Inhibition by cholesterol oxides of NO release from human vascular endothelial cells

Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (Circulation. 1997;95:723-731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 alpha,6 alpha-epoxycholesterol, 19-hydroxycholesterol (60 mu/mL), and alpha-lysophosphatidylcholine (10 mu g/mL) were compared. Pretreatment of HUVECs with cholesterol, 5 alpha,6 alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7 beta-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of L-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.