Identification of important interactions between subchondral bone ...

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CHAPTER 7: PAPER IV Aggrecan degradation in normal, cytokine-induced, and OA cartilage In both bovine and human OA cartilage, the endogenous proteinases activated by MMP-buffer, degraded the aggrecan in to fragments that were able to be detected by the biomarker, AGNx-II. The MMP inhibitor, GM6001, was able to attenuate the levels of AGNx-II in both bovine and human OA cartilage (Table 1). The bovine cartilage pre-cultured with cytokines for 4 and 11 days before metabolic inactivation and incubation in MMP buffer, increased the levels of AGNx- II compared to normal bovine cartilage (Table 1). Furthermore, the human OA cartilage had greatly higher levels of AGNx-II than the bovine samples (Table 1). The cysteine protease inhibitor, E64, did not affect the normal or pre-cultured bovine samples, whereas E64 increased the levels significantly in the human OA samples (Table 1). The proteinases activated by the cysteine buffer in both bovine and human cartilage, also degraded the aggrecan in to fragments that were able to be detected by the biomarker, AGNx-II. 96
CHAPTER 7: PAPER IV The MMP inhibitor, GM6001, was able to attenuate the levels of AGNx-II in normal bovine cartilage, whereas the pre-cultured bovine cartilage was not affected by the inhibitor (Table 2). The bovine cartilage pre-cultured with cytokines for 4 and 11 days before metabolic inactivation and incubation with cysteine buffer, decreased the levels of AGNx-II compared to normal bovine cartilage (Table 2), which was the opposite effects from the one seen in the MMP buffer. Furthermore, the human OA cartilage had levels of AGNx-II as the normal bovine cartilage (Table 2). The cysteine protease inhibitor, E64, did not affect the normal or pre-cultured bovine samples, whereas E64 increased the levels significantly in the human OA samples (Table 2). Discussion The aim of our study was to understand the molecular foundation for the progression of articular cartilage degradation that leads to OA. We analyzed the release of cartilage degradation biomarkers from human OA cartilage by the proteinases reported to play a role in cartilage degradation 5,10 . Furthermore, we investigated how diseased cartilage is altered from normal cartilage with regards to the endogenous proteases, and we were observant to possible masking effects among the proteases. We showed that the cartilage degradation markers, CTX-II, NBC2, and AGNx-II, are released in part by different proteolytic pathways in human OA cartilage, as different proteases generated different biomarkers (fig. 1). The biomarkers were only released by specific metalloproteinases, and none of the supplied cathepsins (fig. 1). Moreover, Cathepsin-B (and a tendency for Cathepsin-K and Cathepsin-L) inhibited the release of CTX-II in cysteine buffer, suggesting either a protective effect by the cathepsins on cartilage degradation or more likely a masking effect (fig. 2) from post-cleavage of CTX-II fragments by the cathepsins. The aggrecan degradation increased in the presence of both MMPs and aggrecanases, although AGNx-II is MMP-specific (fig. 1G), suggesting pre- or post-cleavages by the ADAMTS’ may aid the degradation. In human OA cartilage explants, the collagen type II and aggrecan may already be severely degraded by the increased production of proteases during the disease 10 , so further protease-cleavage ex vivo may be difficult to detect, as specific targets measurable by our biomarkers have already been cleaved and further processed, thereby masking the effect from the MMPs and cysteine proteases we tested. This possible masking effect could be investigated by a follow up study, testing the library of proteases in normal cartilage and comparing the results with those from the present study in human OA cartilage. The endogenous proteases in human OA samples activated in the MMP buffer (Table 1) failed to increase the levels of CTX-II and NBC2. One suggestion could be that the damaged OA cartilage had less chondrocytes and ECM proteins, and thus fewer enzymes present, compared to normal cartilage. This is supported by the increased levels of CTX-II and NBC2, when supplying MMP-7 and MMP-9 (fig.1). However, as we were able to decrease MMP- 97
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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
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97 Nakamura H., Tanaka M., Masuko-H
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139 Garnero P., Ayral X., Rousseau
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CHAPTER 3 Overview of OA models CHA
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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]
Page 73 and 74: after 1 week (Fig. 2F). The additio
Page 75 and 76: use a range of assays ranging from
Page 77 and 78: still some controversy regarding th
Page 79 and 80: CHAPTER 6 CHAPTER 6: PAPER III PAPE
Page 81 and 82: 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: CHAPTER 7: PAPER IV Detection of ke
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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