Introduction. Matrix metalloproteinases (MMP) play a key role in cartilage degradation in osteoarthritis. Statins are a potential suppressor of MMPs. The aim of this research was to assess the efficacy of Pravastatin in suppressing MMP gene and protein expression in an in vitro model. Methods. We stimulated normal human chondrocytes with IL-1b for 6 hours to induce MMP expression and then treated with Pravastatin (1, 5 & 10 mM) for a further 18 hours. Cells stimulated with IL-1b but not treated with Pravastatin served as controls. Real-time PCR was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Staistical analysis was performed using ANOVA. Results. MMP-3 and -9 mRNA expression was reduced at all concentrations tested with a statistically significant trends in reduction (p=0.002 and < 0.001 respectively). Analaysis of culture supernatants revealed that Pravastatin treatment led to a reduction in total MMP activity but not to a statistically significant degree (p=0.07). Conclusion. We conclude that treatment with Pravastatin of stimulated human chondrocytes leads to a down regulation of selected MMP genes and a reduction in MMP enzyme activity. Our results are further evidence that statins may have a role to play in the treatment of osteoarthritis and other disorders of cartilage degradation

The purpose of this study was to evaluate whether AGEs induce annulus fibrosus (AF) cell apoptosis and to further explore the mechanism by which this process occurs. AF cells were treated with various concentrations of AGEs for 3 days. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) and EdU incorporation assays. Cell apoptosis was examined by the Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3 and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by RT-PCR. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by JC-1 staining and DCFH-DA fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggest that AGEs induce rabbit AF cell apoptosis and mitochondrial pathways may be involved in AGE-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions. OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN. Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β. Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s)

Introduction. Osteoarthritis (OA) has historically been thought of as a degenerative joint disease, but inflammation and angiogenesis are increasingly being recognised as contributing to the pathogenesis, symptoms and progression of OA. b-dystroglycan (b-DG) is a pivotal element of the transmembrane adhesion molecule involved in cell-extracellular matrix adhesion and angiogenesis. Matrix metalloproteinases (MMPs) are the main enzymes responsible for cartilage extracellular matrix breakdown and are also implicated in both angiogenesis and b-DG degradation in a number of malignancies. We aimed to investigate the expression and localisation of b-DG and MMP-3, -9, and -13 within cartilage, synovium and synovial fluid and establish their roles in the pathogenesis of OA. Methods. Following ethical committee approval, cartilage, synovium and synovial fluid were obtained from the hip joints of 5 osteoarthritic (patients undergoing total hip replacement) and 5 control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for b-DG expression using Western Blotting and for the distribution of b-DG, MMP-3, -9, and -13 using immunohistochemistry on paraffin embedded tissue. Results. Whilst no significant expression of b-DG was found in cartilage or synovial fluid, b-DG was expressed in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Moreover, b-DG was expressed in endothelium of blood vessels of OA synovium, but not in the normal endothelium. In the endothelium of osteoarthritic synovial blood vessels, b-DG co-localised with MMP -3 and -9. Discussion. Our results demonstrate that b-DG does not act as a cell adhesion molecule binding chondrocytes to the ECM. However, specific immunolocalisation of b-DG within endothelium of inflamed OA blood vessels suggests that b-DG may play a role in angiogenesis associated with OA. Its co-localisation with MMP-3 and -9, previously reported to also have pro-angiogenic roles, may be linked. Further research is required to understand these roles more fully

The purpose of this study was to profile the mRNA expression for the 23 known matrix metalloproteinases (MMPs), 4 tissue inhibitor of metalloproteinases (TIMPs) and 19 ADAMTSs (a disintegrin and metalloproteinase with thrombospontin motif) in Dupuytren's Disease and normal palmar fascia. Dupuytren's Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. The MMPs and ADAMTSs are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs and their inhibition by the TIMPs underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinomata, whereas an imbalance towards inhibition causes e.g. fibrosis. A group of patients with end-stage gastric carcinoma was treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of carcinoma advancement; a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD