Fibronectin fragment mediated cartilage chondrolysis .1. Suppression by anti-oxidants

Fibronectin fragments damage cartilage in vitro by greatly enhancing metalloproteinases and suppressing proteoglycan (PG) synthesis which results in severe cartilage PG depletion. Since reactive oxygen species (ROS) have been implicated in catabolic cytokine action and preliminary data suggested that catabolic cytokines such as TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 are responsible for fibronectin fragment mediated damage selected anti-oxidants (AOs) were tested as inhibitors of cytokine, ROS and fibronectin fragment activity. Damage was measured by depletion of cartilage PG during tissue culture. The AO, N-acetylcysteine (NAC), decreased the extent of cartilage PG depletion caused by TNF-alpha and IL-1 alpha and by the ROS, hydrogen peroxide and superoxide anion, confirming that the cytokines operate through ROS and that ROS can initiate cartilage PG depletion. NAC at 0.1 and 1 mM, totally suppressed PG depletion caused by a highly potent amino-terminal 29-kDa fibronectin fragment (Fn-f) for 14 days in culture. NAC at 10 mM totally blocked Fn-f mediated PG depletion for 21 days and increased the cartilage PG content by 30% above normal levels. Glutathione (10 mu M) and DMSO (1%) were also totally effective while catalase and superoxide decreased Fn-f mediated damage only during the first week and superoxide dismutase alone caused damage after 1 wk. The AOs caused protection by reducing the major catabolic activities of the Fn-f: enhanced release of stromelysin-1 (MMP-3) and suppression of PG and protein synthesis. NAC also decreased normal rates of PG degradation and increased the half-lives of labeled PG in both control and Fn-f treated cartilage. We conclude that the Fn-f mediates cartilage chondrolysis through ROS, consistent with the involvement of catabolic cytokines in the Fn-f mechanism. and that AOs greatly reduce Fn-f mediated cartilage chondrolysis. In an accompanying manuscript we also report that AOs promote reparative responses in Fn-f and cytokine treated cartilage.