Abstract 15038: GAPDH Interaction With APE1 Endonuclease Protects Smooth Muscle Cells Against Cell Death: Potential Role of These Enzymes in Prevention of Atherosclerotic Plaque Destabilization
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 an H2O2-dependent mechanism. We hypothesized that downregulation of GAPDH 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 cell apoptosis (Annexin V assay). A/GAPDH siRNA reduced GAPDH protein by 88±6% and 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 under oxidative stress. Human SMC transfection with pCMV-GAPDH construct increased GAPDH protein (3.5-fold), reduced H2O2-induced cytotoxicity (LDH release assay, 68±5% decrease, P<0.01) and prevented DNA fragmentation (74±4% decrease, P<0.005) vs. pCMV-GFP. Rat SMC constitutively overexpressing GAPDH (1.9-fold) had increased glycolysis (Lactate assay, 54±5% increase), ATP levels (bioluminescent assay, 38±2% increase) and were resistant to H2O2-induced cell death (>90% reduction, TUNEL assay). GAPDH is known to bind APE1 endonuclease (the major DNA repair enzyme) and to stimulate APE1-dependent DNA repair. We found that GAPDH co-immunoprecipitated and co-localized with APE1 in SMC with/without oxidative stress. APE1 specific activity was increased in the GAPDH-APE1 immunocomplex in H2O2-treated GAPDH overexpressing SMC vs. H2O2-treated control cells 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 H2O2-induced cell death potentially via activation of APE1-dependent DNA repair. Vascular SMC apoptosis is known to induce a vulnerable plaque phenotype and to accelerate atherosclerosis. Our data suggest that preservation of GAPDH and APE1 activity in plaque SMC could be a potential strategy to prevent plaque destabilization and subsequent acute coronary events.
- © 2013 by American Heart Association, Inc.