescence Recovery After Photobleaching (FRAP) makes use of the fact that fluorescent molecules lose their ability to emit photons when exposed to repetitive cycles of excitation and emission. By monitoring the levels and rates of fluorescence recovery with time, one can determine kinetic parameters such as the mobile fraction and the diffusion coefficient. Molecular transport in avascular collagenous tissues such as articular cartilage occurs primary via diffusion. New technique of FRAP have shown that diffusional transport of macromolecules is anisotropic in collagenous tissues with higher rates of diffusion along primary orientation if collagen fibers [23]. However, various processes such as membrane flow, molecular interactions and trafficking may simultaneously contribute to the overall recovery kinetics, which makes it difficult to interpret the data obtained [24]. Recently, there has been rapidly increasing use of Fluorescence Correlation Spectroscopy (FCS) for biological applications which is a sensitive technique for measuring dynamic processes (number density, interaction fractions and molecular dynamics) in a fluorescent signal on the nanosecond to second time scales [25,26]. FCS is based on the fluorescence intensity fluctuations associated with molecules passing through a diffraction-limited observation volume created by a focussed laser beam. FCS The autocorrelation function obtained from the intensity fluctuations gives the average diffusion time taken by the molecule to cross the observation volume, along with the average number of molecules present [27]. Sanchez and Gratton used two-photon microscopy in conjunction with fluorescence correlation microscopy [28]. Several works have recently shown that Mesenchymal Stem Cells (MSC) collected from bone marrow can differentiate in vitro into cartilage cells under the effects of transforming growth factor-β (TGF-β), others growth factors or critical transcription factors [29]. The effect of TGF-β, a multifunctional cytokine, has been described as a potent stimulant of the synthesis of the matrix molecules [30]. The mobility of type II TGF-β receptor in the MSC membranes has been compared for MSC TGF-β stimulated or unstimulated cells (Fig. 4). FCS may appear as a more appropriate technique since it analyzes an ensemble of molecules diffusing in the detection volume There is a an increase in the decay time of the FCS curves from before stimulation indicating that receptor diffuse more quickly through the focal volume and are thus shown to have higher mobility after stimulation.

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Figure 4. Confocal image of indirect immuno-labelling of RII collagen receptor (A11017 Invitrogen, Alexa488TM) on Mesenchymal Stem Cells. Fluorescence autocorrelation curves on a single MSC before and after stimulation with TGF-β1 (10 ng/ml TGF-β for 12h). Each curve is the average of five measurements on a single focal spot (target laser) at 488 nm on a SP2-FCS2 Leica Microsystems workstation. Excitation and emission were focused through a 63 x 1.2 NA water immersion. Excitation volume has been calculated as 0.48 ± 0.02 fl.

5. Spectral Microscopy Unlike a fluorescence image, where each point represents the intensity at a different locations (x,y,z) in space, a spectral image is a sequence of image representing the intensity of the 2D plane of the sample at different wavelength (spectral information). Combination of fluorescence spectroscopy with image cytometry is useful in biology by using fluorescent probes to identify and map several fluorophores and to improve the signal-to-noise ratio. The ability to identify a fluorophore signal based on spectral characteristics rather than intensity has significant advantages when trying to separate signals from undesirable background fluorescence and when trying to separate signals from closely related fluorophore. For chondrocytes; spectral imaging has also been used successfully for the detection of early apoptosis by using a JC-1 (5,5’, 6-6’ – tetrachloro-1,1’, 3,3’- tetraethylbenzimidazolocarbocyanine iodide) sensitive mitochondrial potential dye [31]. This ratiometric probe can be used on human cartilage to investigate events involved in programmed cell death as a consequence of disease arthritis. Incubation of chondrocytes from human OA cartilage with JC-1 revealed a fluorescence shift from orange to green with a transmitochondrial potential collapse as obtained during apoptotic processes (Fig. 5).

6. Lifetime Imaging Contrast The fluorescence lifetime of a substance represents an average amount of time the molecule remains in the excited state prior to its return to the ground state, while emitting fluorescence. The advantages and disadvantages of measuring the fluorescence signal in terms of intensity or lifetime have often been discussed [7]. The precise nature of the fluorescence decay can reveal more details about the interactions of the fluorophore with its close surrounding: this parameter can reveal the frequency of collisional


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Figure 5. Mitochondrial transmembrane potential for normal and pathological human chondrocytes. Fluorescence spectra (from 502.5 nm to 632.5 nm, excitation at 488 nm) acquired with a SP2-AOBS-confocal microscope Leica Microsystems (63x/1.2 NA water immersion). of 5,5’, 6-6’ – tetrachloro-1,1’, 3,3’- tetraethylbenzimidazolocarbocyanine iodide (JC-1) in normal chondrocyte (A, B) and knee human osteoarthritic cartilage chondrocyte (C, D).

encounters with quenching agents, the rate of excited state reactions. Multiple decay constants can be the reflection of several distinct surrounding of a fluorophore or of the presence of several conformational states of a molecule. Factors such as ionic strength, hydrophobicity, oxygen concentration, binding to macromolecules can all modify the lifetime of a fluorophore, considered as an indicator of these parameters. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the advantages of lifetime spectroscopy with fluorescence microscopy by revealing the spatial distribution of a fluorescent molecule together with information about its microenvironment. FLIM has been applied with spectral fluorescence to study of articular cartilage and its arthritis disease. By exploiting autofluorescence, the diseased tissue has been detected that were not detectable with the conventional diagnosis [32]. A greater FLIM contrast between the cells and the extracellular matrix has been shown (Fig. 6).

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Figure 6. For lifetime imaging, SPC-730 TCSPC Imaging module (Becker&Hickl, Berlin) was interfaced (signals, Pixel Clock, Frame Sync) to the scan controller of the Leica TCS-AOBS Multiphoton laser scanning microscope. The decay analysis measured by time-correlated single photon counting was performed using the SPCImage software (Becker&Hickl GmbH). (A) Lifetime image of knee human osteoarthritic cartilage chondrocyte labelled with JC-1 (mitochondrial transmembrane potential probe) showing chondrocytes in blue for shorter lifetime and matrix in green-red for longer lifetime (B). The fluorescence decay of JC1 consisted in a minor component (bleu) used to generate a mask for segmentation and to contrast the JC1 fluorescence against the matrix autofluorescence.

7. FRET Imaging Contrast Fluorescence Resonant Energy Transfer (FRET) is a powerful technique for measuring intermolecular distances on a scale of a few nanometers, orientation and dynamic properties. FRET microscopy is typically used to determine binding partners, conformational changes, and proximity or interaction between two molecules. FRET utilizes a pair of fluorophores (donor and acceptor) and takes advantage of long-range dipoledipole interactions, when the excitation spectrum of the acceptor overlaps with the emission spectrum of the donor and the energy is nonradiatively transferred from the acceptor of the donor. This phenomenon only occurs when donor and acceptor are in proximity (1–10 nm) [33]. However, due to the spectral overlap of donor and acceptor excitation/emission, the acceptor is often also excited directly by the donor excitation. Most of pair of fluorophore used in FRET experiment are difficult to separate spectrally by currently available methods based on fluorescence intensity [34]. FRET in vivo studies has remained relatively unexplored due to the thickness of tissue cross sections which can be in part resolved by using two photon excitation [35]. Spectral analysis and FLIM have been combined for better distinction [36]. Autofluorophores in chondrocytes have been characterized by multiphoton, confocal, lifetime and spectral microscopy image in view to subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins [37]. 8. SHG From histological studies have been described different zones of cartilage with increasing depth. Collagen fibers are oriented parallel to the articular surface, then fibers may have many different orientations to progress into the radial zone and starting the bone


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Figure 7. Two-Photon Fluorescence Image (green channel for autofluorescence) combined to Second Harmonic Generation Image (white channel for organized network collagen) at 805 nm (Laser femtoseconde MIRA 900) in osteoarthritic human cartilage (5 µm) showing morphological changes (hyaline, amorphous collagen matrix) with depth near the articular surface (A) and the radial zone (B).

surface (separated by the tide mark) where fibers are oriented orthogonal to the joint surface. Collagen fibers present a high second order non-linear susceptibility, and therefore produced a second harmonic generation (SHG) when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As processes involved in multiphoton fluorescence and SHG are intrinsic properties of the constituent molecules, non-linear microscopy allow submicron resolution images without need for sectioning or staining with dye. By placing bandpass filter at the second harmonic of the excitation wavelength in front of the transmitted light detector, SHG signal with frequency doubled is isolated for a new SHG contrast imaging. SHG is a coherent elastic process which is dependent on the polarization of the incident light, and the greatest intensity for collagen signal is produced when the fibers are oriented parallel to the laser polarization [38]. Both the nonlinear optical microscopy and SHG at 800 nm have been combined to investigate living cells from normal and degenerative cartilage due to macroscopic arrangement of type II collagen [39]. In healthy tissue the fibrous structure of collagen II fibers in the extracellular matrix has been seen along with dark areas representing the sites of chondrocytes [40]. Moreover, chondrocytes and membranes have been identified on the basis of native fluorescence emission spectra and SHG signals from the collagen fibers [41]. Using SHG, it was possible to achieve two-photon excitation images at great depths in strongly (light) scattering collagen membranes (depth up to 300 µm) and cartilage samples (depth up to 460 µm). The SHG images clearly map the distribution of the collagen II fibers within the extracellular matrix while the multiphoton fluorescence mages show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix (Fig. 7). The SHG image clearly demonstrated lacuna occupied by chondrocytes. Conclusion In this work has been presented a panel of studies of microscopy imaging which had tissue cartilage relevance. Conventional imaging with one-photon excitation process

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and organic fluorophores poses several challenges for the visualization of articular tissue, including fluorophore overlap (crosstalk), rate of photobleaching and autofluorescence. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimization of CLSM and deconvolution process. Even if the deconvolution techniques require considerable computing power capacities and extended computation time, they prove very useful in situations of low intensity levels or restricted use of confocal microscopy to improve spatial resolution in multiphoton microscopy. In the future, multiphoton imaging has established itself as an important method for optical microscopy particularly for live cell studies. The feasibility of multi labelling intimal structures by exciting green fluorescent protein variants, multicoloured QDots or organic fluorophores with only one laser wavelength (820 nm) is very useful. In addition to lower photodamage, less background fluorescence is observed since the photon flux is usually too low for nonlinear excitation outside the tiny volume where the laser is focused. Recent technological advances in non-linear optical microscopy have created more powerful imaging as the only strategy currently available for non-invasive, reliable detection (Spectral Microscopy), quantitative (Fluorescence Correlation Spectroscopy) and real time fluorescence (Fluorescence Lifetime Imaging Microscopy) studies in living cartilage tissue. Near-infrared tomography may allow for sufficient tissue penetration to image organized structure (Second Harmonic Generation) combined or not with fluorescence. Future imaging techniques will be designed to answer more specific research questions in tissue cartilage engineering, particularly multimodal approaches by potential combination of scanning multiphoton and arthroscopy (which is actually the standard for assessment for articular cartilage). With the use of green fluorescent protein, it could be possible for long-term to monitor cartilage regeneration after delivery of autologous or mobilization of native cells. The methodological advantages of multimodality imaging (FLIM-MRI) may lead the development of modified probes with a fluorescent or phosphorescent dye characterized with very long lifetime (µs-ms) as obtained by FLIM. The same markers could be very efficient with magnetic resonance imaging as contrast agent (europium, terbium..) [42].

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[9] Grossin L, Cournil-Henrionnet C, Pinzano A, Gaborit N, Dumas D, Etienne S, Stoltz JF, Terlain B, Netter P, Mir LM, Gillet P. Gene transfer with HSP70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J 20(1) (2006), 65-75. [10] Campagnola PJ, Millard AC, Terasaki M, Hoppe PE, Malone CJ, Mohler WA. Three-dimensional high-resolution second-hramonic generation imaging of endogenous structural proteins in biological tissues. Biophys J 82 (2002), 493-508. [11] Dumas D, Gigant C, Presle N, Cipolletta C, Miralles G, Payan E, Jouzeau JY, Mainard D, Terlain B, Netter P, Stoltz JF. The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application, Biorheol 37 (2000), 165-176. [12] Verschure PJ, Van marle J, Van Noorden CFG, Van den Berg WB. The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology. Microscopy Research and technique 37(4) (1997), 285-298. [13] Yansen ES, Krasieva TB, Sun CH, Wong BJF, Sobol EN; Laser scanning confocal microscopy of chondrocytes in an alginate matrix. Dynamics of cartilage matrix formation. Laser Physics 15(11) (2005), 1585-86. [14] Youn I, Choi JB, Cao L, Setton LA, Guilak F. Zonal variations in the three-dimensional morphology of the chondron measured in situ using confocal microscopy. Osteoarthritis Cartilage 14(9) (2006), 889-97. [15] Blanc A, Tran-Khanh N, Filion D, Buschmann MD. Optimal processing method to obtain four-color conocal fluorescent images of the cytoskeleton and nucleus in three-dimensional chondrocyte culture. Journal of Histochemistry & Cytochemistry 53(9) (2005), 1171-1175. [16] Denk W, Stricklen JH, Webb WW. Two-photon fluorescence scanning microscopy, Science 2 (1990), 248-273. [17] Diaspro A, Robello M. Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures, J Photochem Photobiol 55 (2000), 1-8. [18] Koester HJ, Baur D, Uhl R, Hell SW. Ca2+ fluorescence imaging with Pico-anf femtosecond twophoton excitation: signal and photodamage, Biophys J 77 (1999), 2226-2236.0 [19] Helmchen F, Denk W. Deep tissue two-photon microscopy. Nature methods 2 (2005), 932-340. [20] Wong BJF, Wallace V, Coleno M, Benton HP, Tromberg HJ. Two-photon excitation laser scanning microscopy of human, porcine and rabbit nasal septal cartilage Tissue Eng 7(5) (2001), 599-606. [21] Girkin JM, Wokosin DL. Practical Multiphoton Microscopy: Confocal and Two-photon Microscopy: Foundations, Applications and Advances, Edited by Alberto Diaspro. Wiley-Liss, Inc, 2002. [22] Gigant-Huselstein C, Dumas D, Hubert P, Baptiste D, Dellacherie E, Mainard D, Netter P, Payan E, Stoltz JF. Influence of mechanical stress on extracellular matrixes synthesized by chondrocytes seeded onto alginate and hyaluronate-based 3D biosystems. Journal of Mechanics in Medicine and Biology 3 (2003), 59-70. [23] Leddy HA, Haider MA, Guilak F. Diffusional anisotropy in collagenous tissues: fluorescence imaging of continuous point photobleaching. Biophys J 91(1) (2006), 311-6. [24] Marguet D, Lenne PF, Rigneault H, He HT. Dynamics in the plasma membrane: how to combine fluidity and order. The EMBO Journal 00 (2006), 1-12. [25] Hess ST, Webb WW. Focal volume optics and experimental artefacts in confocal fluorescence correlation spectroscopy. Biophys J 30 (2002), 2300-2317. [26] Bacia K, Kim SA, Schwille P. Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3 (2006), 83-89. [27] Schwille P. Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochemistry and Biophysics 34(3) (2001), 383-408. [28] Sanchez SA, Gratton E. Lipid–protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy Acc. Chem. Res 2 (2005), 932-940. [29] Raghunath J, Salacinski HJ, Sales KM, Butler PE, Seifalian AM. Advancing cartilage tissue engineering: the application of stem cell technology. Current Opinion in Biotechnology 16 (2005), 503-509. [30] Redini F, Galera P, Mauviel A, Loyau G and Pujol JP. Transforming growth factor beta; stimulates collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes. FEBS Letters 234(1) (1988), 172-175. [31] Blanco FJ, López-Armada MJ, Maneiro E. Mitochondrial dysfunction in osteoarthritis. Mitochondrion 4(5-6) (2004), 715-728. [32] Talbot, CB, Benninger RPK, De Beule P, Requejo-Isidro J, Elson DS, Dunsby C, Munro I, Neil MA, Sandison A, Sofat N, Nagase H, French PMW, Laver MJ. Application of hyperspectral fluorescence lifetime imaging to tissue autofluorescence: Arthritis. Progress in Biomedical Optics and Imaging – Proceedings of SPIE 5862, (2005), 1-6. [33] Jares-Erijman EA, Jovin TM. FRET imaging. Nat Biotechnol 21(11) (2003), 1387-95.

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[34] Dumas D, Stoltz JF. New tool to monitor membrane potential monitored by FRET Voltage Sensitive Dye (VSD) using Multiphoton Microscopy, Spectral and Fluorescence Lifetime Imaging Microscopy (FLIM). Interest in cell engineering. Clin. Hemorheol. Microcirc 33, (2005), 293-302. [35] Mills, JD, Stone JR, Rubin D, Melon DE, Okonkwo DO, Periasamy A, Helm GA. J. Biomed. Opt 8 (2003), 347-356. [36] Dumas D, Gaborit N, Tran N, Grossin L, Gillet P, Stoltz JF. Spectral and Time-resolved fluorescence imaging microscopies: New modalities of multiphoton applied to tissue or cell engineering. Biorheology 41(3,4) (2004), 459-467. [37] Wachsmann-Hogiu S, Krakow D, Kirilova V, Cohn DH, Bertolotto C, Acuna D, Fang Q, Krivorov N, Farkas DL. Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage. Progress in Biomedical Optics and Imaging. Proceedings of SPIE 5699 (2005), p. 75-81. [38] Stoller, P. Quantitative second-harmonic generation microscopy in collagen. Applied Optics 42(25) (2003) 5209-5219. [39] Yeh AT, Hammer-Wilson MJ, Van Sickle DC, Benton HP, Zoumi A, Tromberg BJ, Peavy GM. Nonlinear optical microscopy of articular cartilage. OsteoArthritis and Cartilage 13 (2005), 345-352. [40] Mansfield JC, Winlove CP, Knapp K, Matcher SJ. Imaging articular cartilage using harmonic generation microscopy. Multiphoton Microscopy in the Biomedical Sciences VI, edited by Ammasi Periasamy, peter T.C.So, Proceedings of SPIE 6089 (2006). [41] Martini J, Tönsing K, Dickob M, Schade R, Liefeith K, Anselmetti D. Two photon laser scanning microscopy on native cartilage and collagen-membranes for tissue–engineering. Proceedings of SPIE. Multiphoton Microscopy in the Biomedical Sciences VI, Ammasia Periasamy, Peter T.C. So, Editors, 6089 (2006). [42] Kahn E, Lizard G, Dumas D, Frouin F, Menetrier F, Stoltz JF, Todd-Pokropek A. Analysis of fluorescent MRI contrast agent behavior in the liver and thoracic aorta of mice.Anal Quant Cytol Histol 26(4) (2004), 233-238.


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XVII Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis Leonardo PUNZI *, Francesca OLIVIERO and Paolo SFRISO Rheumatology Unit, University of Padova, Italy Abstract. Molecular markers or biomarkers have recently received growing attention in osteoarthritis (OA), due to their potential usefulness in early diagnosis, assessment of disease activity and severity, and evaluation of drug effects. In this context, biomarkers are ideals, due to their characteristics of non-invasive and nonexpansive measures. Concerning the diagnosis, no marker seems able to satisfy the needs to diagnose OA in pre-radiological stages and to identify different subsets of OA. Instead, biomarkers are useful in the assessment of disease activity and the prevision of its outcome. In the recent years, stimulated by the recent introduction of high sensitive immunoassays, number of studies have suggested a role of C-reactive protein (CRP) as marker of activity or severity of OA. Furthermore, higher CRP levels predict those patients whose disease will progress over 4 years. Since metalloproteases (MMPs) are highly involved in cartilage degradation, their levels or activities have been investigated to obtain information on OA severity or progression. Both in serum and synovial fluid (SF), the most abundant MMP is MMP-3. It has been proposed that pro-matrix MMP-3 may act as a marker for posttraumatic cartilage degradation. The molecular markers most useful in suggesting synthesis or degradation of cartilage originate from different articular sources such as cartilage, bone and synovial tissue. Serum hyaluronan (HA) is the most commonly used marker of synovial proliferation and hyperactivity, which may reflect the OA evolution. Other useful biochemical markers are serum keratin sulphate (KS), cartilage oligomeric matrix protein (COMP), YKL-40, and urinary C-terminal crosslinking telopeptides of collagen types I and II (uCTX-II). COMP concentration in SF from lavage as well as in serum is an early indicator of radiographic progression at follow up. Furthermore, COMP was the most sensitive test for identifying affected subjects with the genetic form of premature OA. uCTX-II is well correlated with radiological severity of both knee and hip OA and, in addition, the combined measurements of uCTX-II and serum HA seem the best predictor of the structural progression of hip OA. Keywords. Osteoarthritis, Laboratory investigation, Synovial fluid, Biomarkers, Biochemical markers, Molecular markers, Markers of inflammation, Cytokines

Introduction The progress in the knowledge of the pathophysiology of osteoarthritis (OA) has contributed to clarify the role of some substances as putative markers of this process [1]. In * Corresponding Author: Leonardo Punzi, MD, PhD, Rheumatology Unit, Department of Clinical and Experimental Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy, E-mail: [email protected].

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L. Punzi et al. / Biomarkers of Matrix Fragments, Inflammation Markers in Osteoarthritis

OA they may be utilised for several objectives, including diagnosis, assessment of the disease activity, prevision of the outcome, and evaluation of drugs effects. Recently a multidisciplinary group, the NIH-funded Biomarkers Network, has proposed to classify OA markers as “BIPED”, which stands for Burden of Disease, Investigative, Prognostic, Efficacy of Intervention and Diagnostic [2]. However, although a great number of substances are continually proposed, only few among these may be considered as true “disease marker” in OA [1–6]. Biochemical markers or biomarkers available in OA may be classified in “direct” and “indirect” markers, according to Thonar’s suggestion [7]. Direct markers are molecules or fragments which originate principally from joint structures while indirect markers are found in many tissues and produced by many cell types. They have the potential to influence the metabolism not only of chondrocytes but also of synovial cells and other joint cells. Biomarkers may be determined in three different biological fluids: blood, synovial fluid (SF) and urine. Obviously, since serum and urine are commonly available, determinations in these fluids are easier performed than in SF that however, offers information which better reflects local changes occurring in joint affected by OA. To appropriately evaluate the significance of the substances determined in serum or in urine, it should be remember that they may originate from many tissues outside the joints, including non articular cartilage. Furthermore, molecular markers are smaller and have shorter half-lives in the blood circulation than in joint fluid and in addition, due to their complex metabolism, are difficult to be adequately interpreted. Marker concentration in serum or urine may be influenced by the function of organs mainly responsible for the elimination of the molecular fragments, in particular lymph nodes and liver [5,7]. For example, the concentration of a marker of cartilage matrix degradation in SF may depend not only on the rate of cartilage matrix degradation, but also on the rate of clearance from the joint. Furthermore, some biomarkers vary diurnally, thus suggesting that serum and urine sampling need to be carefully standardised [8]. Among the most ambitious objectives of OA markers there is the possibility for an early diagnosis. Since main diagnostic hallmarks in OA are still represented by radiographic changes, which are only evident when disease is established, biochemical markers would be ideal in order to diagnose OA in pre-radiological stages and identify different subsets of OA. In addition, these substances could be also potentially utilised in assessing the value and the significance of new imaging techniques very sensitive in detecting cartilage changes, such as magnetic resonance (MR) and sonography [9]. Unfortunately, no evidences have been until now demonstrated an established utility of markers in satisfying these needs, although interesting studies are in progress. In diagnostic perspectives, the more traditional laboratory features are still helpful. These may be utilised in a “routine” approach, to exclude inflammatory arthropathies or other conditions causing arthritis, and in more specific aims, to investigate the causes mainly responsible for OA or OA-like arthropathies, which may in turn allow to subdivide OA in two classical categories: primary or secondary. In a “routine” fashion, two biological fluids may be analysed: blood and SF, obviously when available (Table 1). Among blood or serum determinations, although no features may be considered as a diagnostic “marker”, erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) may be useful in the basic but essential task of identifying subclinical inflammatory conditions. In fact, although some inflammatory reactions are important in the pathogenesis of OA, this disease is usually considered, at least for classification purposes, as a non inflammatory arthropathy. For this reason, an ESR < 20 mm/h has been included among ACR


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Table 1. Aspects to be considered in the evaluation of laboratory markers in osteoarthritis Biological fluids in which markers are detected

Comments

Serum

a) Due to their complex metabolism, molecular markers may be difficult to interpret adequately b) Frequently molecular markers are smaller and present shorter half-lives in blood circulation than in synovial fluid c) Substances may originate from tissues outside the joints d) Marker concentration in serum may be influenced by the function of the organs mainly responsible for the elimination of the molecular fragments, in particular lymph nodes and liver

Synovial fluid

a) The concentration of a marker of cartilage degradation may depend not only on the rate of cartilage matrix degradation, but also on the rate of clearance from the joint b) The volume of fluid within the joint cannot be accurately measured c) The volume of an effusion can changes rapidly d) Joint aspiration may be performed easily only a few joints e) This determination gives information concerning a single joint

Urine

a) The marker could be metabolised before detection

Table 2. Synovial fluid findings in osteoarthritis. Personal experience on 432 patients (3) Findings

Common features

Observations

Aspect

Clear

Cartilage fragments, cells, crystals or particles may cloud the synovial fluid

Colour

Yellow pale or dark

In some cases, severe OA may be associated with hemartrosis

Viscosity

Normal

Usually decreased in relation to local inflammation

32 (3–130)

Volume exceeding 100 ml is rare

WBC number (x 10 /mm ), mean (± SD) (range)

0.4 (0.7) (0.1–2.7)

Most fluids contain WBC < 1,000/mm3; very rare are those with WBC > 2,000/mm3

PMN, % range

2–10

Value higher than those indicated may be found in patients with concomitant disease

Total protein, mean (± SD)

3.5 (2.7)

Simultaneous serum determination is recommended

Volume, median (range) 3

3

PMN=polymorphonuclear cells

criteria for the classification of OA of the hip [10]. Although no OA criteria include CRP, this test may be useful in detecting an inflammatory condition, in which it is usually found at levels above the limit of normal value commonly fixed at 6 mg/L. However, in rare cases of OA, such as erosive OA, ESR and mainly CRP may be increased, with a value slightly above the normal [11–13]. SF analysis may be useful in OA even in a “routine” approach (Table 2). In particular, among the various possible determinations, white blood cell (WBC) number is crucial, helping to differentiate the “non-inflammatory” SF typical of OA, characterised by a number of WBC < 2,000/mm3, from the “inflammatory” SF of arthritis which ex-

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hibit a WBC number > 2,000/mm3 [14]. Thus, the feature of “non-inflammatory” SF was included in the classification criteria of OA of knee by the ACR [15]. However, the approach to this useful finding offered by the SF analysis should be prudential, due to number of mild inflammatory conditions possibly found in the range between 1,000 and 2,000 WBC/mm3. Thus, while a number of WBC < 1,000/mm3 indicates almost always a “non-inflammatory” SF, a number of WBC between 1,000 and 2,000 WBC/mm3 need to be interpreted according with the clinical context. The assessment of disease activity and the prevision of its outcome are essential for a rational therapeutic approach of OA. To these purposes, several molecular markers are thought to be useful, due to their characteristics of non-invasive and nonexpansive measures.

1. Markers of Inflammation Since the inflammation plays a key role in OA [16], markers of this process may be useful also in OA. They include both systemic markers, such as ESR and CRP, or local measured in SF, such as WBC number and concentrations of total proteins and various enzymes. Cytokines or their receptors, in particular interleukin (IL)-1β, IL-6, tumor necrosis factor (sTNF) and soluble receptors of TNF (sTNFR) and IL-2 (sIL-2R) may be included in this category. C-Reactive Protein In the recent years, stimulated by the recent introduction of high sensitive (hs) immunoassays, a growing number of studies has been dedicated to the role of C-reactive protein (CRP) as marker of activity or severity of OA. Conrozier et al. showed that mean hsCRP was significantly higher in the rapidly destructive than in the slowly progressive hip OA, thus suggesting that rapidly destructive hip OA may be associated with some degree of inflammation [17]. According with this study, Sharif et al. found that low level increases in CRP were associated with progression of hip OA and knee OA [18]. By examining 655 consecutive patients with OA of the knee or hip, Wolfe observed that CRP was elevated in OA compared to healthy individuals and in addition, CRP but not ESR was correlated with clinical severity in patients with OA of the knee or hip [19]. Furthermore, he found that CRP was significantly associated with functional disability, joint tenderness, pain, fatigue, global severity, and depression. In a study by Spector et al., women with knee OA have hsCRP values increased, in comparison with normal population, in a population based, cross sectional study of 845 women in Chingford [20]. Furthermore, higher hsCRP levels predict those patients whose disease will progress over 4 years, suggesting that low-grade inflammation may be a significant aspect of early OA. Stürmer et al. assessed the association between hsCRP and severity and extent of OA in patients with advanced OA, by measuring preoperative hsCRP in serum samples from 770 consecutive patients with hip or knee joint replacement [21]. They observed that hsCRP was associated with severity of pain, but not with the bilateral or the generalised extent of OA. In a study by Punzi et al., hsCRP was increased in erosive OA in comparison with nodal OA and in addition, it was correlated with disease activity, as evaluated by the number of active joints [13].


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Cytokines Although cytokines have tight relationships with CRP and an important role in cartilage metabolism, their determination in blood of OA patients is not useful as disease marker. This may be explained, at least in part, by technical difficulties, including the sensitivity of the assays. Most cytokines are undetectable in serum of non inflammatory arthropathies, with the exception of the receptors sTNFR, sIL-6R and sIL-2R, which however are always found at levels lower than inflammatory arthropathies and thus difficult to interpret [22–27]. Interestingly serum levels of sIL-2R have been found to be increased in erosive OA of the hand, thus suggesting a possible immune disregulation in affected patients [27]. Most proinflammatory cytokines, including IL-1, IL-6, IL-8, TNF and its receptors sTNFRs have been found in SF of OA, even if at low levels, much lower than in rheumatoid arthritis (RA) [23–26,28,29]. IL-1β and TNFα are highly involved in the metabolism of human cartilage [16]. At low concentrations they are thought to inhibit the synthesis of aggrecan and collagen type II; at higher concentrations, they stimulate the production of proteolytic enzymes responsible for the degradation of cartilage matrix. Thus, at low levels found in OA, IL-1 and TNF are probably differently involved in the inhibition of the synthesis and in the promotion of the degradation [16]. However, the clinical significance of their determination is doubtful also in SF. Proteolytic Enzymes In inflammation associated with OA, proteolytic enzymes are probably the mainly responsible for degradation processes seen in OA cartilage. In particular, a central role seems to be played by the metalloproteases (MMP) family, which contains the only mammalian proteinases that can specifically degrade triple-helical collagens at neutral pH [30]. The most classic collagenases MMP-1, MMP-8, and MMP-13 have differing substrate specificities for types I, II, and III collagen, with MMP-13 showing a preference for type II collagen [31]. It has been suggested that MMP-1, produced in the synovium, is the primary collagenase in RA, while MMP-13, produced by chondrocytes, is the most important collagenase in OA. However, several other members of the MMP family have been localized to cartilage or synovium in the arthropathies [32,33]. Another group of proteinases thought to be relevant in the extracellular matrix metabolism is the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family, which contains 19 members involved in collagen biosynthesis as procollagen propeptidases (ADAMTS-2, ADAMTS-3, and ADAMTS-14) [34]. Other members of this family are the so-called aggrecanases (ADAMTS-1, ADAMTS4, ADAMTS-5, ADAMTS-9, and ADAMTS-15), able to degrade the interglobular domain separating G1 and G2 of aggrecan [35,36]. Although it has been recently suggested that aggrecanases are active early in the disease process, followed by increases in MMP activity, the enzyme actually responsible for cartilage aggrecan destruction at any stage in arthritis is unclear. MMP activity is also regulated by a family of 4 specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [37], which may also inhibit ADAMTS. The local balance of MMP and TIMP activities is pivotal in regulating cartilage homeostasis, and that disturbance of this balance, resulting in an excess of MMPs over TIMPs, underlies pathologic cartilage destruction. The relative contribution of any MMP or TIMP to this balance is largely unknown. The most relevant in OA are collagenases 1 (MMP-1) and 3 (MMP-13), and stromelysine (MMP-3). Proinflam-

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matory cytokines stimulate chondrocytes to synthesise MMPs, which are usually produced in non-active forms and are subsequently activated by number of substances locally produced in the joint, including enzymes from the serine- and cysteine-dependent protease families. In particular, a strong activator is the complex plasminogen activator (PA)/plasmin [16]. PA and its inhibitor (PAI) are correlated with IL-1β in the SF of patients with OA [38]. This is not surprising, due to the influence of IL-1β on PA, and in particular on its urokinase-type receptor (uPAR) [39]. This receptor plays an important role in OA cartilage degradation by regulating pericellular proteolysis mediated by serine proteinases. By immunohistochemical analysis Schwab et al. have observed an enhanced expression of uPAR on chondrocytes derived from OA human cartilage compared to non-OA controls [40]. They found an IL-1β-mediated expression of uPAR on chondrocytes and a functional co-localization between uPAR and MMP-9 on IL-1β-stimulated chondrocytes. Due to the key role of MMPs in OA, number of studies have investigated the significance of their determination in the serum or in the SF of OA [41–43]. However, no clear information derived from these studies. One of the reasons could be that the value of MMP determination is relatively limited without the knowledge of the levels of their inhibitors TIMPs, since it has been observed that the effects of MMP mainly depend on the net molar ratio between MMP and TIMP [44]. Both in serum and SF, the most abundant MMP is MMP-3, considered as a cytokine-driven MMP, due to its strong relationships with pro-inflammatory cytokines, in particular TNFα [45]. Marini et al. have studied the amount and activity levels of MMPs and their inhibitors TIMP-1 and TIMP-2 in SF from 56 patients with different degrees of either chondral lesions or knee arthritis identified and classified by arthroscopy [46]. They observed that the degree of cartilage degradation, as seen by arthroscopy, was strictly related to the activity of MMP-2 and MMP-13 and on reduced inhibitory effect of MMP-2 by TIMP-2. In addition, a serine protease weighting about 125 kDa appears only in patients with severe cartilage degradation [46]. In another study, it has been proposed that pro-matrix MMP-3 may act as a marker for posttraumatic cartilage degradation [47]. Since the development of posttraumatic OA is a relatively slow process, the early prevision of OA changes would be crucial for the therapeutic intervention. To this purpose Bobacz et al. investigated the significance of proMMP-3 in SF and serum samples from 259 patients of trauma clinic at the time of arthroscopy [47]. Serum proMMP-3 levels of the total cohort were markedly increased compared to healthy controls. However, the grade of cartilage damage did not correlate with enzyme concentration neither in patients’ serum nor in SF samples, thus suggesting that although proMMP-3 SF concentration was increased early after trauma, its measurement in serum or SF did not reflect the cartilage damage. That of post-traumatic OA is one of the more fascinating field of application of laboratory markers, and in particular molecular markers in OA.

2. Molecular Markers Tissue joint products or molecular markers most useful in suggesting synthesis or degradation of cartilage, originate from different articular sources (Table 3), such as synovial tissue, cartilage and bone. Some derive only from synovial membrane, such as hyaluronan (HA), while some others are markers of both synovial and cartilage metabolism, such as human cartilage glycoprotein-39 (YKL-40).


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Table 3. Matrix markers in OA

SOURCE

MARKERS of SYNTHESIS

MARKERS of DEGRADATION

Serum

PINP, PICP, bone specific ALP, osteocalcin

DPD, PYD, NTX, CTX, ICTP, BSP

Synovial fluid

ND

BSP

Urine

HP

CTX, DPD, PYD, NTX

Serum

CS846, CS3B3, CS7D4, PIICP,PIIANP,YKL-40, TIMPs

CPF, KS5D4, KSAN9P1, COL23/4m, COL 2-1/4N1, CTX-II, 2B4, COMP, MMPs

Synovial fluid

CS846, CS3B3, CS7D4, PIICP, PIIANP, YKL-40, TIMPs

CPF, KS5D4, KSAN9P1, COL23/4m, COL 2-1/4N1, CTX-II, 2B4, COMP, MMPs

Urine

ND

CTX-II, HELIX-II, TIINE

Serum

PICP, PIIINP, HA, YKL-40, COMP, MMPs, TIMPs, Cytokines

PYD, Glc-Gal-PYD, CTX

Synovial fluid

PICP, PIIINP, YKL-40, COMP, MMPs, TIMPs, cytokines

PYD

Urine

ND

CTX-II, Glc-Gal-PYD, PYD

BONE

CARTILAGE

SYNOVIAL MEMBRANE

BSP: bone sialoprotein; Col2-1/4n1: type II collagen denaturation product; Col2-3/4m: type II collagen denaturation product; CS3B3: chondroitin sulphate epitope 3B3; CS7D4: chondroitin sulphate epitope 7D4; CS846: chondroitin sulphate epitope 846; CTX: C-terminal cross-linking telopeptide of type I collagen; CTX-II: C-terminal crosslinking telopeptide of type II collagen; Glc-Gal-PYD: glucosyl-galactosylpyridinoline; HA: hyaluronan; HELIX-II: type II collagen helical peptide I; HP: hydroxyproline; ICTP: carboxy-terminal telopeptide of type I collagen; KS5D4: keratan sulphate epitope 5D4; KSAN9P1: keratan sulphate epitope AN9P1; NTX: N-terminal cross-linking telopeptide of type I collagen; PICP: Procollagen I carboxyterminal propeptide; PIIANP: N-propeptide of type IIA procollagen; PIICP: procollagen type II carboxy-terminal propeptide; PIIINP: type III collagen N-propeptide; PINP: collagen I amino-terminal propeptide; TIINE: collagene type II neoepitope.

Serum HA is the most commonly used marker of synovial proliferation and hyperactivity, which may reflect OA evolution [48,49]. Bruyere et al. observed that HA levels were significantly correlated with 3-year progression in mean joint space width of the femorotibial joint in a 3-year longitudinal study of patients with knee OA [50]. In this ability, HA was similar to serum osteocalcin, but superior to other biochemical markers, such as serum keratin sulphate (KS) and cartilage oligomeric matrix protein (COMP), and urine pyridinoline (PYD) and deoxypyridinoline (DPD). These data were subsequently confirmed by Pavelka et al. [52] who studied the prognostic value of different biochemical markers for morphological progression of early knee OA, by a follow-up period of 2 years. They found that patients with higher basic serum levels of HA had a faster radiological progression. Other biochemical markers, including MMP-9, TIMP and COMP, had no statistically significant prognostic value. HA receptor CD44H and v5 and v6 were studied in SF from patients with primary OA of the knee joint with and without synovitis [53]. SF from OA with synovitis showed significantly higher levels of CD44H and v6, but not v5, than OA patients without synovial

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inflammation. However, CD 44 concentrations do not reflect the OA stage in the Kellgren grading scale. In ECHODIAH study, performed by French authors to determine whether systemic markers of bone, cartilage, and synovium can predict structural progression of hip OA, 10 markers were evaluated: N-propeptides of collagen types I and III, COMP, YKL-40, HA, MMP-1 and MMP-3, CRP, urinary C-terminal crosslinking telopeptides of collagen types I and II (uCTX-II) [54]. Combined measurements of uCTX-II and sHA were the best predictor of the structural progression of hip OA. YKL-40 is a protein with an apparent MW of 42 kDa, originally described as a major gene product of chondrocytes and synovial cells [55–58]. YKL-40 messanger RNA seems expressed in cartilage from RA or OA but not in healthy adult cartilage [59]. In SF of OA, YKL-40 was found to be increased, and correlated with disease severity [60–62]. It has been proposed that another related molecule, chitinase 3-like protein 2 (YKL-39, chondrocyte protein 39) also abundantly secreted by chondrocytes in vitro being about 4% of all secreted proteins, may be a more accurate marker of chondrocyte activation than YKL-40 [63]. Among markers assessing cartilage metabolism, the most interesting are COMP and uCTX-II. It has been suggested that COMP concentration in SF from lavage as well as in serum is an early indicator of radiographic progression at follow up [64–68]. Furthermore, COMP was the most sensitive test for identifying affected subjects with the genetic form of premature OA [69,70]. In this context, a recent study on heritable determinants of COMP was performed on 160 monozygotic and 349 dizygotic twin pairs showed that heritable factors influence serum levels of COMP [71]. Another interesting “direct” marker is AgKS, found almost exclusively in aggrecan, the main noncollagenous constituent of articular cartilage. When the aggrecan molecules are cleaved by proteolitic enzymes, AgKS fragments rapidly diffuse out of the tissues and appear in the body fluid, where they can be measured. AgKS is elevated in the serum of a high percentage of patients with polyarticular OA and is thought to precede clinical evidence of degenerative changes [72]. Furthermore, destabilisation of the knee after injury to a ligament or meniscus leads within hours to a marked increase in the SF concentrations of AgKS, aggrecan core protein and COMP, which often remains elevated for several years [73]. These changes are not usually seen in individuals who present with knee pain without evidence of damage to at least one internal supporting element. Other recent studies have compared many putative markers of OA disease activity and severity in humans [74–77]. In the Garnero’s study, a panel of biochemical markers were measured in a group of 67 patients with knee OA and in 67 healthy controls, and correlated with pain and physical function (WOMAC index) and with quantitative radiographic evaluation of the joint space using the posteroanterior view of the knees flexed at 30 degrees [76]. By univariate analyses, increased urinary glucosyl-galactosyl pyridinoline (Glc-Gal-PYD) and decreased serum osteocalcin were associated with a higher total WOMAC index. Increased urinary CTX-II and Glc-Gal-PYD, and serum type III collagen N-propeptide PIIINP were the only markers which correlated with joint surface area. By multivariate analyses, urinary Glc-Gal-PYD and CTX-II were the most important predictors of the WOMAC index and joint damage [76]. More recently, the same group has proposed the urinary type II collagen helical peptide (HELIX-II) as a new biochemical marker of cartilage degradation in patients with OA and RA [77]. They developed a specific ELISA for HELIX-II which was subsequently utilised to determine the urinary levels in patients and controls. Median urinary HELIX-II levels were increased in knee OA and early RA, compared to controls. In a study by Otterness, 14 serum and urine molecular markers were investigated for an association with


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particular clinical end-points [78]. Thus, baseline clinical assessments were correlated with urinary hydroxylysyl pyrididinoline (HP), swelling of the signal joint was correlated with serum CRP, and change in clinical assessment over the 1 year evaluation with TGFβ. The crosslinks HP, derived from bone and cartilage, and lysylpyridinoline (LP), derived from bone, were both increased in urine from OA patients, in comparison with age-matched controls [78]. Recently, new serum biochemical markers, Coll 2-1 and Coll 2-1 NO2 have been proposed for studying oxidative related type II collagen network degradation in patients with OA and RA [79]. In these disorders, both markers were found to be significantly increased compared to controls, and in RA were higher than in OA. However, no relationship was found between radiological OA severity and the levels of Coll 2-1 and Coll 1-2 NO2 in serum. Interestingly, this latter marker but not Coll 1-2, was correlated with CRP in the sera of OA and RA patients [79]. Influence of Drugs on OA Markers Laboratory findings may be proposed to monitor the therapeutic response in OA. In this context, the most useful seem markers which are better related with the degree of local inflammation or with cartilage degradation. Due to the possible influence of some drugs on the metabolic processes of many molecular markers, SF determinations seem more suitable than those in serum. The most frequently tested drugs are non steroidal anti-inflammatory drugs (NSAIDs) and intra-articularly injected drugs, in particular HA. Among NSAIDs, it has been demonstrated that etodolac reduced the SF levels of prostaglandin (PG)E2 and IL-6 while increasing those of TNFα [80]. This observation is interesting, because confirms in vivo the possibility of a modulation of TNF production by PGE2. In vitro studies have demonstrated that PGE2 may induce elevation of cAMP in mononuclear cells which in turn inhibit TNF and augment IL-6 expression [81,82]. The authors suggest that PG inhibition by etodolac could have consequently lowered cAMP levels, resulting in augmented TNFα and lower IL-6 levels [80]. Our studies on the influence of NSAIDs on SF cAMP are in contrast with such hypothesis, because NSAIDs increased cAMP levels in all SF tested [83,84]. Molecular markers seems unaffected by NSAIDs, as suggested by the observation that they are unable to modify serum level of AgKS when administered orally to patients with OA [7]. To investigate the effects of HA on inflammatory cytokines in SF of patients with knee OA, Sezgin et al. have performed a single blind, placebocontrolled, randomised study [85]. They administered intra-articular HA to 22 patients in the study group and placebo to 19 in the control group. Both HA and placebo caused a significant decrease in IL-6 levels, although more significantly in the study group, while IL-8 and TNF-alpha levels did not change in either group. The ability of HA injections to influence some aspects of SF inflammation, was confirmed by other studies, showing the SF decrease of PGE2, PGF1 alpha and F2 alpha, IL-1β, and MMPs [86–89]. Furthermore, HA seems also to act on chondrocyte metabolism, as suggested by the significant reduction in SF levels of proteoglycan monomers, and an increase in COMP concentration and osteocalcin levels [90]. Among the other intra-articular drugs, a single intra-articular injection of prednisolone markedly reduce serum and SF levels of AgKS [7], and the bisphosphonate clodronate decrease SF levels of prostaglandin E2 [91]. Another category of drugs which have recently received attention in the treatment of OA is that of the so called “chondroprotective” drugs or “structure modifying drugs

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in OA”. Among these drugs, previously known as “symptomatic slow active drugs for OA (SYSADOA)”, the most studied was the glucosamine sulphate, especially after the demonstration-still under discussion-of its ability to reduce symptoms and progression of knee OA in a long term (3 years) study [92]. The clinical effects of these drugs parallel corresponding changes in molecular markers [93]. During their clinical follow-up study for evaluate the effects of glucosamine sulphate, these authors have found that measurements of uCTX-II, enables the identification of OA patients with high cartilage turnover who at the same time are most responsive to therapy with structure modifying drugs. Concerning the possible mechanisms of action of these drugs, McCarthy et al. suggested that glucosamine therapy can improve SF HA content in OA [94].

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XVIII Cartilage Engineering a

J.F. STOLTZ a, M. LOTZ b and J. BUCKWALTER c Groupe d’Ingénierie Cellulaire et Tissulaire et thérapie, UMR CNRS 7563, Faculté de Médecine (Université Henri Poincaré), 54 500 Vandoeuvre Lès Nancy, France ([email protected]) b Scripps Research Inst, Div MEM 161, 10550 N Torrey Pines Rd, La Jolla, CA 92037 ([email protected]) c Department of Orthopaedics § Rehabilitation, University of Lowa Hospitals, 200 Hawkins Drive, Iowa City, IA 52242 ([email protected])

Abstract. The rapid development of tissue engineering today allows us to envisage the clinical use of grafts of chondrocytes, autologous stem cells, biocartilage preparations or gene therapy . However, in spite of the high stakes for orthopedic surgery as well as rhumatology or sports medecine, the answers remain unclear. Clinical research on cell therapy and preparation of biocartilage must continue to be developed in order to better determine the choice of a support matrix (scaffold) the local mechanical forces on the cells (chondrocyte, MSL...).

1. Introduction 1.1. Normal Cartilage Hyaline cartilage in adults is a non-vascularized, non-innervated and highly-specialized type of connective tissue [1]. Its principal function is to protect underlying bone from mechanical and traumatic stresses, to absorb shocks and to provide a virtually frictionless articulating surfacing [3]. It has biochemical and biophysiological properties which allow it to provide elasticity and resistance to the forces of compression. Knee cartilage, with a thickness of between 2 to 4 mm, is thus able to withstand a force of up to 5 times the body’s weight. This tissue consists of a single type of cell, the chondrocyte (approximately 1% of its volume), which is dispersed throughout an extracellular matrix base composed of water (70–80%), a network of collagen fibers (90 to 95% of which are Type II collagen fibers) surrounding a large concentration of proteoglycans (agrecans), the latter being grouped in aggregates of a very high molecular weight. Proteoglycans have particular biochemical and biophysiological properties (negatively charged and attractive of water molecules), creating a strong osmotic pressure which ensures the hydration of the cartilage and maintains the tension within the collagen network. It is the qualitative and quantitative maintenance of this three-dimensional structure that provides cartilage with its functional properties. In this manner, the chondrocytes, nourished by the syno-

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vial liquid dispersed throughout the matrix, conserve their phenotype. The regeneration of the matrix is constant but slow: 1000 days for proteoglycans and 200 years for collagen. Collagen synthesis is even slower and is not significant except in very young subjects. In adults, it appears that collagen synthesis may even decrease with age, causing collagen tissue to deteriorate faster than it is replaced. 1.2. Destruction of Cartilage Cartilage is vulnerable to trauma and diseases which can produce irreversible tissular lesions. The resulting disorganization of the collagen network and the proteoglycans modifies the chondrocytes’ capacities to resist the mechanical stresses to which they are subjected. When cartilage is damaged, its reparative capacity is weak and the lesions irreversible in the majority of cases. Deep losses of cartilage can naturally be replaced by fibrocartilaginous tissue originating from medullary stem cells, but this new tissue is not functional [6]. The evolution of cartilaginous lesions over time is not well known. Nevertheless, according to the majority of published works, the progression towards osteoarthritis seems probable, especially when the lesions are located in weight bearing regions. The prevalence and incidence of losses of cartilage are unknown and a large variety of pathologies could be involved. Cartilaginous lesions which later may require the implantation of chondrocytes are those which occur in patients with osteochondritis or which are secondary to an instability in the knee due to a ligamentary lesion or a problem with the meniscus [3–5]. In young subjects, the most frequent cause is a sportsrelated injury. Cartilaginous lesions are difficult to diagnose because there is no correlation between the symptomology and the state of the cartilage. Because the cartilage is devoid of nerve tissue, there are no early warnings signs of lesions. The symptomology includes pain, swelling, and mobility problems all of which can have a significant negative impact upon the quality of life. The arthroscopy is the standard examination to diagnose and assess the loss of cartilage; at the same time, it can allow its treatment. The use of the MRI remains more controversial. According to some, however, it allows the diagnosis of an increasing number of lesions. For others, its performance is too variable. In addition, there is currently a lack of consensus on which sequences to use.

2. Autologous Chondrocyte Grafts The loss of cartilage in the knee, a weight-bearing zone, in young subjects poses a therapeutic problem. Cartilaginous lesions, most frequently of traumatic origin, are for the most part irreversible due to the weak ability for spontaneous regeneration of cartilage and the probability of a progression of these lesions towards osteoarthritis. Autologous chondrocyte implantation is a recent therapeutic option [6,7]. This method requires an expertise in the culture of chondrocytes on the one hand and good surgical skills on the other. Recent clinical data appears to show encouraging signs of clinical improvement, but these results have not passed the test of time and have been obtained using heterogenous populations and pathologies [4,8–10]. Due to a lack of exhaustive information on undesirable effects, tolerance is difficult to assess. It is not currently possible to evaluate either the risk/benefit relationship of chondrocyte grafts

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or their place in the treatment of deep and isolated chondral lesions. In fact, this new technique is can still be considered as within the domain of clinical research. It is important to note that chondrocyte implantation is has been recognized by the FDA since 1997 as a tissue engineering approach whereas in Europe, the absence of a scaffold results in it being considered as an act of cellular therapy. The potential indications for a chondrocyte graft are: –

– – –

The patient is between 15 and a 50 years of age (maximum) taking into account the level of physical activity and presenting with a traumatic and symptomatic loss of cartilage, preferably of the femoral condyles; after period of non-surgical treatment; the size of the lesion (beyond 8 cm2 the implantation of chondrocyte tissue is not recommended); a healthy mechanical environment (ligaments, meniscus, alignment).

The strict contraindications are: all synovial and inflammatory pathologies and lesions with a diameter of less than 1 cm2.

3. Surgical Alternatives to Autologous Chondrocyte Grafts Various conservation or reparation techniques have been proposed, either by the elimination of microscopic debris (lavage) or by the formation of fibrocartilage by stimulating the stem cells of sub-chondral bone tissue. Other options include the transplantation of autologous osteochondral tissue from a healthy, non weight-bearing region or the antologous transplant of osteochondral cells from a tissue banks, however the latter presents the risk of secondary immunological reactions or the risk, even slight, of the transmission of disease (viruses, prions…).

4. Cartilage Engineering: Towards Biocartilage Currently, numerous research projects are being conducted to develop new treatments for osteochondral lesions. The most innovative ones involve the concept of tissue engineering, which consists of seeding a biomaterial with chondrocytes or progenitor cells in order to prepare a reimplantable cartilaginous matrix in vitro [11–14]. In this technique, the biomaterial must be biocompatible with the cartilage located in the surrounding lesional site. It thus serves as a scaffold for the synthesis of reparative tissue. Even so, the functionality of the newly formed tissue is determined, on the one hand, by the exogenenous supply or endogenous presence of the growth factor at the lesional site, and on the other hand, the mechanical properties of the biomaterial to resist the forces of compression imposed by the movement of the articulation. This new therapeutic direction should eventually provide solutions to such problems as the lack of donors or even the risks of viral transmission. The ideal support matrix, or scaffold, should be biocompatible, non-toxic, biofunctional, biodegradable and easy to use. The inclusion of chondrocytes cultivated in three-dimensional matrix provides an environment that will not only promote cellular differentiation but will also maintain the cells in the lesion. Different supports of synthetic, organic or even hybrid origin have been proposed. Examples of synthetic com-


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ponents include polylactic and polyglycolic acid. Supports of organic origin are composed of fibrin and of collagen fibers, with the collagen fibers being used either in the form of hydrogels or in the form of a sponge. Research has shown that collagen sponges are superior to collagen hydrogels in promoting the proliferation of cells as well as in the synthesis of collagen and proteoglycans. Finally, there is one last type of biomaterial of organic origin which can be used: polysaccaride polymers such as the hydrogels. Hydrogels are materials composed of reticulated polymers which have the particularity of being able to absorb large quantities of water. From a mechanical point of view, the hydrogels present the advantage of using water in the same way as cartilage [15]. Under the force of compression, water pushed out of the hydrogel, which allows the latter to absorb the shock. Then, once the compressive force is released, the water is returns back to its place in the hydrogel, thus allowing it to return to its initial volume. From a biological point of view, the hydrogels provide a three-dimensional environment that is sufficiently porous to allow the proliferation of cells as well as the transportation of nutrients. Among the hydrogels, those containing of sodium alginate constitute the model of reference not only in terms of studies of cellular morphology and the synthesis of collagen and proteoglycans [16], but also in terms of mechanobiology [17,18]. Although sodium alginate is not a natural component of the extracellular matrix, it has a structure similar to that of the glycosaminoglycans of cartilage. It has also been shown that this type of hydrogel ensures the maintenance of chondrocytary phenotype [19]. Biomechanic al parameters are important for the in vitro development of biocartilage as well as for the in vivo fate of implanted cellular scaffold constructs. Reaction to local forces can be conceptualised as: – –

Reaction of loads to the non living materials used (scaffold). Reaction of cells towards physical signal in the microenvironment of tissue.

Cartilage biomechanics varies within the different subsets of cartilage. The development and maintenance of cartilage structure and mechanical characteristics are closely correlated to the effect of mechanical loading. The impact of load on cartilage structure and function is of outer most importance in hyaline cartilage. The histology structure of articular cartilage is deeply influenced by the local mechanical loading of chondrocytes in the different zones, but there is limited information on the physiologies in vivo mechanical environment. Stresses in a normal joint are difficult to determine but evidence from experimental studies indicate that stresses may range from 5 to 20 MPa in animal and human joints. Loads tend to deform cartilage but compression and subsequent deformations are resisted by generation of fluid pressure and restriction of tissue deformation. In other respect intermitted loads created by the movements of joints transfer cyclic hydrostatic pressure in the interstitial fluid. Chondrocyte reactions in response to loading are genes activation that determine the remodelling and repair. Several types of cells have been described for their potential application in the engineering of cartilage tissue such as mature cells (chondrocytes) or immature cells (mesenchymal stem cells). The utilization of each type of cell has its advantages and its disadvantages due to intrinsic biological properties as well as ethical issues. For example, the utilization of autologous chondrocytes in the repair of cartilage can present the problem of their dedifferentiation during the course of their amplification ex-vivo. The necessity of harvesting these cells in a single layer in order to multiply can result in the loss of the chondrocyte phenotype to a fibroblaste phenotype. In order to avoid this phenomenon of dedifferentiation, recent studies have used cells that

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are less mature such as the mesenchymal stem cells (MSC). These cells appear capable of differentiating into numerous tissues including bones, cartilage, tendons, muscles etc. [20]. The mechanisms behind the differentiation of the MSC are poorly understood. However, several studies have demonstrated the importance of using growth factors such as TGF-β1 in single layer cultures as well as the BMP-2, 6 et 7 [21,22]. In addition, several authors have shown the beneficial effects on chondrogenesis and in the quality of the newly-formed matrix when mechanical forces are applied in vitro to mesenchymal stem cells [23–25].

5. Gene Therapy of Cartilage Defects The development of methods for gene transfer has been the object of numerous studies over the last 10 years. However, if the concept of gene therapy for the repair of articular cartilage seems appealing, recent research shows that the application of this technique remains extremely difficult and requires an optimization of the transfer of genes into the cartilage [26–29]. Indeed, the therapeutic success of gene therapy for cartilage involves the delivery and the expression of therapeutic factors at the lesional site. The determining factors are the cellular density (cellularity) of the repaired tissue and the production and maintenance of a matrix rich in Type II collagen and in proteoglycans. The ideal therapeutic agent must also be capable of inducing chondrogenesis, stimulating cellular proliferation and promoting the synthesis of the matrix. Growth factors would appear to satisfy these criteria and thus are ideal agents to be applied during gene therapy (the super family of transforming growth factor beta (TGF – β 1 and 2), bone morphogenetic protein 2 (BMP2), and the fibroblast growth factor family such as FGF-2. Another strategy concerns the applications of transcription factors such as matrix proteins and inhibitors of articular cartilage degeneration. In this case, the transcription factors modulate the expression of the genes involved in chondrogenesis. As such, experimental models have shown the chondrogenic properties of transcription factors such as S0X-9 [30] and cart-1 [31]. Finally, another alternative can be found in the induction of the inhibition of the chondrocyte catabolism. Potential targets in this case would be IL-1β , tumor necrosis factor (TNF) and IL-17 [55]. It is thus a question of inhibiting the production of matrix degradation, inflammation mediators and other mechanisms which lead to cellular death. Treatment requires the ability to deliver therapeutic agents, in the optimal concentrations, over the course of a period that is sufficiently long to allow for the induction of a chondrogenic reponse. Different target cells for gene transfer have been proposed: differentiated chondrocytes, synoviocytes and/or progenitor cells. Chondrocytes are the key target cells for gene transfer. It has thus been proposed to use adenoviral or retroviral vectors, however, their efficacy was only moderate [32]. Other transduction (vector) systems have also been proposed such as the lentivirus or the baculovirus [33]. The synoviocytes are also a target cell of interest because these cells can differentiate into chondrocytes and fill in chondral defects [34]. However, the majority of the studies involving synoviocytes have been conducted in vitro.


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Finally, the utilization of progenitor cells represents an attractive target for gene transfer. The possibility of using stem cells [35–38] and adenoviral or retroviral vectors has been well documented in vitro. It is important to mention that embryonic stem cells have also been considered. In fact, these cells differentiate into chondrocytes in the presence BMP-2 [39].

6. Conclusion The regeneration of cartilage is and will remain a challenge for the development of cell therapy, of tissue engineering and of gene therapy. However, to this day many problems remain to be solved: –

–

Technical problems regarding the definition of supports (scaffolds), cells used current gene transfer techniques and transient expression. In particular, the impact of the biomaterial used remains to be defined. Legal issues with respect to the different regulations in the USA, Europe, etc.

Cartilage engineering can be introduced via cell implantation, biocartilage transplantation or gene therapy. Complementary scientific approaches remain to be developed. Nevertheless, current knowledge permits a certain optimism for the future.

References [1] Dewire P., Einhorn T.A. The joint as an organ in: Osteoarthritis, Diagnosis and medical/surgical Management – 3th edition – ed. by Moskowitz R.W., Howell D.S., Altamn R.D., Buckwalter J.A., Golring V.M. Saunders Compagny Publ (Philadelphia, London) 2001., 49-68. [2] Sellards R.A., Nho S.J., Cole B.J. Chondral injuries. Curr Opin Rheumatol 2002., 14:134-141. [3] Jobanputra P., Parry D., Fry-Smith A., Burls A. Effectiveness of autologous chondrocyte transplantation for hyaline cartilage defects in knees: a rapid and systematic review. Health Technol. Assess 2001., (22)5. [4] Wroble R.R. Articular cartilage injury and autologous chondrocyte implantation. Which patients might benefit? Phys Sport Smed 2000., 8:43-49. [5] Corvol M.T. La thérapie cellulaire dans ses applications cliniques: thérapie cellulaire du cartilage, présent et future. J Soc Biol 2001., 195:79-782. [6] Brittberg M., Lindahl A., Nilsson A., Ohlsson C., Isaksson O., Peterson L. Treatment of deep cartilage defects in the knee with antilogous chondrocyte transplantation. N Engl J Med 1994., 331:889-895. [7] Brittberg M., Peterson L., Sjögren-Jansson E., Tallheden T., Lindahl A. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg Am 2003., 85-A (Sup 3):109-15. [8] King P.J., Bryant T., Minas T. Autologous chondrocytes implantation for chondral defects of the knee: indications and technique. J Knee Surg 2002., 15:177-84. [9] Knutsen G., Engebretsen L., Ludvigsen T.C., Drogset J.O., Grøntvedt T., Solheim E. Autologous chondrocyte implantation compared with microfracture in the knee: a randomized trial. J Bone Joint Surg Am 2004., 86-A:455-464. [10] Minas T. Antilogous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop related Res 2001., 391S:S349-61. [11] Cancedda R., Dozin B., Giannoni P., Quarto R. Tissue engineering and cell therapy of cartilage and bone. Matrix Biol. 2003., 22:81-91. [12] Fragonas E., Valente M., Pozzi-Mucelli M., Toffanin R., Rizzo R. Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate. Biomaterials, 2000., 21:795-801. [13] Corkill P.H., Fitton J.H., Tighe B.J. Towards a synthetic articular J Biomater Sci Polym ED. 1993., 4:615-630. [14] Stoltz J.F., De Isla N., Huselstein C., Bensoussan D., Muller S., Decot V. Mechanobiology and Cartilage engineering: The under lying pathophysiological phenomena. Biorheology 2006., 43:171-180.

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[15] Hauselmann H.J., Aydelotte M.B., Schumacher B.L., Kuettner K.E., Gitelis S.H., Thonar E.J. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix, 1992., 12:116-129. [16] Wong M., Siegrist M., Wang X., Hunziker E. Development of mechanically stable alginate/chondrocyte constructs: effects of glucuronic acid content and matrix synthesis. J Orthop Res, 2001., 19:493-499. [17] Ragan P.M., Chin V.I., Hung H.H., Masuda K., Thonar E.J. et al. Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system. Arch Biochem Biophys. 2000., 383:256-264. [18] Gigant-Huselstein C., Dumas D., Hubert P., Baptiste D., Dellacherie E. et al. Influence of mechanical stress on extracellular matrixes synthezed by chondrocytes seeded on to alginate and hyaluronate-based 3D biosystems. J Mechanics in Medicine and Biology, 2003., 3:59-70. [19] Miralles G., Baudoin R., Dumas D., Baptiste D., Hubert P., Stoltz J.F., Dellacherie E. Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage. J Biomed Mater Res, 2001., 57:268-278. [20] Roufosse C.A., Direkze N.C., Otto W.R., Wright N.A. Circulating mesenchymal stem cell. Int J Biochem Cell Biol., 2004., 36:585-597. [21] Sekiya I., Colter D.C., Prockop D.J. BMP-6 enchances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun. 2001., 284:411-418. [22] Schmitt B., Ringe J., Haupl T., Notter M., Manz R. et al. BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. Differentiation, 2003., 71: 567-577. [23] Angele P., Yoo J., Smith C., Mansour J., Jepsen K.J. et al. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J Orthop Res. 2003., 21:451-457. [24] J.F. Stoltz. Mechanobiology cartilage and chondrocyte 2006 vol 4-447pp (multiauthors monograph). In: serie Biomedical and Health Research vol 68. IOS Press Published (Amsterdam). [25] Stoltz J.F., Netter P., Gigant-Huselstein C. Muller S., Dellacherie E., Gillet P. Mecanobiologie et cartilage. Bull Acad Nath Med 2005., 189:1803-1816. [26] Cucchiarini M., Madry H. Gene therapy for cartilage defects. Gene med 2005., 7:1495-1509. [27] Lind M., Bünger C. Orthopaedic applications of gene therapy. International orthopaedics (SICOT) 2005., 29:205-209. [28] Evans C.H., Robbins P.D. Potential treatment of osteoarthritis by gene therapy. Rheum Dis Clin North Am 1999., 25:333-334. [29] Evans C.H., Gouze J.N., Gouze F., Robbins P.D., Ghivizzani S.C. Osteoarthritis gene therapy Gene Therapy 2004., 11: 379-389. [30] Bi W., Deng J.M., Zhang Z. Sox 9 is required for cartilage formation. Nat Genet 1999., 22:85-89. [31] Zhao G.Q., Eberspaecher H. The gene for the homeodomain containing protein cart-1 is expressed in cell that have a chondrogenic potential during embryonic development. Mech Dev 1994., 48:245-254. [32] Hirshmann F., Verhoeyen E., with D. Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein. Osteoarthritis Cartilage 2002., 10:109-118. [33] Li Y., tew S.R., Russell A.M. Transduction of passaged human articular chondrocytes with adenoviral, retroviral and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Engineering 2004., 10:643-651. [34] Hunziger E.B., Rosenberg L.C. Repair of partial – thickness defects in articular Cartilage: cell recruitment from the synovial membrane J Bone Joint Surg Am 1996., 78:721-733. [35] Calberg A.L., Pucci B., Rallapalli R. Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene Transfer of BMP-2. Differentiation 2001., 67:128-138. [36] Hiraoka K., Grogan S., Olee T., Lotz M. Mesenchymal progenitor cells in adult human articular Cartilage Biorheology 2006., 43: 447-454. [37] Campbell J.J., Lee D.A., Bader D.L. Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells. Biorheology 2006., 43:455-470. [38] Turgeman G., Pittman D.D., Muller R., Kurkalli B.G., Zhou S., Pelled G., Peyser A., Zilberman Y., Moutsatsos I.K., Gazit D. Engineered human mesenchimal stem cells: a novel platform for skeletal cell mediated gene Therapy J Gene Med 2001., 3:340-251. [39] Rosen V., Nove J., Song J.J. Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between Cartilage and bone cell phenotype J Bone Miner Res 1994., 9:1759-1768.


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XIX Therapeutics and Osteoarthritis J. BUCKWALTER, M. LOTZ and J.F. STOLTZ

This whole chapter on therapeutics in osteoarthritis involves a large field that must be subdivided according to the therapeutic class of agents and it would be quite presumptuous to summarize in a few pages the whole action of these drugs in osteoarthritis at a time where numerous publications in many top journals have cast some doubt on the tolerance of some of these drugs. We used simple methods to retrieve most of the information about therapeutics used in osteoarthritis (OA). We extracted data from MEDLINE and from the private databases of the companies marketing the main drugs used in OA. We crossed on a 1:1 comparison all the current used drugs in OA with the following items: inflammation, osteoarthritis, IL-1 beta, prostaglandins, Nitric Oxide, iNOS, cyclooxygenase-2, metalloproteinases, transcription factors and others items such as IL-6, IL-8, PPAR, NF kappa and AP-1, MAPK P38, JNK, MMP-3, MMP-9 and MMP-13, ICAM and VCAM, HSF-1 and HO. As several drugs were used concomitantly in the same study, we considered that all the data were respectively attributable to each of the drug provided the results were individually consistent. We excluded all the data concerning the relation between COX-2 and coxibs as the relevancy of this association seemed quite obvious. We attributed a quotation to each selected peer-reviewed journal including both clinical and fundamental journals. With this method, we included in our report data coming from 70 articles and we excluded 26 journals from our final report. In a first part, we will try to show that among non steroidal anti-inflammatory drugs (NSAIDs), different mechanisms of action and sometimes different outcomes for the same drug were observed. Thus, although NSAIDs have been used for many years in osteoarthritis and globally against all forms of inflammatory processes, their impact in osteoarthritis is the matter of a continuing debate which has been lately extended beyond efficacy to the safety margins of these drugs in this indication. The second part of this chapter was centered on a heterogenous group of molecules called disease-modifying drug for osteoarthritis (DMOADs), which encompasses both DMOADs with a validated mechanism of action and nutraceuticals the action of which is not clear enough to consider this latter group of molecules as fully efficient drugs with a validated mechanism of action. Among these products, many of which are found as OTC drugs, such different dosages are observed that it is barely difficult to find evidence of a therapeutic efficacy and of a scientific reproducibility.

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Non Steroidal Anti-Inflammatory Drugs (NSAIDs) NSAIDs consist of a large group of agents with different mechanisms of action and the pharmacological studies in this class present an inconsistent quality. These molecules have a demonstrated anti-prostaglandins effect. This action is however highly variable according to the given molecule and some NSAIDs: – – – –

Have several mixed mechanisms of action, Have been evaluated by studies of inconsistent quality, Have different mechanisms of action according to the species, Have for most of them no pharmacological selectivity but the COX-2.

However it seems that NSAIDs reduce PGE-2 release through various mechanisms: – – – –

Inhibition of COX-1 and COX-2, Inhibition more centered on COX-2 than on COX-1, such as meloxicam, Selective inhibition of COX-2 such as coxibs or nimesulide, Inhibition of the COX/LOX pathways such as licofelone.

They have a more inconsistent effect on the other factors involved in the inflammatory process in osteoarthritis. It seems that they do not play a role against inflammatory cytokines and some could even increase IL-1 and TNF alpha synthesis. However the outcomes of many studies are sometimes contradictory. Thus, while tiaprofenic acid exhibits an inhibiting effect of IL-1 in one study (Pelletier JP et al. 1993), it has a stimulating action on IL-1 in two other studies (Weithman KH et al. 1997, Vignon E et al. 1998). Piroxicam would have a stimulating effect on IL-1 in human (Hernvann A et al. 1996) but naproxen would induce an inhibition of IL-1 in animal models (Cicala C et al. 2000). Thus all these data do not yield a consistent conclusion. Other NSAIDs could intervene in inhibiting IL-6 production such as aceclofenac or indomethacin, and aceclofenac could stimulate IL-1Ra while piroxicam and aspirin could have a reverse action. Therefore, the global mechanism of action of NSAIDs on inflammatory cytokines is difficult to apprehend and it would be possible that according to the type of molecule, the species and the used pharmacological model, the outcomes were significantly different. NSAIDs can have an effect on the transcriptional factors, mainly NF kappa B (such as meloxicam), but there are very few studies tending to demonstrate an impact on these factors. Celecoxib would inhibit NF kappa B while stimulating AP-1, an action which could explain partly the renal side effects of the coxibs in comparison with meloxicam which inhibits NF kappa B and AP-1. NSAIDs can inhibit or not NO according to the type of molecule. Aceclofenac and aspirin decrease NO production, as well as meloxicam and celecoxib. However other studies reported contradictory results. NSAIDs would have a stimulating effect on MMP1 production (such as naproxen), but other studies report that NSAIDs would inhibit MMPs production through suppression of NF kappa B and AP-1 such as meloxicam. All these mechanisms of action still remain non conclusive. At last, PPARs have been seldom fully evaluated although nimesulide could decrease COX-2 synthesis through an effect on PPARs. In conclusion, there are many contradictory studies with NSAIDs which have a definite anti-prostaglandins action. They also have an impact on COX-1 and COX-2

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which is more or less selective according to the evaluated agent. However, it seems they tend to stimulate anti-inflammatory cytokines and metalloproteases which could explain their potential for maintaining a chronic inflammatory process and therefore a slow degradation within the joint. Table 1 summarizes the mechanisms of action of NSAIDs. Table 1. Effects of NSAID on the inflammatory chain reaction in osteoarthritis

AINS Celecoxib

ILTNF 1bêta alpha

PG

NO Cox-2¶ MMP TF Others Study Type

–

Celecoxib ++

+

+

Celecoxib +++

+

+

Celecoxib

+

Rofécoxib # Diclofenac

+

+

Diclofenac

+

Diclofenac

+ /– –

Indomethacin

+

Indomethacin

+

Indomethacin

–

Indomethacin*

+

Indomethacin

–

Indomethacin

+ –

+

Aceclofenac

+

Aceclofenac

+

Aceclofenac**

–

+ +

Meloxicam Meloxicam

+

+

Indomethacin

Aceclofenac

+

–

Diclofenac

Indomethacin

+/–

+ +

–

+

Animal in vitro Human in vitro Human in vitro Human in vitro Animal in vitro Animal in vitro Human in vitro Human in vitro Human in vitro Animal in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro Animal in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro

Authors

Patten C et al. 2004 Takahashi T et al. 2004 Takahashi T et al. 2004 Mastbergen SC et al. 2005 Niederberger E et al. 2003 Patten C et al. 2004 Sanchez C et al. 2002 Smith RL et al. 1995 Borderie D et al. 2001 Patten C et al. 2004 Sanchez C et al. 2002 Lindsey HB et al. 1990 Hernvann A et al. 1996 Massicotte F et al. 2002 Lader CS et al. 1998 Yin H et al. 2005 Herman JH et al. 1994 Mathy-Hartert M et al. 2002 Sanchez C et al. 2002 Maneiro E et al. 2001 Maneiro E et al. 2001 Blanco FJ et al. 1999 Rainsford KD H/A in vitro et al. 1997

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Table 1. (Continued.)

AINS

ILTNF 1bêta alpha

PG

NO Cox-2¶ MMP TF Others Study Type

Meloxicam

+

Meloxicam***

+

Meloxicam §

+

Meloxicam

+

+

Meloxicam

+

+

Nimesulide

+

Nimesulide

+

Nimesulide

+

+

Nimesulide ||

–

Nimesulide §§

+

Nimesulide

+

Nimesulide

+

Piroxicam

+

Piroxicam

+

Piroxicam

+

–

Piroxicam

+

Naproxen

+

Naproxen*

+

+

+

Naproxen

+

Ibuprofen

+

Tenidap

+

Tenidap Tiaprofenic acid Tiaprofenic acid Tiaprofenic acid

+

–

Piroxicam

Naproxen

+

+ + – –

– +

Animal in vitro Human in vitro Human in vitro Animal in vitro Animal in vivo Human in vitro Human in vitro Human in vitro Human in vivo Human in vitro Human in vitro Animal in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro Human in vitro Animal in vitro Human in vitro Human in vitro Human in vitro Animal in vitro Human in vitro

Authors

Yin H et al. 2005 Asano K et al. 2006 Li LC et al. 2002 Engelhardt G et al. 1996 Engelhardt G et al. 1996 Sanchez C et al. 2002 Fahmi H et al. 2001 Di Battista JA et al. 2001 Manicourt DH et al. 2005 Kalajdzic T et al. 2002 He W et al. 2002 Futaki N et al. 1994 Maneiro E et al. 2001 Lindsey HB et al. 1990 Hernvann A et al. 1996 Maneiro E et al. 2001 Herman JH et al. 1994 Hernvann A et al. 1996 Massicotte F et al. 2002 Cicala C et al. 2000 Martel-Pelletier J et al. 2004 Smith RL et al. 1995 Pelletier JP et al. 1993 Griswold DE et al. 1993 Pelletier JP et al. 1993 Weithman KH H/A in vitro et al. 1997 Human in Vignon E et al. vitro 1998

291


Table 1. (Continued.) ILTNF 1bêta alpha

AINS

PG

Ketoprofen

+

Licofelone

+

Licofenone +

+

NO Cox-2¶ MMP TF Others Study Type –

Licofenone §§§

+ +

Authors

Animal in Jarvinen TA et al. vitro 1996 Human in Martel-Pelletier J vitro et al. 2004 Animal in Lajeunesse D vivo et al. 2004 Human in Marcouiller P vitro et al. 2005

Red: stimulating effect of the study drug on the evaluated factor Blue: inhibiting effect of the study drug on the evaluated factor Green: intermediate effect of the study drug on the evaluated factor Grey: effect non evaluated in the corresponding study

+ ++ +++ * ** *** § §§ §§§ # || ¶

Diminution of IGF-1 with licofenone. Diminished production of MAPK p38 with celecoxib. Diminished production of MAPK p38 and p44/42 after induction by NO. Diminution of IL-6 with indomethacin. 46-fold increase of IL-Ra with aceclofenac and of IL-1R with tenidap. Inhibition of NF kappa B and AP-1 with meloxicam. Inhibition of NF kappa B and AP-1 with meloxicam and diminution of ICAM expression. Inhibition of PPAR agonistic stimulation on COX-2 expression and synthesis. Effect of licofen both on COX and LOX pathways. Rofecoxib induced opposite results on transcription factors; inhibiting effect on NF kappa B and stimulation of AP-1, with stimulation of iNOS. Nimesulide induced a diminution in serum levels of MMP-3 and MMP-13 opposed to ibuprofen. In some studies, the COX-2 have not been the main target of the study. Therefore they are not indicated as COX-2 inhibitors.

Disease-Modifying Drug for Osteoarthritis (DMOADs) DMOADs represent a difficult field to apprehend, as all molecules have not been subjected to the same constraints of development and if some have followed the gold standard of development for a pharmaceutical agent, the vast majority has not been properly evaluated. When they have been studied, they have been evaluated as nutraceuticals, which implies they have not gone through the long process of developing a pharmaceutical agent. Therefore the quality of the results suffers from a lack of consistency of the pharmacological studies. Some of these studies have only been recently conducted in order to justify a recent use of these products as DMOADs.

292


Chondroitin sulfate was pharmacologically evaluated only recently. The different studies, both in human and in animal models, showed an inconstant inhibitory effect on prostaglandins, NO and metalloproteases. However, this agent would only have an impact on stromelysin (MMP-3) and MMP-9. To date, the few completed studies cannot conclude definitely this agent has an inhibitory effect on any factors of the inflammatory chain in osteoarthritis. The avocado/soybean unsaponifiable mixtures have more consistent effects on prostaglandins (PGE-2) and metalloproteases (MMP-3), an irregular effect on interleukin-1, interleukin-6 and interleukin-8. However, when considered separately, these compounds have no more any positive impact. A lack of proper pharmacological studies might explain these data. As far as hyaluronic acid products are concerned, only one formulation has been able to exhibit a positive effect on some components of the inflammatory chain in osteoarthritis. However all these studies yield different results depending on what type of model has been studied, human or animal, as in man only an inconstant inhibitory effect on interleukin-6 has been shown, this specific study presenting scarce information on the animal model to be fully conclusive (Sezgin M et al. 2005). In animal models a positive effect on ICAZM-1 and VCAM-1 has been found, with again a lack of information to conclude to something else than a pure mechanical effect of these products in osteoarthritis. Glucosamine hydrochloride has an effect on prostaglandins, NO, metalloproteases (MMP-3) and transcriptional factors. These studies have been conducted both in human and in animal models. However, the suppressed metalloproteases production in one of these studies has only been obtained in normal chondrocytes without any induction of pharmacological or mechanical stress. In another study, despite the positive comments of the authors in their conclusion (Gouze JN et al. 2002), no data were given and the effect on the transcriptional factors was only observed for NF kappa B without effect on AP-1. In such experimental conditions, drawing a conclusion about glucosamine hydrochloride having any positive impact on inflammation in osteoarthritis is difficult. On the contrary, diacerhein, or its metabolite, rhein, have been fully evaluated by many pharmacological studies, consisting of different models, both in animal and in human. Its consistent and well established anti-interleukin-1 effect has been observed across species. This effect would be associated to an inhibitory effect all along the inflammatory chain in osteoarthritis. Thus it was shown that: – – – – –

diacerhein had no anti-prostaglandin effect, diacerhein was able to inhibit transcriptional factors through a potential JNK-dependent effect, in particular during AP-1 inhibition, diacerhein was able to decrease NO production in inhibiting iNOS (in a stimulated-chondrocytes model), diacerhein could reduce metalloproteases production, diacerhein would present an anti-TNF alpha effect through HSF-1 and HO-1, i.e. through the heat shock proteins pathway.

This targeted mechanism of action could explain its positive effect on the lowgrade inflammatory mechanisms as observed in osteoarthritis models. DMOADs effects are summarized in Table 2.

293


Table 2. Effects of DMOAD on the inflammatory chain reaction in osteoarthritis

Drugs

IL1bêta

TNF alpha

Chondroitin sulphate Chondroitin sulphate * Chondroitin sulphate Chondroitin sulphate ** AS § *** AS §§

PG NO Cox-2 MMP TF Others Study Type

+ +

+ +

+/–

+

+

+

Hyaluronic acid #

+ +

+

+

+

Hyaluronic acid

+

+

Hyaluronic acid

+

Hyaluronic acid

+

Hyaluronic acid ## Hyaluronic acid ||

+ +

Glucosamine |||

+

Glucosamine &

+

Glucosamine

+

+

+ +

+

+ +

Glucosamine +

+

Glucosamine ++

+

Glucosamine Diacerein

+

Diacerein

+

Diacerein

+

Diacerein

+

Diacerein

+

+

+

+

+ +

Diacerein +++ Diacerein

+ +

Diacerein Diacerein

+

+ +

+

Authors

Animal in Neil KM et al. vitro 2005 Human in Monfort J et al. vitro 2005 Human in Chan PS et al. vitro 2005 Animal in Chou MM et al. vivo 2005 Human in Henrotin YE et al. vitro 1998 Human in Henrotin Y et al. vitro 2003 Animal in Greenberg DD vitro et al. 2006 Animal in Monfort J et al. vitro 2005 Animal in Diaz-Gallego L vivo et al. 2005 Human in Fioravanti A et al. vitro 2005 Human in Karatay S et al. vitro 2004 Human in Sezgin M et al. vitro 2005 Human in Nakamura H et al. vitro 2004 Human in Largo R et al. vitro 2003 Animal in Fenton JI et al. vitro 2002 Animal in Gouze JN et al. vitro 2002 Animal in Gouze JN et al. vitro 2001 Animal in Fenton JI et al. vitro 2000 Human in Martel-Pelletier J vitro et al. 1997 Human in Martel-Pelletier J vitro et al. 1998 GigantHuman in Huselstein C et al. vitro 2002 Animal in Martin G et al. vitro 2004 Human in Martel-Pelletier J vitro et al. 1997 Animal in Lin S et al. vitro 2003 Animal in Mendes AF et al. vitro 2002 Animal in Tamura T et al. vitro 2001 Animal in Tamura T, vitro Ohmori K 2001

294


Table 2. (Continued.) IL1bêta

Drugs

TNF alpha

PG NO Cox-2 MMP TF Others Study Type

Diacerein

+

+

Diacerein ++++

+

+

Diacerein // Diacerein

Human in vitro

+

+

+ +

Authors

+

Yaron M et al. 1999 Stoltz JF et al. A/H in vitro 2006 (in press) Human in Mistry D et al. vitro 2006 (in press) Human in Sanchez C et al. vitro 2003

Red: stimulating effect of the study drug on the evaluated factor Blue: inhibiting effect of the study drug on the evaluated factor Green: intermediate effect of the study drug on the evaluated factor Grey: effect non evaluated in the corresponding study

* ** § ***

Inhibitory effect of CS on MMP-3 (stromelysin). Inhibitory effect of CS on MMP-9. AS means avocado/soybean unsaponifiable mixtures. AS: strong inhibitory effect of AS on MMP-3, IL-6, ILM-8 and PGE-2; however each isolated compound of this mixture had no effect. Partial effect on IL-1. §§ This study showed an effect on nearly any element of the inflammatory chain in osteoarthritis. No confirmation was obtained in any other study. # Only one formulation of hyaluronic acid exhibited an effect (Hyalgan). ## Inhibitory effect on ICAZM-1 and VCAM-1. || Inhibitory effect on IL-6; however, lack of information on this model. ||| Of note, MMPs production was only suppressed in normal chondrocytes. & No numbers were indicated in this study. + Effect only on NF kappa B. No effect on AP-1. ++ Inhibitory effect on MMP-3. +++ Inhibitory JNK-dependent effect of rein on AP-1. ++++ Inhibitory effect of diacerein on iNOS production in stimulated chondrocytes. // Inhibitory HSF-1 via HO-1 induced effect of diacerein on TNF alpha.

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299

Author Index Aigner, T. Berenbaum, F. Bianchi, A. Blanco, F.J. Buckwalter, J. Ding, L. Dumas, D. Gabay, O. Galteau, M.-M. Goldring, M.B. Gómez, R. Gómez-Reino, J.J. Gosset, M. Gualillo, O. Guo, D. Homandberg, G.A. Isla, N.D. Jimenez, S.A. Jouzeau, J.-Y. Kirchmeyer, M. Lago, F. Lago, R. Lajeunesse, D.

219 v, 163 77 192 280, 287 56 254 163 77 118 43 43 163 43 56 56 254 143 77 77 43 43 206

López-Armada, M.J. 192 Lotz, M. 182, 280, 287 Malemud, C.J. 99 Martel-Pelletier, J. 3, 206 Martin, J.A. 239 Oliviero, F. 267 Otero, M. 43 Pedersen, D.R. 239 Pelletier, J.-P. 3, 206 Piera-Velazquez, S. 143 Punzi, L. 267 Rego, I. 192 Riquelme, B. 254 Sandell, L.J. 118 Sfriso, P. 267 Smith, R.L. 14 Stoltz, J.F. vii, 254, 280, 287 Terkeltaub, R.A. 31 Thedens, D.R. 239 van den Berg, W. 219 van der Kraan, P. 219 Werkmeister, E. 254




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