Abstract P216: Nicotinamide Nucleotide Transhydrogenase Regulates Mitochondrial Redox Balance and Endothelial Function in Response to Angiotensin II

Abstract

Hypertension (HTN) involves increased peripheral resistance to blood flow mediated by changes in vascular tone. Endothelial dysfunction is an initiating step in HTN and mitochondrial reactive oxygen species (ROS) contribute to endothelial dysfunction. Nicotinamide nucleotide transhydrogenase (NNT) is a critical enzyme in the regulation of mitochondrial NADPH levels and can impact mitochondrial redox status. We have demonstrated that the loss of NNT in C57Bl/6J mice is associated with a more severe hypertensive phenotype in response to Ang II characterized by mitochondrial and vascular dysfunction driven by vascular ROS production in vivo and in vitro . Little is known regarding the role of NNT human endothelial cells, but based on previous studies we hypothesize that the loss of NNT promotes mitochondrial and cellular ROS production and disrupts normal endothelial • NO function. In human aortic endothelial cells (HAEC) treatment with 500nM Ang II for two hours significantly increased NNT expression (225 + 1 3%) and glutathione peroxidase 2 (Gpx2) activity (17.81 + 0 .75 vs 29.06 + 2 .53 nmol/min/ug protein) consistent with NNT’s role in driving the mitochondrial antioxidant systems. Conversely, siRNA knockdown of NNT inhibited Gpx2 activity (18.32 + 1.17 vs 13.16 + 0 .7 nmol/min/ug protein) and increased mitochondrial superoxide production (0.12 + 0 .01 vs 0.17 + 0 .01 nmol/mg protein) in response to Ang II. These changes in the redox balance of the mitochondria contribute to mitochondrial dysfunction where the loss of NNT led to a marked decrease in the respiratory control ratio (RCR) of the mitochondria (6.42 + 0 .17 vs 3.83 + 0 .1) of Ang II treated cells. Ultimately, this shift to a pro-oxidative mitochondrial redox phenotype in these cells conspired to inhibit Ang II-induced • NO production (35.60 + 1 .28 vs 30.10 + 1 .24 pmol/mg protein). Our data indicate that the absence of NNT expression increases the steady state levels of mitochondrial ROS through increased ROS production and decreased antioxidant activity, which is contributes to reduced • NO production and endothelial dysfunction. These studies highlight NNT as a novel regulator of mitochondrial redox tone that may critically impact the ability of vascular ROS to contribute to the development of HTN.