Identification of important interactions between subchondral bone ...

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AGNx-II ng/mL CTX-II ng/mL NBC2 ng/mL 2.4 1.8 1.2 0.6 Vehicle MMP1 MMP3 MMP buffer ** MMP7 MMP9 MMP13 CHAPTER 7: PAPER IV A B C E 15 10 5 12000 9000 6000 3000 Vehicle Vehicle MMP1 MMP1 MMP3 MMP buffer *** MMP3 MMP7 * * MMP9 MMP13 MMP buffer * MMP7 MMP9 ADAMTS4 ADAMTS4 MMP13 ADAMTS5 ADAMTS5 * ADAMTS4 94 * ADAMTS5 NBC2 ng/mL CTX-II ng/mL 1.6 1.2 0.8 0.4 200 150 100 50 Vehicle Vehicle Vehicle Cysteine buffer CatK * CatB CatL Cysteine buffer CatK CatB CatL Cysteine buffer Fig. 1. Collagen type II and aggrecan degradation are mediated by different pathways. Human OA cartilage explants were incubated in MMP and cysteine buffer in the presence or absence of different metalloproteinases [100 ng/ml] and cysteine proteases [100 ng/ml] (Cat = cathepsin). Biomarkers for collagen type II degradation (CTX-II and NBC2) and aggrecan degradation (AGNx-II) were measured after 3, 6 and 9h. Data are shown as accumulated for the three measurements. N=4. AGNx-II ng/ml D F 400 300 200 100 CatK CatB CatL CatS CatS CatS
CHAPTER 7: PAPER IV Detection of key proteases to degrade human OA cartilage ex vivo; evaluated by an aggrecan degradation marker In human metabolic inactivated OA cartilage incubated in MMP buffer, MMP-3 and -7, and ADAMTS-4 and -5 all increased aggrecan degradation (AGNx-II) compared to non-stimulated vehicle (fig. 1E). Addition of the MMP inhibitor, GM6001, to the proteases attenuated this release of the aggrecan fragments (data not shown). We did not observe any changes in the release of AGNx-II fragments by MMP-1, -9 and -13, compared to vehicle (fig. 1E). The cathepsins did not affect the levels of AGNx-II significantly; however, we observed a tendency towards a decrease in the levels of AGNx-II compared to vehicle (fig. 1F). Collagen type II degradation in normal, cytokine-induced, and OA cartilage In normal bovine cartilage, the endogenous proteinases activated by the MMP-buffer, degraded the collagen type II in to fragments that were able to be detected by the biomarker, CTX-II. The MMP inhibitor, GM6001, was able to attenuate CTX-II (Table 1). The bovine cartilage pre- cultured with cytokines for 4 and 11 days before metabolic inactivated and incubated in MMP buffer, increased the levels of CTX-II compared to normal bovine cartilage, which were also attenuated in the presence of GM6001. However, the human OA cartilage did not generate CTX- II fragments as in the bovine samples. The cysteine protease inhibitor, E64, did not affect the CTX-II levels. The biomarker, NBC2, was not detectable in the MMP buffer (Table 1). The proteinases activated by the cysteine buffer in both bovine and human cartilage, generated fragments that could be detected by both biomarkers of collagen type II degradation; CTX-II and NBC2 (Table 2). The MMP inhibitor, GM6001 showed a tendency to decrease CTX-II levels (P=0.057) in normal bovine cartilage, whereas the same inhibitor showed a tendency to increase CTX-II levels in pre-cultured bovine cartilage (11 days) and in human OA cartilage (Table 2). Additionally, GM6001 increased NBC2 in human OA and bovine normal and pre-cultured cartilage. The cysteine protease inhibitor, E64, decreased the levels of CTX-II in human OA and bovine normal and pre-cultured (11 days) cartilage. E64 also decreased the levels of NBC2 in normal bovine cartilage, whereas the cysteine protease inhibitor increased the levels of NBC2 in bovine cartilage pre-cultured with cytokines for 4 days (Table 2). The bovine cartilage pre-cultured with cytokines for 4 and 11 days before metabolic inactivated and incubated in cysteine buffer, decreased the levels of CTX-II compared to normal bovine cartilage (Table 2), which was the opposite effects from the one seen in MMP buffer (Table 1). Furthermore, the human OA cartilage had CTX-II levels corresponding to the cartilage pre- cultured with cytokines for 11 days (Table 2). Moreover, the levels of NBC2 were also lower for the pre-cultured cartilage (11 days) compared with the normal cartilage. However, the human OA cartilage had greatly higher levels of NBC2 compared to bovine cartilage in general (Table 2). 95
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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
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]
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: Bovine articular cartilage experime
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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