Abstract 13447: GAPDH Protects Vascular Smooth Muscle Cells Against Oxidant-Induced Cell Death: Potential Role 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 expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured SMC via a redox-sensitive mechanism. We hypothesized that GAPDH downregulation mediates SMC apoptosis. OxLDL (80 ug/ml) or H2O2 (220 uM, 16h) decreased GAPDH protein by 78±5% and 55±5%, respectively (immunoblotting with GAPDH antibody) and induced TUNEL-positive DNA fragmentation and cell apoptosis (Annexin V assay). Koningic acid, a specific inhibitor of GAPDH activity, potentiated H2O2-induced cytotoxicity (4.0±0.3-fold increase, P<0.01, LDH release assay) and DNA fragmentation induced by OxLDL (2.5±0.2-fold, P<0.05) or by H2O2 (8.5±0.6-fold, P<0.001). Anti-GAPDH siRNA (50 nM) reduced GAPDH protein by 88±6% and potentiated OxLDL- and H2O2-induced DNA fragmentation (1.9±0.2 and 7.0±0.9-fold increase, respectively) suggesting that maintenance of GAPDH activity is critical for DNA integrity and cell survival under oxidative stress. SMC transfection with pCMV-GAPDH increased GAPDH protein (3.5-fold), reduced H2O2-induced cytotoxicity (68±5% decrease, P<0.01) and prevented DNA fragmentation (74±4% decrease, P<0.005) vs. pCMV-GFP. GAPDH is known to bind Ape1 endonuclease (the major DNA repair enzyme) and to stimulate Ape1-dependent DNA repair. H2O2-induced GAPDH downregulation correlated with reduced Ape1 activity in SMC. Alpha-smooth muscle actin-positive and mainly apoptotic SMC in mouse atherosclerotic plaque fibrous cap had increased levels of oxidative stress marker (43±10% increase in N-tyrosine staining, P<0.05), reduced GAPDH levels (48±5% decrease, P<0.05), and elevated DNA oxidation (22±5% increase in 8-oxo-2’-deoxyguanosine staining, P<0.05) vs. medial SMC. 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. Plaque SMC have increased oxidative stress, reduced GAPDH and damaged DNA. These data demonstrate that GAPDH is critical for protection against SMC cell death and plaque destabilization.
- © 2012 by American Heart Association, Inc.