Identification of important interactions between subchondral bone ...

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Biomarkers Downloaded from informahealthcare.com by Panum Biblioteket on 08/31/10 For personal use only. Biomarkers, 2010; 15(3): 266–276 original article Aggrecanase- and matrix metalloproteinase- mediated aggrecan degradation is associated with different molecular characteristics of aggrecan and separated in time ex vivo Suzi Hoegh Madsen, Eren Ufuk Sumer, Anne-Christine Bay-Jensen, Bodil-Cecilie Sondergaard, Per Qvist, and Morten Asser Karsdal Nordic Bioscience A/S, Herlev, Denmark Introduction abstract Aggrecan is one of the first proteins to be depleted from articular cartilage in early osteoarthritis. We investigated the molecular differences between matrix metalloproteinase (MMP)- and aggrecanase-mediated aggrecan degradation, as a consequence of their distinct time-dependent degradation profiles. Cartilage degradation was induced by cytokine stimulation in bovine articular cartilage explants and quantified by a dye-binding assay and immunoassays. The size of degradation fragments was analysed by Western blot. Cytokine stimulation resulted in the early release of aggrecanase-mediated aggrecan degradation fragments. In contrast, MMP-mediated aggrecan degradation began only at day 16 and continued to day 21. Western blot analysis showed that glycosylated high-molecular-weight 374 ARGSVI fragments appeared at day 7, in contrast to deglycosylated low-molecular-weight 342 FFGVG fragments which were detected at day 21. Aggrecan degradation may be divided into two different pools, a high-molecular-weight aggrecanasemediated pool, and a low-molecular-weight MMP-mediated pool. This may have implications for the development of intervention strategies for OA. Keywords: Proteoglycans; cartilage; osteoarthritis; ELISA; biomarkers; aggrecanases; MMPs Cartilage is normally maintained by a balance between catabolic and anabolic processes. However, in joint degenerative diseases, such as osteoarthritis (OA), the rate of cartilage degradation exceeds the rate of formation, resulting in a net loss of cartilage matrix (Behrens et al. 1989, Felson & Neogi 2004). The extracellular matrix (ECM) consists mainly of collagen type II, which forms a fibrous network that entraps proteoglycans of which aggrecan is the most predominant (Hardingham & Fosang 1995, Kiani et al. 2002). Aggrecan is one of the ECM proteins to be degraded in OA (Caterson et al. 2000), primarily by ADAMTSs (a disintegrin and metalloproteinase domain with thrombospondin motifs) (Glasson et al. 2005, Struglics et al. 2006) and matrix metalloproteinases (MMPs) (Fosang et al. 1996, Lark et al. 1997, Struglics et al. 2006). Aggrecanase activity leads to the release of glycosaminoglycans (GAGs), although it is not known if MMPs also play a role in aggrecan GAG loss (Sandy 2006, Struglics et al. 2006). As a consequence of aggrecan loss, the cartilage exhibits altered matrix properties such as loss of fixed negatively charged density of the tissue and thereby lower water content and elasticity (Roughley & Lee 1994). A deeper understanding of the molecular mechanisms involved in proteoglycan depletion may aid the development of efficient diseasemodifying osteoarthritic drugs (DMOADs). The core protein of aggrecan has three globular domains: G1, G2 and G3. G1 and G2 are separated by Address for Correspondence: Suzi Hoegh Madsen, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. Tel: + 45 44 52 52 52. Fax: + 45 44 52 52 51. E-mail: shm@nordicbioscience.com (Received 03 August 2009; revised 01 December 2009; accepted 01 December 2009) ISSN 1354-750X print/ISSN 1366-5804 online © 2010 Informa UK Ltd DOI: 10.3109/13547500903521810 http://www.informahealthcare.com/bmk 78
Biomarkers Downloaded from informahealthcare.com by Panum Biblioteket on 08/31/10 For personal use only. the interglobular domain (IGD), which is highly sensitive to proteolysis (Flannery et al. 1992, Hardingham & Fosang 1995). G2 and G3 are heavily saturated with highly negatively charged GAGs (Flannery et al. 1992, Hardingham & Fosang 1995) (Figure 1). The G2 domain is unique to aggrecan, and sandwich enzyme-linked immunosorbent assays (ELISAs) of aggrecan degradation have been developed against peptides with the G2 and the neoepitopes 342 FFGVG or 374 ARGSV (Karsdal et al. 2007, 2008) generated by MMPs (Flannery et al. 1992) and ADAMTS (Tortorella et al. 1999), respectively. The ADAMTS-specific 374 ARGSV site is located between the 342 FFGVG site and the G2 domain. The 342 FFGVG-G2 fragments assayed in the present study represent only MMP-mediated degradation, which has not already been processed by ADAMTSs. The 374 ARGSV-G2 represents fragments, which theoretically may have been pre-processed by MMPs at the 342 FFGVG site (Figure 1), although the available evidence suggests that this is very unlikely (Mercuri et al. 2000). Studies with ex vivo cultures have shown that cytokinestimulated cartilage releases collagen and proteoglycan fragments to the conditioned medium (Sondergaard et al. 2006a). These fragments can be detected by novel ELISA assays, which measure neoepitopes cleaved by specific proteases. These degradation patterns resemble the molecular mechanisms seen in the pathogenesis of OA (Caterson et al. 2000). Our group has previously shown that cytokine-stimulated cartilage releases products with specific neoepitopes from collagen type II and aggrecan (Karsdal et al. 2007, 2008, Rousseau et al. 2008, G1 MMP IPEN 341 342 FFGV AF-28 ADAMTS Aggrecanase- and MMP-mediated aggrecan degradation 267 IGD sGAG TEGE 373 374 ARGS BC-3 Sondergaard 2006a). Furthermore, we also showed that MMP-2 and -9 activity increased over time in cytokinestimulated cartilage explants (Sondergaard 2006a), which is important when investigating the difference between aggrecanase- and MMP-mediated degradation of aggrecan. Different research groups have suggested that articular cartilage aggrecan may be present in the tissue in two or more metabolic pools (Ilic et al. 1995, Lark et al. 1997, Lohmander et al. 1973, Mok et al. 1994, Struglics et al. 2006). However, currently, the temporal and spatial molecular degradation of aggrecan has not been clarified, and various contradictory theories and models have been proposed by different investigators (Fosang et al. 2000, Lark et al. 1997, Sandy 2006, Struglics et al. 2006). It has been proposed by Fosang et al. (2000) that aggrecan degradation by ADAMTSs and MMPs occurs in independent locations and with different kinetics. More recently Struglics et al. (2006) analysed human synovial fluids and concluded that ADAMTSs and MMPs act sequentially on the same substrate molecule. A review of the area by Sandy (2006) attempted to clarify these controversies. On the basis of available evidence, Sandy (2006) concluded that ADAMTSs and MMPs degrade different aggrecan structures. Specifically it was suggested that ADAMTSs act primarily on full-length or G1-G2-chondroitin sulfate1 aggrecan by a sequential process which removes the chondroitin sulfate-bearing domains and lastly at the Glu 373-374 Ala bond in the IGD. In contrast it was proposed that MMPs degrade only the calpain (calcium-dependent cysteine protease) product G2 G3 F-78-HRP F-78-HRP Figure 1. Protease-mediated neoepitopes in aggrecan. Aggrecan molecule with three globular domains G1, G2 and G3. The interglobular domain (IGD) is highly sensitive to proteolysis. Beneath the aggrecan molecule are the 342 FFGVG-G2 and 374 ARGSV-G2 fragments mediated by matrix metalloproteinase (MMP) and ADAMTS, respectively. The antibodies BC-3 and AF-28 recognize the neoepitopes and F-78 is the detection antibody binding to the G2 domain. The domain between G2 and G3 is heavily saturated with highly negatively charged glycosaminoglycans (GAGs). 79
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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
Page 29 and 30: CHAPTER 2: Introduction number of a
Page 31 and 32: CHAPTER 2: Introduction Cysteine pr
Page 33 and 34: CHAPTER 2: Introduction include car
Page 35 and 36: 2.4 The effect of Glucocorticoids C
Page 37 and 38: 2.5 References for CHAPTER 2 1 WebM
Page 39 and 40: 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 83 and 84: Biomarkers Downloaded from informah
Page 91 and 92: CHAPTER 7 CHAPTER 7: PAPER IV PAPER
Page 93 and 94: Introduction CHAPTER 7: PAPER IV Ca
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
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Page 115: Biochemical Markers of Joint Tissue
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