Nicotinamide nucleotide transhydrogenase regulates redox balance in atherosclerosis

项目摘要 动脉粥样硬化是一种进行性慢性血管壁炎性疾病，受氧化应激的调节 在疾病发展过程中。内皮功能障碍是一个关键的，启动步骤， 动脉粥样硬化的发展和越来越多的证据表明线粒体活性氧 (ROS)作为内皮功能障碍和血管炎症的重要贡献者。烟酰胺核苷酸 转氢酶（NNT）是一种重要的酶，在线粒体NADPH水平的调节 它可以通过调节线粒体对许多代谢途径产生重大影响， 氧化还原平衡我们实验室的初步数据支持这一概念，表明NNT表达 与健康对照相比，动脉粥样硬化患者的内皮细胞中的β-淀粉样蛋白减少。这些数据 与我们实验室先前发表的数据一致，表明AAV 8-PCSK 9和高脂饮食 治疗导致缺乏NNT的C57 B1/6 J小鼠中斑块负荷增加。利用小发夹的最新研究 人主动脉内皮细胞中NNT的RNA敲低表明过氧化氢的产生增加， 与线粒体NADPH减少和下游抗氧化酶受损有关 谷胱甘肽过氧化物酶和谷胱甘肽还原酶。此外，这些细胞中NNT的丢失也导致了 增加的粘附分子表达和炎性细胞运输表明NNT在 调节线粒体平衡可以在调节动脉粥样硬化斑块发展中起关键作用。我们 已经使用一种新的内皮特异性敲除小鼠在体内证实了这些观察结果， NNT促进在经受AAV 8-PCSK 9的小鼠的主动脉弓中增强的粘附分子表达， 部分颈动脉结扎和高脂饮食在紊乱的流动诱导的斑块发展的模型中。基础上 根据这些发现，我们认为NNT活性的丧失有助于线粒体的促氧化作用， 通过增强线粒体ROS而加剧动脉粥样硬化进展的表型 产生、内皮功能障碍和血管炎症。为了验证这一假设， 提出将确定i）NNT是否抑制线粒体ROS产生并保留线粒体 人血管内皮细胞中的抗氧化活性和氧化代谢; ii）如果NNT表达降低， 增加线粒体ROS产生刺激Nox活性，这有助于降低NO生物利用度 和促进内皮活化;和iii）如果内皮NNT严格调节线粒体氧化还原平衡 和血管功能。拟议研究的数据将确定NNT为 线粒体功能和ROS产生的主要调节因子，其缺失加剧了 动脉粥样硬化通过促进内皮功能障碍和血管炎症，导致斑块增加 发展

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.