Identification of important interactions between subchondral bone ...

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CHAPTER 7: PAPER IV Metalloproteinase and cysteine protease profiling in normal and Osteoarthritic cartilage Suzi Hoegh Madsen 1 , Kim Henriksen 3 , Søren Tvorup Christensen 2 , Morten Asser Karsdal 1 , and Anne-Christine Bay-Jensen 1 In review in Proteome Science 1 Cartilage Biology and Biomarkers R&D, Nordic Bioscience A/S, Herlev, Denmark 2 Department of Molecular Biology, University of Copenhagen, Copenhagen, Denmark 3 Bone Biology R&D, Nordic Bioscience A/S, Herlev, Denmark ___________________________________________________________________________ Abstract Treatment for cartilage degenerating diseases is either analgesic or joint replacement. Thus, the discovery and development of new treatments are needed. Proteases, such as metalloproteinases and cathepsins, play a major role in the net degradation of cartilage, and are therefore a natural target for drug development. By testing the ability of a library of proteases to degrade cartilage explants, and by inhibiting one type of protease, we aim to find the proteases responsible for collagen type II and aggrecan degradation and possible masking effects by other proteases. The cysteine inhibitor, E64, and the proteases; MMP-3 and -7, and ADAMTS-4 and -5, all increased aggrecan degradation (AGNx-II), in human metabolic inactivated osteoarthritic (OA) cartilage. The MMP inhibitor, GM6001, abrogated all the increased aggrecan degradation. Collagen type II was degraded with MMP-7 (CTX-II) and MMP-9 (NBC2), which both could be inhibited by GM6001. Human OA cartilage cultured in buffer optimal for cysteine proteases showed an increase in levels of CTX-II and NBC2 fragments when incubated with GM6001. Furthermore, we observed altered levels of the biomarkers between normal and pre- cytokine-stimulated bovine cartilage, indicating an induced alteration of the endogenous proteases. In human OA cartilage, the degradation markers are released in part by different proteolytic pathways. Inhibition of cysteine proteases unmasks the levels of MMP-mediated aggrecan degradation in OA cartilage. Inhibition of MMPs also unmasks the levels of the collagen type II degradation mediated by non-MMPs. The role of endogenous proteases probably depends on the stage of OA, as we did not observe the same release of biomarkers in normal versus cytokine-stimulated and OA cartilage. 90
Introduction CHAPTER 7: PAPER IV Cartilage turnover is a complex process in which proteases play a prominent role in both health and disease. The extracellular matrix (ECM) in healthy articular cartilage is expressed and regulated by the chondrocytes, which include expression of matrix proteins, proteases, and protease inhibitors 1 . One of the major mediators of cartilage destruction in osteoarthritis (OA) is the metalloproteinases; matrix metalloproteinases (MMPs) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS’) 2 . In few words, the destruction of ECM in OA results from initial aggrecan degradation by ADAMTS’ and subsequently degradation by diverse MMPs that continues to degrade the major components of the ECM, e.g. collagen type II and aggrecan 2,3,4 . However, cysteine proteases are also essential players in the proteolytic cleavage of ECM in OA, which are synthesized both by the chondrocytes and synovial cells in response to cytokines and growth factors 5 . Currently, treatment of cartilage degenerative diseases is either analgesic or joint replacement. Thus, the discovery and development of new treatments are needed. Since proteases - such as MMPs and cathepsins - play a major role in the net degradation of cartilage, they are a natural target for drug development. Early treatment with protease inhibitors to either reduce inflammation and/or reduce aggrecan/collagen loss may have the potential to reduce the onset of future idiopathic or posttraumatic osteoarthritis. However, to target the right protease at the right time during the progression of OA, a better understanding of the proteolytic processes is needed. ECM-neoepitope biomarkers are great tools for investigating the proteolytic processes 6 . The collagen type II degradation marker, C-terminal telopeptide degradation fragment (CTX-II) 7 and the aggrecan degradation marker, AGNx-II 8 , are both MMP-specific. The novel collagen type II intra-helical degradation markers, NBC2, are supposed to represent cathepsin-specificity. However, the biomarker will be evaluated in the present study. In this study, we identified which MMPs, ADAMTS’ and cathepsins were able to generate CTX-II, NBC2, and AGNx-II fragments using an in vitro and ex vivo model of human OA cartilage. Such data could provide information on the specificity of the different proteases for cleavage of collagen and aggrecan, and also inform us about the proteolytic pathways for each biomarker release. Furthermore, the endogenous proteases in normal bovine cartilage were compared with cytokine-stimulated bovine and human OA cartilage. The cytokines, oncostatin M (OSM) and tumor necrosis factor-α (TNF-α), are inducers of cartilage degradation in vivo and in vitro 9 , and would allow us to induce a controlled catabolic system towards the one seen in human OA cartilage. I.e. induction of aggrecanases after 4 days with cytokine stimulation and induction of MMPs after 11 days with cytokine stimulation 3 . By inhibiting one type of protease, we aimed to find the proteases responsible for collagen type II and aggrecan degradation and possible masking effects by other proteases. 91
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F A C U L T Y O F S C I E N C E U N
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Supervisors University of Copenhage
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Preface Preface The work presented
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Contents Contents Abstract ........
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Abstract Abstract Osteoarthritis (O
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Sammendrag på dansk Sammendrag på
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CHAPTER 1 Objectives CHAPTER 1: Obj
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CHAPTER 2 Introduction CHAPTER 2: I
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2.2 Biology of the joint CHAPTER 2:
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CHAPTER 2: Introduction Also osteob
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CHAPTER 2: Introduction follows nor
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CHAPTER 2: Introduction When the jo
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CHAPTER 2: Introduction Cartilage i
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2.3 The pathology of Osteoarthritis
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CHAPTER 2: Introduction number of a
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CHAPTER 2: Introduction Cysteine pr
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CHAPTER 2: Introduction include car
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2.4 The effect of Glucocorticoids C
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2.5 References for CHAPTER 2 1 WebM
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51 Lorenz H. and Richter W. Osteoar
Page 41 and 42: 97 Nakamura H., Tanaka M., Masuko-H
Page 43 and 44: 139 Garnero P., Ayral X., Rousseau
Page 45 and 46: CHAPTER 3 Overview of OA models CHA
Page 47 and 48: CHAPTER 3: Overview of OA models of
Page 49 and 50: 3.3 Ex vivo controls CHAPTER 3: Ove
Page 51 and 52: 19 Brandt K.D. Animal models of ost
Page 53 and 54: CHAPTER 4 CHAPTER 4: PAPER I PAPER
Page 55 and 56: 266 Cartilage 2(3) therapy for oste
Page 57 and 58: 268 Cartilage 2(3) Figure 1. Charac
Page 59 and 60: 270 Cartilage 2(3) Figure 4. The ca
Page 61 and 62: 272 Cartilage 2(3) Figure 5. Cytoki
Page 63 and 64: 274 Cartilage 2(3) Figure 6. The an
Page 65 and 66: 276 Cartilage 2(3) supports the inc
Page 67 and 68: 278 Cartilage 2(3) release and rece
Page 69 and 70: CHAPTER 5 CHAPTER 5: PAPER II PAPER
Page 71 and 72: leading to fragility fractures [12]
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Page 79 and 80: CHAPTER 6 CHAPTER 6: PAPER III PAPE
Page 81 and 82: Biomarkers Downloaded from informah
Page 91: CHAPTER 7 CHAPTER 7: PAPER IV PAPER
Page 95 and 96: Bovine articular cartilage experime
Page 97 and 98: CHAPTER 7: PAPER IV Detection of ke
Page 99 and 100: CHAPTER 7: PAPER IV The MMP inhibit
Page 101 and 102: CHAPTER 7: PAPER IV The pre-stimula
Page 103 and 104: CHAPTER 8 CHAPTER 8: General discus
Page 105 and 106: Future perspectives CHAPTER 8: Futu
Page 107 and 108: Appendix I Co-author declarations f
Page 109 and 110: Appendix I 107
Page 115: Biochemical Markers of Joint Tissue
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