Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT

Objective - Oxidative stress ( OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 ( HO- 1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/ Akt may modulate HO- 1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO- 1 in OS- induced apoptosis of human aortic smooth muscle cells ( HASMCs). Methods and Results - HASMCs were transduced with retroviral vectors expressing HO- 1, Akt, or GFP and exposed to H2O2. Cell viability was assessed by MTT assay. OS was determined by CM- H2DCFDA fluorescence, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling ( TUNEL), caspase- 3 activity, and Bcl- 2/ Bad levels. Mitochondrial membrane potential (Delta psi(m)) was assessed by fluorescence- activated cell sorter ( FACS) using JC- 1. HO- 1 reduced H2O2- induced OS and apoptosis. Akt knockdown removed the protective effect of HO- 1 on Delta psi(m) during exposure to H2O2. Conversely, HO- 1 knockdown removed the protective effect of Akt on Delta psi(m). Inhibition of PI3K- Akt reduced induction of HO- 1 protein expression by H2O2 and blocked its anti-apoptotic effects. The Akt- mediated upregulation of HO- 1 was dependent on activation of HO- 1 promoter by Nrf2. Conclusion - HO- 1 and Akt exert codependent cytoprotective effects against OS- induced apoptosis in HASMCs. These findings may have implications for the design of novel therapeutic strategies for plaque stabilization.