APOPTOSIS AND ACTIVATION OF THE SPHINGOMYELIN-CERAMIDE PATHWAY INDUCED BY OXIDIZED LOW-DENSITY LIPOPROTEINS ARE NOT CAUSALLY RELATED IN ECV-304 ENDOTHELIAL-CELLS

Oxidized low density lipoproteins (oxLDL) are thought to play a centra l role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Esc argueil-Blanc, I., Meilhac, O., Pieraggi, M. T., Amal J. F., Salvayre, R., Negre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 3 31-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide fo rmation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early ac tivation of the sphingomyelin-ceramide pathway, as shown by both sphin gomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chl oromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-pro tease inhibitors (serpins), blocked the oxLDL-induced ceramide generat ion but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Co nversely, treatment of endothelial cells by bacterial sphingomyelinase , under conditions effectively generating ceramide, did not induce apo ptosis, In contrast, short-chain permeant C-2- and C-6-ceramides elici ted apoptosis of ECV-304. However, the mechanisms of apoptosis trigger ed by C-2-ceramide and by oxLDL were (at least in part) different, bec ause C-2 ceramide-induced apoptosis was calcium-independent, whereas o xLDL-induced apoptosis was calcium dependent. In conclusion, it is sug gested that oxLDL-induced apoptosis is calcium-dependent but independe nt of the activation of the sphingomyelin-ceramide pathway and that th e toxic effect of short chain permeant ceramides is calcium-independen t and does not mimic the effect of natural ceramides induced by oxLDL.