Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.

Objective: Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21–24‐kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin‐1β (IL‐1β) induce features of premature senescence of articular chondrocytes through up‐regulation of caveolin 1 expression.Methods: Caveolin 1 expression was investigated in human OA cartilage by real‐time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL‐1β and H₂O₂ induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes.Results: In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL‐1β and H₂O₂ up‐regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence‐associated β‐galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down‐regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL‐1β and H₂O₂ up‐regulated p53 and p21 and down‐regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress.Conclusion: Our findings suggest that IL‐1β and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress‐induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down‐regulation.

Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein α-1 subunit [CapZα-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZα-1. As a result, frequencies of autoantibodies to non-citrullinated CapZα-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZα-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZα-1 is relevant to RA. The antibody titers to the citrullinated CapZα-1 were significantly higher than those to the non-citrullinated CapZα-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZα-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZα-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.

Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis – including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production – under hypoxic conditions. We speculated that HIF-1α may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1α mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1β and H₂O₂ induce the expression of HIF-1α in OA chondrocytes under normoxic and hypoxic conditions (O₂ <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1α-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1α mRNA was higher in the degenerated regions than in the intact regions. Both IL-1β and H₂O₂ accelerated mRNA and protein levels of HIF-1α in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1α expression. HIF-1α-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1α-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1α expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1β, and oxidative stress induce the expression of HIF-1α in chondrocytes. Our results suggest an important role of stress-induced HIF-1α in the maintenance of chondrocyte viability in OA articular cartilage.

Objective: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1β and TNFα. Methods: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 μg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), IFNγ, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. Results: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1β, or TNFα increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1β, and TNFα did not induce M-CSF, GM-CSF, IFNγ, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1β, but less potent than that of TNFα. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1β, and greatly inhibited induction by TNFα. Conclusion: IL18, IL1β, or TNFα can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1β, but less potent than TNFα.

Recent reports have shown that statin (HMG-CoA reductase inhibitors) may have the potential to inhibit inflammatory arthritis. More recently, the idea that chondrocyte aging is closely associated with the progression of cartilage degeneration has been promulgated. Here, we demonstrate the potential of statin as protective agents against chondrocyte aging and degeneration of articular cartilage during the progression of osteoarthritis (OA), both in vitro and in vivo. The OA-related catabolic factor, IL-1β induced marked downregulation of cellular activity, expression of a senescent biomarker, specific senescence-associated β-galactosidase activity and shortening of the cellular lifespan in chondrocytes. In contrast, treatment with statin inhibited the IL-1β-induced production of cartilage matrix degrading .enzymes (metalloprotease-1 and -13) and cellular senescence in of chondrocytes in vitro. In addition, this statin accelerated the production of cartilage matrix proteoglycan in chondrocytes. The in vivo study was performed on the STR/OrtCrlj mouse, an experimental model which spontaneously develops an osteoarthritic process. In this mouse model, treatment with statin significantly reduced the degeneration of articular cartilage, while the control knee joints showed progressive cartilage degeneration over time. These findings suggest that statin may have the potential to prevent the catabolic stress-induced chondrocyte disability and aging observed in articular cartilage. Our results indicate that statin are potential therapeutic agents for protection of articular cartilage against the progression of OA.