LOW-DENSITY-LIPOPROTEIN STIMULATED PEROXIDE PRODUCTION AND ENDOCYTOSIS IN CULTURED HUMAN ENDOTHELIAL-CELLS - MECHANISMS OF ACTION

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor s on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-de nsity lipoprotein (LDL) levels have been investigated. EC were incubat ed with 240 mg/dl LDL cholesterol and cellular H2O2 production and end ocytotic activity measured in the presence and absence of the arachido nic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic aci d, 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 change s similar to those induced by high LDL concentrations. EC incubated wi th 1-25 uM arachidonic acid had increased H2O2 production and heighten ed endocytotic activity. Likewise, EC pre-loaded with [H-3]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [H-3]arachidonic acid. The pho spholipase A(2) inhibitors, 4-bromophenacyl bromide and 7,7-dimethylei cosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic aci d contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to o ur understanding of endothelial perturbation, which has been hypothesi zed to be a major contributing factor in the pathogenesis of atheroscl erosis.