Abstract 19986: Rotenone-Induced Mitochondrial Oxidative Stress Corrupts Tube Formations in Human Coronary Artery Endothelial Cells by Inhibiting mTOR Signaling Pathway
The function of the heart is highly dependent on oxidative energy that is generated in mitochondria. A by-product of mitochondrial bioenergetic activity is generation of reactive oxygen species. We have recently observed increased mitochondrial oxidative stress in Zucker obese fatty rats (ZOF), a rat model of human metabolic syndrome. A striking feature of the ZOF rat is abrogation of coronary collateral growth in response to ischemia. These results suggested that mitochondrial oxidative stress negatively impacts coronary collateral growth. To gain insight into mechanisms underlying the effects of mitochondrial oxidative stress, we used an in vitro model system of angiogenesis (tube formation) in the setting of mitochondrial oxidative stress. Accordingly, we treated human coronary artery endothelial cells (HCAECs) with either 50 ng/ml vascular endothelial growth factor (VEGF), 2 uM Rotenone (Rot) or VEGF and Rot, respectively, in incubators supplemented with either 20.0% (normoxic) or 2.5% (hypoxic) oxygen for 48 hr. VEGF induced robust tube formation under normoxic and hypoxic conditions; however, rotenone completely inhibited formation of tubes in the presence of VEGF during normoxia or hypoxia (n=4 [8 wells per experiment]) (p<0.05 vs VEGF treatment). Inhibition of tube formation was also associated with significant increased in hydrogen peroxide, but not cytosolic or mitochondrial-derived superoxide. Immunoblot analyses showed significant inhibition of the mammalian target of rapamycin (mTOR)-mediated phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E BP1) (n=3; p<0.05) and ribosomal p70 S6 kinase (S6K1) of HCAECs cultured in both normoxic and hypoxic conditions with Rot treatment, which is consistent with decreased protein synthesis. In addition Rot treatment also activated upstream mTOR negative regulators, Akt and AMP-activated protein kinase α (AMPKα). Our data suggest that the negative effects of mitochondrial oxidative stress on coronary collateral growth may be mediated by corrupted mTOR signaling, which leads to impairments in new protein synthesis that is necessary for the growth of blood vessels.
- © 2010 by American Heart Association, Inc.