Two distinct calcium-dependent mitochondrial pathways are involved in oxidized LDL-induced apoptosis

Objective - Oxidized low-density lipoprotein (oxLDL) - induced apoptosis of vascular endothelial cells may contribute to plaque erosion and rupture. We aimed to clarify the relationship between the oxLDL- induced calcium signal and induction of apoptotic pathways. Methods and Results - Apoptosis was evaluated by biochemical methods, including studies of enzyme activities, protein processing, release of proapoptotic factors, chromatin cleavage, and especially by morphological methods that evaluate apoptosis/ necrosis by SYTO- 13/ propidium iodide fluorescent labeling. The oxLDL- induced sustained calcium rise activated 2 distinct calcium- dependent mitochondrial apoptotic pathways in human microvascular endothelial cells. OxLDLs induced calpain activation and subsequent Bid cleavage and cytochrome C release, which were blocked by calpeptin. Cyclosporin- A inhibited cytochrome C release, possibly by inhibiting the opening of the mitochondrial permeability transition pore ( mPTP). Calcineurin, another cyclosporin- sensitive step, was not implicated, because oxLDLs inhibited calcineurin and FK- 506 treatment was ineffective. Cytochrome C release in turn induced caspase- 3 activation. In addition, oxLDLs triggered release and nuclear translocation of mitochondrial apoptosis-inducing factor through a mechanism dependent on calcium but independent of calpains, mPTP, and caspases. Conclusions - OxLDL-induced apoptosis involves 2 distinct calcium- dependent pathways, the first mediated by calpain/ mPTP/cytochrome C/caspase-3 and the second mediated by apoptosis-inducing factor, which is cyclosporin- insensitive and caspase- independent.