Protective Role of Activating PPARγ in Advanced Glycation End Products-Induced Impairment of Coronary Artery Vasodilation Via Inhibiting P38 Phosphorylation and Reactive Oxygen Species Production.

Advanced glycation end products (AGEs) can damage voltage-gated K+ (Kv) channels and attenuate coronary artery vasodilation, but the underlying mechanisms remain unclear. The aim of this study was to investigate the role and potential mechanism of PPAR gamma in AGEs-induced Kv 1 channels impairment. We used both primary rat coronary smooth muscle cells (CSMCs) in vitro and Zucker Diabetic Fatty (ZDF) rat model in vivo. Over-expression of the Pparg gene by lentivirus vector (LV-Pparg) was used to transfect CSMCs for upregulation PPAR gamma. Kv 1.2 and Kv 1.5 currents were measured by patch clamp. The vascular tone of coronary artery was evaluated by isometric force measurements. The proteins expression of Kv1.2 and Kv1.5 channel were detected by western blot. PPAR gamma was detected by immunofluorescence and western blot. Oxidative stress markers including superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) were detected by enzyme linked immunosorbent assay (ELISA). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) and total p38 expression were detected by western blot. The intracellular ROS levels were measured by the fluorescent dye 2',7'- dichlorofluorescein diacetate (DCFDA) and a cellular ROS assay kit. We found that activating PPAR gamma via LV-Pparg (100 MOI, 5 x 10(8) TU/mL) prevented AGEs (100 mu g/mL) -mediated impairment of Kv 1.2 and Kv 1.5 channels activity and improved the reduction of Kv 1.2 and Kv 1.5 protein expression in CSMCs. Isometric force measurements showed that activating PPAR gamma by pioglitazone (10 mg/kg/d, intragastric administration) improved the impairment of coronary artery vasodilation, and western blot analysis showed that activating PPAR gamma increased the Kv 1.2 and Kv 1.5 protein expression, while inhibiting PPAR gamma by GW9662 (10 mg/kg/d, intraperitoneal injection) attenuated these effects in ZDF rats. Furthermore, LV-Pparg overexpression PPAR gamma attenuated NADPH oxidase activity, which was shown as the reduction of the NOX2 and p22phox expression by western blot analysis, decreased the MDA production and increased the SOD and GPx activities by ELISA, finally led to reduce AGEs-mediated ROS production. Moreover, activating PPARy by LV-Pparg inhibited AGEs-induced phosphorylation of p38 MAPK, by which could reduce AGEs-mediated NOX2, p22phox expression and ROS production, while CSMCs treatment with 513203580 (10 mu mol/L), a p38 MAPK inhibitor, attenuated these effects. Activating PPARy plays a protective role in AGEs-induced impairment of coronary artery vasodilation by inhibiting p38 phosphorylation to attenuate NOX2 and p22phox expression and further decrease oxidative stress induced by ROS overproduction.