Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7 beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall

Oxidized low density lipoproteins (LDLs) play a central role in atheroscler osis, and their toxicity is due, at least in part, to the formation of oxys terols that have been shown to induce apoptosis in various cell types. As 7 beta-hydroxycholesterol and 7-ketocholesterol are the major oxysterols fou nd in oxidized LDLs, we have investigated and compared the mode of cell dea th, apoptosis versus necrosis, that they induce in the cells of the vascula r wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To thi s end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle:cells, MRCS human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7 beta-hydroxycholeste rol or 7-ketocholesterol (concentration range, 5 to 80 mu g/mL). In all cel ls, both 7 beta-hydroxycholesterol and 7-ketocholesterol exhibited toxic ef fects characterized by a loss of cell adhesion and an increased permeabilit y to propidium iodide. In oxysterol-treated endothelial and smooth muscle c ells,typical features of apoptosis were revealed: condensed and/or fragment ed nuclei were detected by fluorescence microscopy after staining with Hoec hst 33342, oligonucleosomal DNA fragments were visualized in situ in the ce ll nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contra st, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei we re never revealed, and no DNP fragmentation was observed either by the TUNE L method or by DNA analysis on agarose gel, indicating that these oxysterol s induced necrosis in these cells but not apoptosis. In addition, acetylate d Asp-Glu-Val-L-aspartic acid aldehyde tan inhibitor of Asp-Glu-Val-L-aspar tic acid-sensitive caspases) prevented 7 beta-hydroxycholesterol- and 7-ket ocholesterol-induced cell death in HUVECs and smooth muscle cells but not i n fibroblasts. Thus, 7 beta-hydroxycholesterol and 7-ketocholesterol have d ual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fi broblasts.