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American Journal of Pathology, Vol 148, 1625-1638, Copyright © 1996 by American Society for Investigative Pathology
REGULAR ARTICLES |
G Lizard, V Deckert, L Dubrez, M Moisant, P Gambert and L Lagrost
Laboratoire de Biochimie des Lipoproteines INSERM CJF 93/10, Faculte de Medecine, Dijon, France.
Cholesterol oxides have a wide range of cytotoxic effects on vascular cells. Therefore, 7-ketocholesterol, 7 beta-hydroxycholesterol, 19- hydroxycholesterol, cholesterol 5 alpha, 6 alpha-epoxide, and 25- hydroxycholesterol, identified in various foodstuffs and human tissues, were chosen to compare and characterize the mode of cell death they induce, apoptosis or necrosis, on bovine aortic endothelial cells. The toxic potency differed from one compound to another, and 7 beta- hydroxycholesterol and 7-ketocholesterol exhibited the most potent effects. Cytotoxicity was accompanied by a decreased number of adherent cells, an increased number of non-adherent cells, and an enhanced permeability to propidium iodide. By electron and fluorescence microscopy performed after staining with Hoechst 33342, apoptotic cells with fragmented and condensed nuclei were identified mainly among non- adherent cells. By flow cytometry, cells with a lower DNA content than cells in the G0/G1 phase were apparent, giving a characteristic sub-G1 peak. Quantification of apoptosis evaluated either by the proportion of apoptotic cells identified by fluorescence microscopy after staining with Hoechst 33342 or by the percentage of cells present in the sub-G1 peak indicated that the ability of cholesterol oxides in inducing apoptosis was in the following order: 7 beta-hydroxycholesterol > 7- ketocholesterol > 19-hydroxycholesterol > cholesterol 5 alpha, 6 alpha- epoxide > 25-hydroxycholesterol. By using electrophoresis on agarose gel, typical internucleosomal DNA fragmentations were detected; they were no longer observed when bovine aortic endothelial cells were simultaneously incubated with 0.5 mmol/L zinc chloride, known to inhibit Ca2+/Mg2+-dependent endonucleases. None of the cholesterol- oxide-induced apoptotic features described above were noted with cholesterol. It is concluded that cholesterol oxides constitute a new class of cholesterol derivatives that can induce cell death by apoptosis in cultured endothelial cells.
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