Cell death in osteoarthritis.

To date, most studies examining cell death during the development of osteoarthritis (OA) have focused on death of chondrocytes and have primarily examined advanced stages of the disease. Very good evidence suggests that chondrocyte death does occur at some point in the pathogenesis of OA and that it can be due to apoptosis, necrosis, or some combination of the two. Chondrocyte death can be induced by mechanical injury, loss of extracellular matrix, loss of growth factors, or excessive levels of reactive oxygen species. Although therapy specifically targeting cell death in human OA has not been reported, preclinical studies in animal models have provided early evidence that inhibition of caspases might slow OA-like changes in articular cartilage. Because of potential unwanted side effects from agents systemically inhibiting cell death, treatments specifically targeting cell death in OA will likely need to be delivered locally and in a manner that prevents systemic absorption. Inhibition of cell death in OA likely will not be a sole therapeutic target but rather a desired effect of interventions designed to reverse the catabolic-anabolic imbalance occurring in OA joint tissues.

Differential calcium independent regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by interleukin-1beta and transforming growth factor-beta in Peyronie's plaque fibroblasts.

PURPOSE: Peyronie's disease is a fibrotic disorder of the tunica albuginea characterized by the localized formation of an inelastic plaque. We characterized matrix metalloproteinases and TIMPs (tissue inhibitors of matrix metalloproteinase) in Peyronie's disease tissue. MATERIALS AND METHODS: Matrix metalloproteinases and TIMPs were investigated in Peyronie's disease plaque tunica removed from patients with stable Peyronie's disease. Immunological methods were used to characterize the matrix metalloproteinases and TIMPs produced by cell cultures stimulated with transforming growth factor-beta or interleukin-1beta (PreproTech, Rocky Hill, New Jersey). Enzyme activity was quantified with a fluorescent substrate and correlated with mRNA levels using real-time polymerase chain reaction. RESULTS: Interleukin-1beta significantly induced (p <0.01) matrix metalloproteinase-1, 3, 10 and 13 protein production, endogenous matrix metalloproteinase-13 activity (12-fold) and matrix metalloproteinase-13 mRNA expression (11.2-fold) through a Ca(2+) independent mechanism in cultured fibroblasts. Transforming growth factor-beta stimulation failed to induce any detectable matrix metalloproteinase protein production or activity and conditioned culture medium even had the capacity to inhibit (p <0.01) the activity of purified recombinant human matrix metalloproteinase-13. Intact Peyronie's disease plaques were highly enriched with TIMP-1 to 4 compared to donor matched perilesional tunica. CONCLUSIONS: These data show that, while interleukin-1beta strongly induces matrix metalloproteinase expression, transforming growth factor-beta strongly induces TIMP expression without any effect on matrix metalloproteinases and may represent an important downstream biochemical mechanism that leads to the progression of Peyronie's disease. The localized accumulation of TIMPs together with decreased matrix metalloproteinase activity in the Peyronie's disease lesion may be the biochemical consequence of the transforming growth factor-beta over expression that has been reported in many fibrotic disorders, including Peyronie's disease.

Endogenous production of reactive oxygen species is required for stimulation of human articular chondrocyte matrix metalloproteinase production by fibronectin fragments.

The objective of the present study was to determine if reactive oxygen species (ROS) are required as secondary messengers for fibronectin fragment-stimulated matrix metalloproteinase (MMP) production in human articular chondrocytes. Cultured cells were stimulated with 25 microg/ml of the alpha5beta1 integrin-binding 110-kDa fibronectin fragment (FN-f) in the presence and absence of various antioxidants including Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP). FN-f stimulation significantly increased intracellular levels of ROS in articular chondrocytes. Pretreatment of cells with 250 microM MnTBAP or 40 mM N-acetyl-L-cysteine, but not inhibitors of nitric oxide synthase, completely prevented FN-f-stimulated MMP-3, -10, and -13 production. MnTBAP also blocked FN-f-induced phosphorylation of the MAP kinases and NF-kappaB-associated proteins and blocked activation of an NF-kappaB promoter-reporter construct. Overexpression of catalase, superoxide dismutase, or glutathione peroxidase also inhibited FN-f-stimulated MMP-13 production. Preincubation of chondrocytes with rotenone, an inhibitor of the mitochondrial electron transport chain, or nordihydroguaiaretic acid (NDGA), a selective 5-lipoxygenase inhibitor, partially prevented FN-f-stimulated MMP-13 production and decreased MAP kinase and NF-kappaB phosphorylation. These results show that increased production of ROS but not nitric oxide as obligatory secondary messengers in the chondrocyte FN-f signaling pathway leads to the increased production of MMPs, including MMP-13.

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin-1beta and with chondrocyte resistance to insulin-like growth factor 1.

OBJECTIVE: To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin-1beta (IL-1beta) and the responsiveness of isolated chondrocytes to stimulation with insulin-like growth factor 1 (IGF-1). METHODS: The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age-related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0-7 for the presence of OA-like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL-1beta. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF-1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA-related joint replacement surgery were also immunostained for nitrotyrosine and IL-1beta. RESULTS: The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL-1beta also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL-1beta was absent, suggesting that some age-related oxidative damage is independent of IL-1beta. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL-1beta, the response to stimulation with IGF-1 was significantly reduced. In some samples from older adult monkeys, IGF-1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL-1beta. CONCLUSION: Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF-1 stimulation, but it is unlikely to be the sole cause of IGF-1 resistance in these chondrocytes.

The alpha5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro.

OBJECTIVE: Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors. METHODS: Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis. RESULTS: Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the alpha5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the alpha5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 microg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of alphav and alpha1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of alpha5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to alpha5 inhibition was associated with apoptosis. CONCLUSION: These results demonstrate that chondrocyte survival signals are transmitted via the alpha5beta1 FN receptor. Inhibition of matrix survival signals mediated by alpha5beta1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from alpha5beta1.

Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species.

OBJECTIVE: Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS). METHODS: Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH- and NADPH-dependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L-cystine- and glutathione (GSH)-depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histone-associated DNA fragments. RESULTS: Treatment of chondrocytes with peroxynitrite (ONOO-), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) resulted in apoptotic cell death at concentrations of 0.5 mM, 1.0 mM, and 0.5 mM, respectively. In contrast, treatment of chondrocytes with diazeniumdiolates (or the "NOC" compounds, NOC-5 and NOC-12) at concentrations as high as 2.0 mM did not cause cell death. Furthermore, NOC-5 and NOC-12, at all concentrations tested (0.125-2.0 mM), could prevent cell death caused by oxidative stress. Selective ROS scavengers protected against cell death caused by either SIN-1 or ONOO-; however, no protection could be afforded against the cytotoxicity of SNP with any of the ROS scavengers tested. CONCLUSION: These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated.