Differential sensitivity to NO-induced apoptosis between anterior cruciate and medial collateral ligament cells

Abstract

It is well known that the anterior cruciate ligament (ACL) of the knee joint has poorer healing responses than the medial collateral ligament (MCL). Nitric oxide (NO) induces free radicals and plays a key role in the induction of apoptosis in various wound-healing models. We hypothesized that the poor healing response of the ACL may be ascribed to high susceptibility to apoptosis, and we investigated the difference in susceptibility to apoptosis between ACL and MCL cells after treatment with sodium nitroprusside, a NO donor. Apoptosis was evaluated by phase contrast microscopy, electron microscopy, DNA gel electrophoresis, and flow cytometric analysis. Although morphological changes and DNA ladders were observed in both ACL and MCL cells after 2mM sodium nitroprusside treatment, ACL cells were more prone to apoptosis at 1 mM. Based on flow cytometric analysis, DNA fragmentation at ImM sodium nitroprusside was significantly greater in ACL cells than in MCL cells (58.6%±1.6% vs. 11.9%±2.2%). Caspase-3 inhibitor (Ac-Asp-Glu- Val-Asp-CHO) and caspase-9 inhibitor (Ac-Leu-Glu-His- Asp-CHO) completely inhibited this DNA fragmentation. In conclusion, the ACL and MCL cells exhibit essential differences, and the differential sensitivity to NO-induced apoptosis between the ACL and MCL cells may be a reflection of these differences.