A Caspase Inhibitor Decreases Oxidized Low-Density Lipoprotein-Induced Apoptosis in Bovine Endothelial Cells

Abstract

Background. Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Oxidized low density lipoprotein (oxLDL) is a putative cause of atherogenesis. We examined the effect of oxLDL on endothelial cell (EC) apoptosis and the ability of a caspase antagonist to inhibit oxLDL-induced EC injury.Methods. Bovine ECs were plated at a concentration of 5.0 × 105 cells/ml and exposed to LDL oxidized by ultraviolet radiation at a concentration of 100 μg oxLDL/ml for 20 h. Some ECs were pretreated with an irreversible caspase inhibitor (ZVAD). Samples were analyzed histologically. Apoptosis was measured using the Annexin V assay (flow cytometry) which detects phosphatidylserine on plasma membranes and confirmed by TUNEL assay (flow cytometry). Statistical assessments were performed using ANOVA.Results. ECs treated with LDL were morphologically similar to untreated cells. Cells treated with oxLDL demonstrated cytoplasmic shrinkage, plasma membrane blebbing, chromatin condensation, and loss of adhesion. These effects were diminished after pretreatment with the caspase inhibitor ZVAD. The Annexin V assay showed: (a) cells exposed to LDL had a 12 ± 1% apoptosis rate, (b) exposure to oxLDL induced apoptosis in 30 ± 0.3% of the cells, and (c) pretreatment with the caspase inhibitor ZVAD decreased the oxLDL-induced apoptosis to 16 ± 1% (P < 0.05). This decrease in apoptosis was also reflected by an increase in the percentage of alive cells from 34 ± 7% after oxLDL exposure to 55 ± 6% after apoptosis inhibition with ZVAD. TUNEL assay demonstrated a 2.5-fold reduction in mean fluorescence intensity between cells treated with oxLDL alone and those treated with ZVAD, suggesting a significant decrease in apoptosis in the latter group.Conclusions. We conclude that treatment of bovine ECs with oxLDL induces apoptosis which can be significantly reduced by a specific caspase inhibitor.