Objective. Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age-related changes in mitochondria.Methods. Mitochondrial function was evaluated by analyzing respiratory chain enzyme complexes and citrate synthase (CS) activities as well as changes in mitochondrial membrane potential (⌬⌿m). The activities of mitochondrial respiratory chain complexes (complex I: rotenone-sensitive NADH-coenzyme Q 1 reductase; complex II: succinate dehydrogenase; complex III: antimycin-sensitive ubiquinol cytochrome c reductase; and complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from OA and normal cartilage. ⌬⌿m was measured by JC-1 using flow cytometry. Statistical analysis was performed using the Mann-Whitney U test and Student's t-test as well as several models of multiple linear regression.Results. OA articular chondrocytes had reduced activities of complexes II and III compared with cells from normal cartilage. However, the mitochondrial mass was increased in OA. Cultures of OA chondrocytes contained a higher proportion of cells with de-energized mitochondria. We found no relationship between mitochondrial function and donor age either in normal or in OA chondrocytes. Conclusion. These findings suggest the involvement of mitochondrial function in the pathophysiology of OA. Cartilage degradation by OA and cartilage aging may be two different processes.Osteoarthritis (OA) is a degenerative joint disease that is characterized by articular cartilage degradation. The final phase of OA seems to reflect a failure of the reparative process, resulting in degradation of the matrix, cell death, and total loss of cartilage integrity. The chondrocyte is the only cell type that is present in mature cartilage, and it is responsible for repairing the damaged tissue (1,2).Mitochondria are complex organelles that oxidize a wide range of metabolic intermediates. It has been reported that in living cells, these organelles have a characteristic appearance that depends on the tissue type and the oxidative state of the cell. Multienzyme complexes located both in the inner mitochondrial membrane and in the mitochondrial matrix oxidize tricarboxylic acid-cycle intermediates derived from primary nutrients. ATP is generated by the activity of an electrogenic proton pump that spans the inner mitochondrial membrane. The energy for synthesis of ATP is derived from two components: a pH gradient and the electric potential across the mitochondrial membrane.Mitochondrial impairment and defective oxidative phosphorylation have been linked to some human disorders (3,4). Mitochondria are important in regulating cell survival (5-9), and the classic signs of cell death

Background:The role of DNA methylation in musculoskeletal diseases is poorly understood. Results: Methylation of specific CpG sites in the MMP13 and IL1B promoters correlates strongly with gene activity in human cartilage. Conclusion: Specific CpG site methylation inhibits MMP13 transactivation by HIF-2␣. Significance: Our findings offer new insight on the impact of epigenetic control of a joint disease that can affect adults throughout life.

Objective: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes. Materials and methods: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Dym). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ 1 reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Dym was measured by 5,59,6,69-tetracholoro-1,19,3,39-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP ) was used as an NO compound donor. Results: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Dym and induced apoptosis. Conclusions: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.

Objective Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma–Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 2 mM GlcN, (v) cultured with 1.0 μM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 1.0 μM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1β and MMP13 proximal promoter were quantified by pyrosequencing. Result IL1β expression was enhanced over 580-fold in articular chondrocytes treated with IL-1β and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1β expression by 3-fold. In the presence of BAY, IL-1β induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the −256 CpG site in the IL1β promoter was 65% in control cultures and decreased to 36% in the presence of IL-1β/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1β/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. Conclusion We demonstrate for the first ti...

Objective To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. Methods Expression of iNOS was quantified by quantitative reverse transcriptase–polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. Results The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at −289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at −5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. Conclusion These findings demonstrate the association between demethylation of specific NF-κB–responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.