GAPDH prevents oxidant‐induced apoptosis in smooth muscle cells via upregulation of APE1/Ref‐1 endonuclease (1093.1)

Abstract

We have shown that oxidized low density lipoprotein (OxLDL) co‐localized with apoptotic smooth muscle cells (SMC) in atherosclerotic plaque and that OxLDL downregulated the major glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in cultured SMC via H2O2‐dependent mechanism. We hypothesized that GAPDH downregulation mediates SMC apoptosis.OxLDL (80 ug/ml) or H2O2 (220 uM, 16h) decreased GAPDH protein (immunoblotting, 78±5% and 55±5% decrease, respectively) and induced TUNEL‐positive DNA fragmentation and apoptosis (Annexin V assay). GAPDH downregulation with siRNA potentiated OxLDL‐ or H2O2‐induced DNA fragmentation (1.9±0.2 and 7.0±0.9‐fold increase, respectively) suggesting that maintenance of sufficient GAPDH levels is critical for DNA integrity and cell survival. GAPDH transient overexpression with pCMV‐GAPDH construct decreased H2O2‐induced cytotoxicity (LDH release assay, 68±5% decrease) and DNA fragmentation (74±4% decrease). SMC constitutively overexpressing GAPDH had increased glycolysis, ATP levels and these cells were resistant to H2O2‐induced cell death (>90% reduction) showing that GAPDH protects SMC against oxidant‐induced apoptosis. We have found that GAPDH was co‐immunoprecipitated and had nuclear co‐localization with APE1/Ref‐1 endonuclease, major DNA repair enzyme. APE1 endonuclease activity was increased in H2O2‐treated GAPDH overexpressing SMC vs.control SMC suggesting that GAPDH preserved APE1 activity under oxidative stress.In summary, GAPDH downregulation mediates oxidant‐induced SMC apoptosis and forced expression of GAPDH protects SMC from oxidant‐induced cell death potentially via stimulation of APE1‐dependent DNA repair. Since SMC apoptosis induces vulnerable plaque phenotype and accelerates atherosclerosis, our data suggest that preservation of GAPDH and APE1 activity in plaque SMC could represent novel therapy to prevent plaque destabilization and atherogenesis.Grant Funding Source: Supported by American Heart Association