Objective-Oxidative stress is proposed as an important factor in osteoarthritis (OA). We therefore investigated the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA.Methods-SOD expression was determined by real-time polymerase chain reaction and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulfite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX™ and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression.Results-All three SOD were abundantly expressed in human cartilage but were markedly down-regulated in end-stage OA cartilage, especially SOD2. In the Dunkin Hartley guinea pig spontaneous OA model SOD2 expression was decreased in the medial tibial chondyle cartilage prior to, and following, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes gave an increase in ROS but a decrease in collagenase expression.Conclusion-This is the first comprehensive expression profile of all SOD genes in cartilage and importantly, using an animal model, we show that a reduction in SOD2 is associated with the earliest stages of OA. We found that a decrease in SOD2 associates with an increase in ROS and but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

Osteoarthritis is a degenerative joint disease characterized by a progressive and irreversible loss of the articular cartilage, due in main part to the cleavage of type II collagen within the matrix by the enzyme matrix metalloproteinase (MMP)13. Here, we examined the methylation status of MMP13 promoter and report the demethylation of specific CpG dinucleotides within its promoter in osteoarthritic compared to normal cartilage, which correlates with increased MMP13 expression. Of the promoter CpG sites examined, the -104 CpG was consistently demethylated following treatment of human articular chondrocytes with 10 μM DNA-methyltransferase inhibitor 5-aza-2'-deoxycytidine, again correlating with increased MMP13 expression. Methylation of the -104 CpG site resulted in reduced promoter activity in the chondrosarcoma cell line SW1353 as shown by CpG-free luciferase reporter. Using electrophoretic mobility shift assays, we identified CREB as the regulating factor able to only bind to the MMP13 promoter when the -104 CpG is demethylated, and confirmed this binding by chromatin immunoprecipitation. Finally, we demonstrated that CREB induces MMP13 expression only following treatment of SW1353 with 0.5 μM Ca(2+) ionophore A23187. In summary, the -104 CpG is demethylated in osteoarthritic cartilage, correlating with the elevated MMP13 expression and cartilage destruction, providing a highly novel link between epigenetic status and arthritic disease.

Objective. To examine the ability of a broadspectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro. Cartilage destruction in osteoarthritis (OA) is mediated by the action of proteinases from the matrix metalloproteinase (MMP) and ADAMTS families. The pathology of OA has been proposed to be driven by chondrocytes, because these cells are a major source of MMP expression within the OA joint (1). Metalloproteinase expression is regulated at the transcriptional level, which is in part mediated through changes in protein acetylation catalyzed by enzymes from the histone acetyltransferase and histone deacetylase (HDAC) families. These enzymes not only can modulate the nucleosomal interaction of DNA and histone proteins but also can reversibly acetylate both nuclear and cytoplasmic proteins, with a concomitant change in function (2). Methods. A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity

This present study is indicative of a pseudo-septic acute inflammatory reaction in response to local accumulation of hylan G-F 20 with the activation of complement and local invasion of pro-inflammatory cells.

Objective Although not considered a classical infl ammatory arthropathy, osteoarthritis (OA) is often associated with lowgrade synovitis. The stimuli required to establish infl ammation in OA synovium are as yet unclear, but there is emerging evidence to suggest activation of the complement pathway may have a role in this process. The aim of this study is to better describe the role that complement activation may play in the establishment or perpetuation of synovial infl ammation in early and advanced OA. Methods Gene expression of complement regulatory proteins was determined in RNA extracted from the synovium of 14 early and 28 advanced stage OA patients using real-time PCR. Immunohistochemistry was used to assess the level of C3 and membrane attack complex (MAC) C5b-9 protein deposition as well as expression of the MAC inhibitor CD59 in early and advanced stage OA synovium. In order to investigate the effect of oxidative stress, OA fi broblast-like synoviocytes (FLS) were treated with H 2 O 2 and subsequently exposed to normal human serum. CD59 expression was then assessed by fl uorescent immunocytochemistry and the level of intact C3 in the supernatant determined by western blot. Results The expression of properdin, a positive regulator of the alternative complement pathway, and CD59, an inhibitor of MAC formation, were signifi cantly upregulated in early OA synovium compared with advanced stage OA synovium by 2.2-fold (p=0.03) and 2

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Objectives: To investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA. Methods: Knee articular cartilages from OA patients were analysed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module domain (Link_TSG6) on cytokine-induced glycosaminoglycan loss in OA cartilage explants were compared. Mesenchymal stem/stromal cell (MSC)-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1β- or TNF-stimulated ADAMTS4, ADAMTS5 and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model and cartilage damage and tactile allodynia were assayed. Results: TSG-6 is predominantly associated with chondrocytes in regions of cartilage damage and its expression is negatively correlated with MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at dose-dependently inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants; about 50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Similarly, Link_TSG6 displayed more potent effects in 3D pellet cultures, suppressing aggrecanase and collagenase gene expression. Link_TSG6 treatment reduced touch-evoked pain and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA. Conclusions: Native TSG-6 is associated with a low catabolic chondrocyte phenotype in OA cartilage. Link_TSG6, which has enhanced chondroprotective activity compared to the full-length TSG-6 protein, demonstrates potential as a disease modifying OA drug (DMOAD) and warrants further investigation and development.

returned to control levels when synovial fibroblasts were co-incubated with PGF 2a inhibitor AL8810 whereas inhibition of TGFb signaling with SB505124 had no effect. aSMA gene expression and collagen production were unaffected by inhibition of TGFb or PGF 2a signaling. The increased synovial fibroblast migration in response to FCM could not be counteracted by SB505124, but was partially counteracted by AL8810. Synovial fibroblast proliferation in response to FCM was unaffected either by SB505142 or AL8810. Conclusions: These results indicate that infrapatellar fat can contribute for the development of synovial fibrosis by increasing collagen production, PLOD2 gene expression, cell proliferation and cell migration; all characteristics of a fibrotic process. Based on our results, not TGFb but the more recently discovered pro-fibrotic factor PGF 2a seems partly responsible for the observed effects.