TRP channels in regulation of vascular tone

描述(申请人提供)：血流产生的切应力是血管张力最重要的生理调节因素之一。Flow刺激血管内皮细胞释放血管扩张因子，随后松弛底层的平滑肌，这种反应通常被称为流动介导的扩张(FMD)。在人类冠状动脉小动脉(HCA)中，FMD是两种完全独立的扩张因子释放的结果：非冠状动脉疾病(CAD)患者的一氧化氮(NO)和冠心病患者的活性氧物种(ROS)，特别是线粒体电子传递链中的过氧化氢(H_2O_2)。然而，剪切如何导致这两个不同的松弛因子的释放仍未解决。目前的提议将检验一个中心假设，即瞬时受体电位香草素4(TRPV4)通道是两种不同调节的松弛因子(NO和线粒体衍生的过氧化氢)释放的共同机制，这些松弛因子与人类冠脉微循环中的FMD有关。我们进一步提出，信号级联发生在小窝内，这些小窝拥有质膜和线粒体之间的新的相互作用。我们将使用一种结合分子生物学、电生理学和荧光/电子成像技术以及体外血管反应性评估的综合方法，对分离的HCA和培养的内皮细胞进行研究。基因工程小鼠也将被用来为人类数据提供更明确的证实。提出了三个具体目标。目的1将确定FMD是否需要在患有或不患有冠心病的患者的HCA中使用内皮细胞TRPV4。我们将测试TRPV4的药理抑制和siRNA下调对冠心病和非冠心病患者HCA血流诱导的钙内流、ROS/NO释放和血管扩张的影响。在目标2中，我们将研究内皮TRPV4通道是否与小凹相关，以及这种关联是否对剪切诱导的TRPV4激活是必要的。我们将测试与TRPV4激活有关的三个小窝相关信号事件：即TRPV4易位、小窝蛋白-1调节和磷脂酶A2-环氧二十碳三烯酸的激活。在目标3中，我们将确定剪切是否通过涉及腔泡TRPV4和邻近线粒体的局部钙信号增加线粒体ROS，以及这一过程是否受到NO的负调控。这项拟议的研究将首次将内皮细胞TRPV4、小窝和线粒体联系起来，作为人类口蹄疫的基本信号组件。我们期待这项提议的结果将大大增加我们对人类冠脉微循环中涉及FMD的复杂信号机制的理解，并可能导致治疗CAD和/或其他心血管疾病的新的治疗靶点。 与公共健康相关：这项拨款提案旨在研究一种新的信号机制，通过这种机制，剪切力--一种由血液流动产生的机械力--会导致人类的血管扩张。具体地说，我们研究了位于血管内皮细胞表面膜上的钙离子通道(TRPV4)是否为人类冠状动脉微血管剪切性扩张提供了必要的信号成分。血流或剪切诱导的扩张是调节血管张力和局部血流的最重要的调节因素之一。因此，这项建议的发现将有助于我们理解冠状动脉血流在正常和疾病状态下是如何调节的，并可能为冠状动脉疾病和/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.