Chondrocyte cell death, aging, and the involvement of reactive oxygen species

Reactive oxygen species (ROS) are endogenously produced by all eukaryotic cells, and when overproduced, can result in cell death through the oxidative damage of obligatory cellular proteins. Because articular chondrocytes normally survive until the death of an individual and chondrocyte cell death has been associated with the pathogenesis of osteoarthritis (OA), the main focus of the present thesis was to determine conditions by which oxidative stress is toxic to cultured cells, and to determine the correlation between the accumulation of oxidative damage and aging in humans.; Chondrocytes were sensitive to cell death caused by treatment with the oxidant ONOO−, but not by the free radical nitric oxide (NO). In separate experiments, freshly isolated chondrocytes cultured at low density under serum-free conditions undergo rapid cell death when plated on coverslips coated with poly-L-lysine through the overproduction of endogenous ROS. This observed cell death can be completely prevented by the addition of either inhibitors of specific protein kinase C (PKC) isoforms or by antioxidants. Direct quantification of ROS production confirmed the increased production of ROS when plated on poly-L-lysine. Through results obtained from time-course experiments, ROS production precedes the activation of several different isoforms of PKC, and treatment with antioxidants could selectively inhibit the activation of PKC-βI. Western blot analysis confirmed the ROS-dependent activation of PKC-βI on poly-L-lysine.; Immunohistochemical data presented here provided evidence that 3-nitrotyrosine (3-NT) immunostaining, a marker for oxidative stress, is higher in sections obtained from older donors when compared to sections from younger donors. Our results also showed that chondrocytes derived from older donors are more susceptible to cell death induced by the oxidant SIN-1. Moreover, cells from older donors have more intracellular oxidized glutathione and a higher ratio between oxidized to reduced glutathione. Because inhibitor studies demonstrated a clear role for glutathione as an important antioxidant defense mechanism against cell death induced by oxidative stress, an age-related imbalance in glutathione may lead to spurious chondrocyte cell death. Cell death induced by an age-related increase in ROS production may be paramount in the initiation and progression of certain age-related diseases including OA.