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提示(包括营养过剩)的信号中发挥着重要作用。线粒体信号在很大程度上是由电子传递链的组成部分调节产生的活性氧物种(ROS)所介导的。生理信号转导依赖于解偶联蛋白-2(UCP2)对膜电位的控制，并通过线粒体分裂和融合之间的适当平衡来维持线粒体蛋白质和线粒体DNA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，慢性能量过剩会造成ROS增加的恶性循环，从而触发线粒体碎裂和UCP-2反应不足，从而进一步增加ROS并损害内皮功能。在这个项目中，我们将在肥胖患者和健康志愿者的新鲜分离的动脉内皮细胞中，将一氧化氮信号和内皮依赖性血管扩张与线粒体功能的相关方面联系起来，这些细胞暴露在两种能量过剩的人类模型中。我们的初步数据显示，肥胖患者收集的内皮细胞中eNOS信号转导受损，UCP2减少，线粒体碎裂，裂变蛋白Fis1增加。我们的项目有三个特定的目标：目标1，我们将收集肥胖患者的动脉内皮细胞，测量线粒体ROS、网络范围、UCP-2、Mfn2和Fis1的表达，并将结果与手臂内皮依赖性血管扩张和新鲜分离的细胞中eNOS的激活相关联。我们还将确定沉默FIS1或过度表达UCP-2或Mfn2是否会恢复eNOS的激活。在目标2中，我们将确定动力学改变和UCP-2是否有助于Intralipid输注(能量过剩)引起的内皮功能障碍，在目标3中，我们将确定这些机制是否有助于卧床休息(能量需求减少)导致的内皮功能障碍。我们预计肥胖患者细胞内ROS增加，网络碎裂，UCP2减少，eNOS激活受损。如果Intralipid和卧床休息诱导了肥胖的内皮表型，如果过度表达UCP-2或沉默Fis1逆转了内皮功能障碍，我们将有强有力的证据表明这些机制在人类肥胖的内皮功能障碍的发病机制中起作用。

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.