Low-Density Lipoprotein Stimulated Peroxide Production and Endocytosis in Cultured Human Endothelial Cells: Mechanisms of Action

Abstract

The effects of arachidonic acid metabolism and NADPH oxidase inhibitors on the hydrogen peroxide (H₂O₂) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H₂O₂ production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H₂O₂ generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1–25 uM arachidonic acid had increased H₂O₂ production and heightened endocytotic activity. Likewise, EC pre-loaded with [³H]arachidonic acid when exposed to increasing LDL levels (90–330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [³H]arachidonic acid. The phospholipase A₂ inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H₂O₂ production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H₂O₂ generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.