Mevastatin reduces cartilage degradation in rabbit experimental osteoarthritis through inhibition of synovial inflammation Y. Akasaki, M.D., S. Matsuda,

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Mevastatin reduces cartilage degradation in rabbit experimental osteoarthritis through inhibition of synovial inflammation Y. Akasaki, M.D., S. Matsuda, M.D., Ph.D., K. Nakayama, M.D., Ph.D., S. Fukagawa, M.D., H. Miura, M.D, Ph.D., Y. Iwamoto, M.D., Ph.D. Osteoarthritis and Cartilage Volume 17, Issue 2, Pages 235-243 (February 2009) DOI: 10.1016/j.joca.2008.06.012 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Effects of mevastatin on gene expressions of MCP-1 and MMP-3 in chondrocytes and synoviocytes. Messenger RNA expressions of MCP-1 and MMP-3 in chondrocytes and synoviocytes were stimulated by IL-1β. Incubation with mevastatin at three different concentrations did not affect gene expression of chondrocytes in the presence of IL-1β (A). However, Incubation with 10μM and 50μM mevastatin inhibited IL-1β stimulation of MMP-3 gene expression in synoviocytes (B). MCP-1 gene expression with 50μM mevastatin was also not stimulated by IL-1β. Each RT-PCR was performed in duplicate and yielded almost identical results. The data was a representative result from three independent experiments. Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effects of mevastatin on the productions of MCP-1 and MMP-3 in chondrocytes and synoviocytes. MCP-1 and MMP-3 productions in chondrocytes and synoviocytes were stimulated by IL-1β. Mevastatin at 50μM slightly reduced the IL-1β-induced productions of MCP-1 and MMP-3 from chondrocytes, although not significantly. In contrast, incubation with 50μM mevastatin significantly inhibited IL-1β stimulation of MCP-1 and MMP-3 productions (n=5). Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effects of mevastatin on cell viability in chondrocytes and synoviocytes. Incubation with mevastatin at three different concentrations for 24h did not affect cell viability in both chondrocytes and synoviocytes (n=5). Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Histological findings of articular cartilage in an untreated knee and a mevastatin (0.1mg/ml)-treated knee. Safranin-O staining (A and B) and TUNEL staining (C and D) were performed. The center of the cartilage lesion on the femoral condyle in an untreated knee and mevastatin treated knee are shown in (A and C) and (B and D), respectively. (Original magnification 200×). Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemical findings in synovial tissue from a control knee (A and C) and a mevastatin (0.1mg/ml)-treated knee (B and D). Sections were immunostained with an antibody against CD68. Round shape cells were CD68 positive, whereas fibrous cells were not. (Original magnification 100× in A and B; 400× in C and D). E: The mean numbers of positively staining cells per field (n=8). Values are the means ±SD (*P<0.05). Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Comparative mRNA expression in control knees and mevastatin (0.1mg/ml)-treated knees. There was no significant difference in gene expression between mevastatin treated and control cartilage (A). In contrast, messenger RNA levels of MCP-1, IL-1β, MMP-3, and MMP-13 were significantly decreased in the mevastatin treated synovial tissue compared to the control synovial tissue (B) (n=8). Values are the means ±SD (*P<0.05). Osteoarthritis and Cartilage 2009 17, 235-243DOI: (10.1016/j.joca.2008.06.012) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions