TRP Channels In The Regulation of Vascular Tone

 描述(申请人提供)：血管动态平衡严重依赖于内皮释放的因子，其中最突出的是一氧化氮(NO)、前列环素(PGI2)和一类内皮衍生的超极化因子。血管疾病及其危险因素的存在会改变这些血管内皮细胞因子的平衡。我们以前的研究已经证明，冠状动脉疾病(CAD)患者的冠状动脉(HCA)会发生这种变化，层流或剪切的生理刺激会诱导内皮细胞产生过氧化氢(H_2O_2)以引起扩张；然而，在没有疾病的情况下，同样的刺激释放的NO作为主要的扩张因素。在血流介导的扩张(FMD)中，剪切如何导致两种不同因子的释放仍不完全清楚。虽然双氧水和非扩张型HCA都有不同或相反的非血管舒缩作用，但对血管壁的稳态和动脉粥样硬化的倾向有不同的影响。这个项目的总体目标是阐明导致这两种FMD介质释放的细胞内途径，并根据已发表的和初步的数据展开，这些数据表明瞬时受体潜在香草素(TRPV)通道在血管扩张因子的内皮产生中起关键作用。这一建议的总体假设是，HCAs的FMD涉及TRPV通道从健康(TRPV1/2；TRPV1和2)到疾病(TRPV4；TRPV4型)的功能切换。这种转换的结果是，扩张的介体从NO变成了过氧化氢。我们提出了三个具体目标来检验这一假设。目的1研究冠心病患者冠状动脉FMD中内皮细胞TRPV通道参与的频谱。目的2试图确定在非冠心病组和冠心病组中，血流诱导的TRPV激活和产生不同的内皮扩张器因子的信号通路。我们将继续寻求初步数据，表明花生四烯酸(AA)在HCAs的FMD中作为TRPV4的内源性激活剂，可能是通过TRPV4蛋白上潜在的新的花生四烯酸识别序列(ARS)。我们还将测试这一创新假设，即一氧化氮和过氧化氢介导的TRPV通道变构调节加强了非冠心病和冠心病患者血管扩张剂释放的两条不同途径。最后，目标3将确定TRPV4是否作为分子开关参与内皮扩张器因子和氧化还原平衡的改变。对新鲜分离的人类冠状动脉和内皮细胞的研究将采用多方面的方法，包括分离的血管反应性、钙成像、膜片钳记录Trp通道活性、生化方法(如配体结合分析)以及包括基因过度表达和蛋白质突变在内的分子技术。基因工程小鼠也将被用于证实人类数据，并支持因果关系和活体意义。我们期望这项拟议工作的成功将提供新的转换和机制方面的见解，使我们了解在冠心病中扩张器因子和血管氧化应激的转归，并影响我们对冠状动脉粥样硬化的理解。

 DESCRIPTION (provided by applicant): Vascular homeostasis is critically dependent upon factors released from the endothelium, the most prominent of which are nitric oxide (NO), prostacyclin (PGI2), and a class of endothelium-derived hyperpolarizing factors. The presence of vascular disease and its risk factors can change the balance of these endothelial factors. Our previous studies have demonstrated that such a change in human coronary arterioles (HCAs) from subjects with coronary artery disease (CAD), where the physiological stimulus of laminar flow or shear induces endothelial production of hydrogen peroxide (H2O2) to elicit dilation; in the absence of disease, however, the same stimulus releases NO as the main dilator factor. How shear induces the release of two distinct factors in flow-mediated dilation (FMD) remains incompletely understood. While both H2O2 and NO dilate HCAs, each has different or opposing non-vasomotor effects on vessel wall homeostasis and propensity for atherosclerosis. The overall goal of this project is to elucidate the intracellular pathways responsible for the releaseof these two mediators of FMD, expanding upon published and preliminary data indicating a crucial role for transient receptor potential vanilloid (TRPV) channels in endothelial production o vasodilator factors. The overall hypothesis of this proposal is that FMD of HCAs involves a functional switch of TRPV channels from health (TRPV1/2; TRPV type 1 and 2) to disease (TRPV4; TRPV type 4). As a result of this switch, the mediator of dilation changes from NO to H2O2. We propose three specific aims to test this hypothesis. Aim 1 will determine the spectrum of endothelial TRPV channel involvement in FMD of coronary arteries in the absence and presence of CAD. Aim 2 attempts to define the signaling pathways responsible for flow-induced TRPV activation and production of disparate endothelial dilator factors in non-CAD versus CAD. We will pursue preliminary data suggesting that arachidonic acid (AA) serves as an endogenous activator of TRPV4 in FMD of HCAs, likely via a potentially novel arachidonate recognition sequence (ARS) on TRPV4 protein. We will also test the innovative hypothesis that NO- and H2O2-mediated allosteric regulation of TRPV channels reinforces two distinct pathways of vasodilator release in non-CAD versus CAD. Finally, aim 3 will determine whether TRPV4 serves as a molecular switch contributing to the change of endothelial dilator factors and redox balance. Studies will be conducted on freshly isolated human coronary vessels and endothelial cells using a multifaceted approach incorporating isolated vessel reactivity, Ca2+ imaging, patch-clamping recording of TRP channel activity, biochemical methods such as ligand binding assays, and molecular techniques including gene overexpression and protein mutagenesis. Genetically engineered mice will also be used to corroborate human data and to support causality and in vivo significance. We expect that success of the proposed work will provide new translational and mechanistic insights into the pathways responsible for the transition of dilator factors and vascular oxidative stress in CAD and impact our understanding of coronary atherosclerosis.