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 central role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Escargueil-Blanc, I., Meilhac, O., Pieraggi, M. T. , Arnal J. F., Salvayre, R., Nègre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 331-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide formation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-protease inhibitors (serpins), blocked the oxLDL-induced ceramide generation but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Conversely, treatment of endothelial cells by bacterial sphingomyelinase, under conditions effectively generating ceramide, did not induce apoptosis. In contrast, short-chain permeant C2- and C6-ceramides elicited apoptosis of ECV-304. However, the mechanisms of apoptosis triggered by C2-ceramide and by oxLDL were (at least in part) different, because C2-ceramide-induced apoptosis was calcium-independent, whereas oxLDL-induced apoptosis was calcium-dependent. In conclusion, it is suggested that oxLDL-induced apoptosis is calcium-dependent but independent of the activation of the sphingomyelin-ceramide pathway and that the toxic effect of short chain permeant ceramides is calcium-independent and does not mimic the effect of natural ceramides induced by oxLDL.
INSERM U-466 and the Biochemistry Department, Institut Louis Bugnard, CHU Rangueil, 31054 Toulouse Cedex, France.
I
I
Escargueil-Blanc
N
N
Andrieu-Abadie
S
S
Caspar-Bauguil
R
R
Brossmer
T
T
Levade
A
A
Nègre-Salvayre
R
R
Salvayre
D017209:Apoptosis; D001161:Arteriosclerosis / Q000209:etiology; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism* / Q000633:toxicity; D004730:Endothelium, Vascular / Q000187:drug effects*; D006801:Humans; D006868:Hydrolysis; D008077:Lipoproteins, LDL / Q000633:toxicity*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000633:toxicity; D014471:Umbilical Veins / Q000166:cytology
D002518:Ceramides; D008077:Lipoproteins, LDL; C064769:N-acetylsphingosine; D015842:Serine Proteinase Inhibitors; D013109:Sphingomyelins; C099252:oxidized low density lipoprotein; C101954:N-caproylsphingosine; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine; D002118:Calcium