INDUCTION OF APOPTOSIS IN ENDOTHELIAL-CELLS TREATED WITH CHOLESTEROL OXIDES

Cholesterol oxides have a wide range of cytotoxic effects on vascular cells, Therefore, 7-ketocholesterol, 7 beta-hydroxycholesterol, 19-hyd roxycholesterol, cholesterol 5 alpha, 6 alpha-epoxide, and 25-hydroxyc holesterol, identified in various foodstuffs and human tissues, were c hosen to compare and characterize the mode of cell death they induce, apoptosis or necrosis, on bovine aortic endothelial cells. The toxic p otency differred from one compound to another, and 7 beta-hydroxychole sterol and 7-ketocholesterol exhibited the most potent effects. Cytoto xicity was accompanied by a decreased number of adherent cells, an inc reased number of non-adherent cells, and an enhanced permeability to p ropidium iodide, By electron and fluorescence microscopy performed aft er staining with Hoechst 33342, apoptotic cells with fragmented and co ndensed nuclei were identified mainly among non-adherent cells. By flo w cytometry, cells with a lower DNA content than cells ill the G0/G1 p hase were apparent, giving a characteristic sub-G1 pent. Quantificatio n of apoptosis evaluated either by the proportion of apoptotic cells i dentified by fluorescence microscopy after staining with Hoechst 33342 or by the percentage of cells present in the sub-G1 peak indicated th at the ability of cholesterol oxides in inducing apoptosis was in the following order: 7 beta-hydroxycholesterol > 7-ketocholesterol > 19-hy droxycholesterol > cholesterol 5 alpha, 6 alpha-epoxide > 25-hydroxych olesterol, By using electrophoresis on agarose gel, typical internucle osomal DNA fragmentations were detected they were no longer observed w hen bovine not-tie endothelial cells were simultaneously incubated wit h 0.5 mmol/L zinc chloride, known to inhibit Ca2+/Mg2+-dependent endon ucleases. None of the cholesterol-oxides constitute apoptotic features described above were noted with cholesterol. It is concluded that cho lesterol oxides constitute a new class of cholesterol derivatives that can induce cell death by apoptosis in cultured endothelial cells.