Hydrogen peroxide induced apoptosis of endothelial cells concomitantly with cycloheximide.

Abstract

We examined whether or not hydrogen peroxide induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by a propidium iodide assay. Under serum free conditions, treatment of the endothelial cells with hydrogen peroxide (H2O2) for 6 hours induced cytotoxicity (51Cr release) in a dose-dependent manner (10 micromol/l-1 mmol/l). Under the condition containing 10% serum, H2O2 did not induce cytotoxicity even at the highest concentration (1 mmol/l). However, concomitant treatment of endothelial cells with cycloheximide at a dose of 10 microg/ml elicited endothelial cell apoptosis of by 15.6+/-1.7% at 6 hours after administration, even under the 10% serum condition. In addition, endothelial cell apoptosis due to H2O2 and cycloheximide was completely inhibited by zD-dcb (50 micromol/l), an inhibitor of caspase. 1 mmol/l of 4, 4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), which is a chloride bicarbonate exchanger blocker, partially inhibited the H2O2 and cycloheximide-induced endothelial cell apoptosis. On the other hand, cytotoxicity of endothelial cells due to H2O2 under serum free conditions was not inhibited by DIDS. These data suggested that hydrogen peroxide could induce endothelial cell apoptosis or cell membrane injury (51Cr release) in the presence or absence of an inhibitor of protein synthesis.