Nicotinamide nucleotide transhydrogenase regulates redox balance in atherosclerosis

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PROJECT ABSTRACT Atherosclerosis, a progressive chronic inflammatory disease of the vessel wall, is regulated by oxidant stress throughout the course of disease development. Endothelial dysfunction is a critical, initiating step in the development of atherosclerosis and increasing evidence implicates mitochondrial reactive oxygen species (ROS) as an important contributor to endothelial dysfunction and vascular inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is emerging as an important enzyme in the regulation of mitochondrial NADPH levels which can have a significant impact on a number of metabolic pathways through the regulation of mitochondrial redox balance. Preliminary data from our laboratory supports this concept demonstrating that NNT expression is reduced in the endothelium of atherosclerotic patients when compared to healthy controls. These data are consistent with previously published data from our laboratory indicating that AAV8-PCSK9 and high fat diet treatment led to increased plaque burden in C57Bl/6J mice that lack NNT. Recent studies utilizing small hairpin RNA knockdown of NNT in human aortic endothelial demonstrates increased hydrogen peroxide production that is associated with reductions in mitochondrial NADPH and impairment of downstream antioxidant enzymes glutathione peroxidase and glutathione reductase. Furthermore, the loss of NNT in these cells also led to increased adhesion molecule expression and inflammatory cell trafficking suggesting that NNT’s role in regulating mitochondrial balance can play a critical role in modulating atherosclerotic plaque development. We have confirmed these observations in vivo using a novel endothelial specific knockout mouse where the loss of NNT promotes enhanced adhesion molecule expression in the aortic arch of mice subjected to AAV8-PCSK9, partial carotid ligation, and high fat diet in a model of disturbed flow induced plaque development. Building upon these findings, we propose that the loss of NNT activity contributes to a pro-oxidative mitochondrial phenotype that exacerbates the progression of atheroscelrosis by enhancing mitochondrial ROS production, endothelial dysfunction, and vascular inflammation . To test this hypothesis, studies are proposed that will determine i) if NNT inhibits mitochondrial ROS production and preserves mitochondrial antioxidant activity and oxidative metabolisim in human vascular endothelial cells; ii) if reduced NNT expression and increased mitochondrial ROS production stimulates Nox activity that contributes to reduced NO bioavailibilty and promotes endothelial activation; and iii) if endothelial NNT critically regulates mitochondrial redox balance and vascular function in mice treated with high fat diet. Data from the proposed studies will identify NNT as a master regulator of mitochondrial function and ROS production whose absence exacerbates the development of atherosclerosis by promoting endothelial dysfunction and vascular inflammation, leading to increased plaque development.