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Cyclooxygenase-2 (Cox-2) Upregulation Is Cytoprotective In H2O2-Induced Free Radical Injury In Rat Aortic Smooth Muscle Cells: Synergistic Effects Of Simvastatin

Xiaodong Wen, Wesley T Myers, Qixu Zhang, Kenneth J. Woodside, Glenn C Hunter ∙ The Univ. of Texas Medical Branch, Galveston, TX

Objectives: The oxidation of low density lipoprotein (LDL) cholesterol and prostaglandins have been implicated in determining the stability of atherosclerotic plaque. Statins reduce the production of reactive oxygen species (ROS) and increase the resistance of LDL to oxidation. In this study, we examined COX-2 expression in human carotid plaque by immunohistochemistry (IHC) and characterized the effects of H2O2 and simvastatin (SIM) on COX-2 expression in rat aortic smooth muscle cells (RASMCs). Methods: COX-2 localization in carotid plaque was analyzed by IHC and protein levels determined by Western immunoblotting. RASMCs were treated with varying concentrations of H2O2 and SIM. The COX-2 inhibitor NS398 and COX-2 siRNA were used to inhibit COX-2 activity or knockdown COX-2 expression. Levels of the COX-2 end-products PGE2 and PGI2 were determined by ELISA and apoptosis detected by caspase-3 assay and DNA fragmentation. Results: COX-2 was detected in macrophages and smooth muscle cells in complex carotid lesions with increased expression in plaque, compared to adjacent reference intima by IHC and Western blotting. H2O2 induced time and dose (0.1~0.3 mM) dependent up-regulation of COX-2 expression in RASMCs, which was synergistically increased by SIM (5 μM). H2O2 + SIM induced COX-2 upregulation was accompanied by a 2-fold increase in PGE2 and 3-fold increase in PGI2, which was inhibited by pre-treatment with NS398. Treatment of RASMCs with H2O2 + SIM induced caspase-3 cleavage and activity and increased DNA fragmentation. Pretreatment with NS398 or transfection with COX-2 siRNA produced additional increases in RASMC H2O2 + SIM-induced apoptosis. Conclusions: COX-2 expression is increased in complex carotid plaque. Inhibition or knockdown of COX-2 activity or expression accelerates ROS-induced RASMC apoptosis suggesting a cytoprotective effect of COX-2. The increase in smooth cell apoptosis induced by ROS + high dose (5 μM) SIM may adversely influence plaque stability.