Introduction Increasing evidence suggests vascular oxidative stress is involved in the development of endothelial dysfunction and cardiovascular disease (CVD). Mitochondria are implicated as a source of vascular superoxide production in a number of CVDs, including coronary artery disease (CAD) and type 2 diabetes (T2D). It has also been proposed that mitochondrial reactive oxygen species (mtROS) activate AMP-activated protein kinase (AMPK) in endothelial cells. We investigated mtROS production in intact saphenous veins and primary saphenous vein endothelial cells (HSVECs) from both patients with advanced CAD and healthy control subjects.
Methods Portions of saphenous vein were obtained from patients undergoing coronary artery bypass grafting (n=54) and elective varicose vein removal (n=19), and HSVECs isolated and cultured from selected vessels. Endothelial function was assessed by relaxation to calcium ionophore A23187 and vascular superoxide production measured using lucigenin-enhanced chemiluminescence. Gene expression was assessed by qRT-PCR and AMPK activity assayed via radioactively-labelled ATP incorporation.
Results Endothelium-dependent vasodilation was impaired in patients with CAD compared to control subjects (maximum relaxation, 43±16 vs 62±16%; p=0.001) and in patients with both CAD and T2D relative to those with CAD alone (34±11 vs 47±16%; p=0.008). Vascular superoxide production was greater in patients with CAD (0.57 (0.39;1.01) nmol/mg/min) than in control subjects (0.42 (0.26;0.64) nmol/mg/min; p=0.008). At a cellular level, significantly increased HSVEC SOD2 expression (ΔCt, −6.51±0.81 vs −3.9±1.2; p=0.044) and AMPK activity (0.062±0.011 vs 0.022±0.006 nmol/min/mg; p=0.007) were observed in patients with CAD and T2D relative to controls. The mitochondria-targeted antioxidant, MitoQ10 significantly reduced AMPK activity in HSVECs from patients with CAD and T2D (0.056±0.004 vs 0.009±0.01 nmol/min/mg; p=0.016) but not in HSVECs from patients with CAD alone, where the effects of MitoQ10 were non-significant.
Conclusions These findings suggest that mitochondria contribute to increased superoxide production and endothelial dysfunction in patients with CAD and T2D. In addition, results support a novel, mtROS-mediated mechanism for AMPK activation in the endothelium of patients with CAD and T2D and a role for the kinase in oxidative stress defence. Mitochondria-targeted antioxidants, used in combination with pharmacological activators of AMPK, may therefore have enhanced potential in prevention and treatment of CAD in patients with T2D.