INDUCTION OF SIMILAR FEATURES OF APOPTOSIS IN HUMAN AND BOVINE VASCULAR ENDOTHELIAL-CELLS TREATED BY 7-KETOCHOLESTEROL

Cholesterol oxides have numerous cytotoxic effects and those oxidized in the C7 position hare been shown to induce apoptosis in bovine aorti c endothelial cells (BAEC). The aim of the present study was to determ ine whether apoptosis also occurs in human vascular endothelial cells (HUVEC) treated with 7-ketocholesterol. To this end, cultured BAEC and HUVEC were incubated for 48 h with 7-ketocholesterol (concentration r ange 5-80 mu g/ml) and the characteristics of cell death mere assessed by various methods: counting of adherent and non-adherent cells; anal ysis of DNA fragmentation pattern; and morphological study by light, f luorescence, and electron microscopy. The 7-ketocholesterol treatment was accompanied by a decrease in the number of adherent cells and an i ncrease in the number of non-adherent cells. Apoptotic cells, recogniz ed by fragmented and/or condensed nuclei after staining with Hoechst 3 3342 or Giemsa, were mainly detected among non-adherent cells, and aga rose gel electrophoresis revealed a typical internucleosomal DNA fragm entation among 7-ketocholesterol-treated cells. The DNA fragmentation mas no longer detected a hen HUVEC and BAEC were simultaneously incuba ted with 0.5 mmol/l zinc chloride, which is known to inhibit Ca2+/Mg2-dependent endonucleases. Finally, the ultrastructural abnormalities o bserved by electron microscopy in both 7-ketocholesterol-treated HUVEC and BAEC were remarkably similar and were mainly characterized by con densed chromatin, altered mitochondria, disturbed organization of the cytoskeleton, and vacuoles containing myelin figures and/or cell debri s; apoptotic bodies mere also frequently detected. It is concluded tha t 7-ketocholesterol constitutes a potent inducer of apoptosis in endot helial vascular cells of both bovine and human origin, suggesting that cholesterol oxides may be involved in the early steps of the atherosc lerotic process in humans. (C) 1997 John Wiley & Sons, Ltd.