OXIDIZED LDLS INDUCE MASSIVE APOPTOSIS OF CULTURED HUMAN ENDOTHELIAL-CELLS THROUGH A CALCIUM-DEPENDENT PATHWAY - PREVENTION BY AURINTRICARBOXYLIC ACID

Oxidized LDLs are thought to play a central role in atherogenesis. Amo ng their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly o xidized LDLs elicited massive apoptosis in both primary and immortaliz ed cultures of endothelial cells as shown by characteristic morphologi cal and biochemical changes. Cytoplasmic and nucleic modifications (eg , chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified b y ultracentrifugation of chromatin fragments, evaluated in situ by usi ng the TUNEL (Terminal transferase-mediated dUTP-biotin nick end label ing) procedure, and Visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and d elayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by in hibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced b y oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process wit hout blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by in hibiting cellular endonucleases by aurintricarboxylic acid.