TRP channels in regulation of vascular tone

描述（申请人提供）：血流产生的剪切应力是血管张力最重要的生理调节因子之一。血流刺激血管内皮细胞释放血管舒张因子，随后放松下层平滑肌，这种反应通常被称为血流介导的舒张（FMD）。在人类冠状动脉（HCA）中，口蹄疫是由两种完全独立的扩张因子的释放引起的：无冠状动脉疾病（CAD）受试者的一氧化氮（NO）和CAD患者的活性氧（ROS），特别是来自线粒体电子传递链的过氧化氢（H2O2）。然而，剪切如何诱导这两种不同的松弛因子的释放仍未得到解决。本研究将验证一个中心假设，即瞬时受体电位香草样蛋白4 （TRPV4）通道是人类冠状动脉微循环中负责FMD的两种不同调节的放松因子（NO和线粒体来源的H2O2）释放的共同机制。我们进一步提出，信号级联发生在细胞质膜和线粒体之间的新相互作用的小泡内。研究将在分离的HCA和培养的内皮细胞上进行，采用综合的方法，结合分子生物学、电生理学和荧光/电子成像技术，并在体外评估血管反应性。基因工程小鼠也将被用于提供对人类数据更明确的佐证。提出了三个具体目标。目的1将确定患有或不患有CAD的患者的HCA是否需要内皮细胞TRPV4。我们将测试TRPV4的药理抑制和siRNA下调对CAD和非CAD受试者HCA血流诱导的Ca2+进入、ROS/NO释放和血管舒张的影响。在目的2中，我们将研究内皮TRPV4通道是否与小泡相关，以及这种关联是否对剪切诱导的TRPV4激活至关重要。我们将测试三种与TRPV4激活有关的小窝相关信号事件：即TRPV4易位、小窝蛋白-1调节和磷脂酶a2 -环氧二碳三烯酸激活。在目的3中，我们将确定剪切是否通过涉及空泡TRPV4和邻近线粒体的局部Ca2+信号通路增加线粒体ROS，以及这一过程是否受到NO的负调控。拟议的研究将首次将内皮TRPV4、小泡和线粒体作为人类口蹄疫的重要信号成分联系起来。我们期望这一建议的结果将大大增加我们对口蹄疫在人类冠状动脉微循环中复杂的信号机制的理解，并可能为CAD和/或其他心血管疾病的治疗提供新的治疗靶点。

DESCRIPTION (provided by applicant): Shear stress generated by blood flow is one of the most important physiological regulators of vascular tone. Flow stimulates vascular endothelial cell to release vasodilator factors that subsequently relax underlying smooth muscle, a response often known as flow-mediated dilation (FMD). In human coronary arterioles (HCA), FMD results from the release of two entirely independent dilator factors: nitric oxide (NO) in subjects without coronary artery disease (CAD), and reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2) derived from the mitochondrial electron transport chain, in CAD patients. However, it remains unsolved how shear induces the release of these two distinct relaxing factors. The present proposal will test a central hypothesis that the transient receptor potential vanilloid 4 (TRPV4) channel serves as a common mechanism for the release of two otherwise diversely regulated relaxing factors (NO and mitochondria-derived H2O2) responsible for FMD in the human coronary microcirculation. We further propose that the signaling cascade occurs within caveolae that host novel interactions between the plasma membrane and mitochondria. Studies will be conducted on isolated HCA and cultured endothelial cells using an integrated approach incorporating molecular biology, electrophysiology and fluorescence/electron imaging techniques with in vitro assessment of vessel reactivity. Genetically engineered mice will also be used to provide more definitive corroboration of the human data. Three specific aims are proposed. Aim 1 will determine whether FMD requires endothelial TRPV4 in HCA from patients with or without CAD. We will test the effects of pharmacological inhibition and siRNA downregulation of TRPV4 on flow-induced Ca2+ entry, ROS/NO release, and vasodilation in HCA from CAD and non-CAD subjects. In aim 2, we will examine whether endothelial TRPV4 channels are associated with caveolae and whether this association is essential for shear-induced TRPV4 activation. We will test three caveolae-associated signaling events contributing to TRPV4 activation: namely, TRPV4 translocation, caveolin-1 regulation, and phospholipase A2-epoxyeicosatrienoic acids activation. In aim 3, we will determine whether shear increases mitochondrial ROS through localized Ca2+ signaling involving caveolar TRPV4 and adjacent mitochondria and whether this process is negatively regulated by NO. The proposed research will, for the first time, link endothelial TRPV4, caveolae, and mitochondria as essential signaling components for FMD in humans. We expect the outcomes of this proposal will substantially increase our understanding of the intricate signaling mechanisms involved in FMD in the human coronary microcirculation, and may lead to new therapeutic targets for the treatment of CAD and/or other cardiovascular disorders. PUBLIC HEALTH RELEVANCE: This grant proposal is designed to study a novel signaling mechanism by which shear stress, a mechanical force generated by blood flow, causes blood vessel dilation in humans. Specifically we examine whether a calcium ion channel (TRPV4) located on the cell surface membrane of vascular endothelial cells serves an essential signaling component for shear-induced dilation in human coronary microvessels. Flow or shear- induced dilation is one of the most important regulators of vascular tone and regional blood flow. Therefore the findings of this proposal will importantly contribute to our understanding of how coronary blood flow is regulated in normal and disease states, and may lead to new therapeutic targets for the treatment of coronary artery disease and/or other cardiovascular disorders.