Mitochondrial Dynamics and UCP2 - Endothelial Dysfunction in Human Obesity

描述（由申请人提供）： 最近的研究强调，内皮细胞中的线粒体在响应环境线索（包括营养过剩）的信号传导中起重要作用。线粒体信号传导在很大程度上是通过电子传递链的组分调节活性氧（ROS）的产生来介导的。生理信号传导依赖于解偶联蛋白-2（UCP 2）对膜电位的控制，以及通过线粒体分裂和融合之间的适当平衡来保持线粒体蛋白和mtDNA的完整性，以维持正常的线粒体网络（线粒体动力学）。肥胖与体内能量供需失衡有关。我们假设，慢性能量过剩会导致ROS增加的恶性循环，引发线粒体断裂和UCP-2反应不足，进一步增加ROS并损害内皮功能。在这个项目中，我们将涉及一氧化氮信号和内皮依赖性血管舒张线粒体功能的相关方面，在新鲜分离的动脉内皮细胞从肥胖患者和健康志愿者暴露于两个人类模型的能量过剩。我们的初步数据显示，在从肥胖患者收集的内皮细胞中，eNOS信号传导受损，UCP 2减少，线粒体碎片化和裂变蛋白Fis 1增加。我们的项目有3个具体目标：对于目标1，我们将从肥胖患者中收集动脉内皮细胞，并测量线粒体ROS、网络范围和UCP-2、Mfn 2和Fis 1的表达，并将结果与手臂中的内皮依赖性血管舒张和新鲜分离的细胞中的eNOS活化相关。我们还将确定是否沉默Fis 1或过表达UCP-2或Mfn 2恢复eNOS激活。在目标2中，我们将确定改变的动力学和UCP-2是否有助于Intraperoid输注诱导的内皮功能障碍（能量过剩），在目标3中，我们将确定这些机制是否有助于卧床休息诱导的内皮功能障碍（能量需求减少）。我们预计在肥胖患者的细胞中ROS增加，网络碎片化，UCP 2减少，eNOS活化受损。如果Intraperoid和卧床休息诱导肥胖内皮表型，并且如果过表达UCP-2或沉默Fis 1逆转内皮功能障碍，我们将有强有力的证据表明这些机制有助于人类肥胖中内皮功能障碍的发病机制。

DESCRIPTION (provided by applicant): Recent studies have emphasized that mitochondria in endothelial cells play an important role in signaling in response to environmental cues, including nutrient excess. Mitochondrial signaling is mediated in large part by regulated production of reactive oxygen species (ROS) by components of the electron transport chain. Physiological signaling depends on control of membrane potential by uncoupling protein-2 (UCP2) and preserved integrity of mitochondrial proteins and mtDNA via an appropriate balance between mitochondrial fission and fusion to maintain normal mitochondrial networks (mitochondrial dynamics). Obesity is associated with an imbalance between energy supply and demand in the body. We hypothesize that chronic energy excess creates a vicious cycle of increased ROS that triggers mitochondrial fragmentation and an inadequate UCP-2 response that further increases ROS and impairs endothelial function. In this project, we will relate nitric oxide signaling and endothelium-dependent vasodilation to relevant aspects of mitochondrial function in freshly isolated arterial endothelial cells from obese patients and from healthy volunteers exposed to two human models of energy excess. Our preliminary data show impaired eNOS signaling, decreased UCP2, mitochondrial fragmentation, and an increase in the fission protein Fis1 in endothelial cells collected from obese patients. Our project has 3 specific aims: For Aim 1, we will collect arterial endothelial cells from obese patients and measure mitochondrial ROS, network extent, and expression of UCP-2, Mfn2, and Fis1 and relate the findings to endothelium- dependent vasodilation in the arm and to eNOS activation in the freshly isolated cells. We will also determine whether silencing Fis1 or over-expressing UCP-2 or Mfn2 restores eNOS activation. In Aim 2, we will determine whether altered dynamics and UCP-2 contribute to endothelial dysfunction induced by Intralipid infusion (energy excess), and in Aim 3, we will determine whether these mechanisms contribute to endothelial dysfunction induced by bed rest (decreased energy demand). We anticipate increased ROS, network fragmentation, decreased UCP2, and impaired eNOS activation in cells from obese patients. If Intralipid and bed rest induce an obese endothelial phenotype and if over- expressing UCP-2 or silencing Fis1 reverses endothelial dysfunction, we will have strong evidence that these mechanisms contribute to the pathogenesis of endothelial dysfunction in human obesity.