TRP Channels In The Regulation of Vascular Tone

 描述（由申请人提供）：血管稳态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（PGI 2）和一类内皮源性超极化因子。血管疾病及其危险因素的存在可以改变这些内皮因子的平衡。我们以前的研究已经证明，这种变化在人类冠状动脉（HCA）的受试者与冠状动脉疾病（CAD），其中层流或剪切的生理刺激诱导内皮细胞产生过氧化氢（H2 O2）引起扩张;在没有疾病的情况下，然而，相同的刺激释放NO作为主要的扩张因子。剪切力如何诱导血流介导的扩张（FMD）中两种不同因子的释放仍不完全清楚。虽然H2 O2和NO都扩张HCA，但各自对血管壁稳态和动脉粥样硬化倾向具有不同或相反的非血管扩张作用。本项目的总体目标是阐明负责FMD的这两种介质的释放的细胞内途径，扩展已发表的和初步的数据，这些数据表明瞬时受体电位香草酸（TRPV）通道在血管舒张因子的内皮生产中起关键作用。该建议的总体假设是HCA的FMD涉及TRPV通道从健康（TRPV 1/2; TRPV 1型和2型）到疾病（TRPV 4; TRPV 4型）的功能转换。由于这种转换，扩张的介质从NO变为H2 O2。我们提出了三个具体目标来检验这一假设。目的1将确定是否存在CAD时，内皮TRPV通道参与冠状动脉FMD的频谱。目的2试图确定非CAD与CAD中负责血流诱导的TRPV激活和不同内皮扩张因子产生的信号通路。我们将追求的初步数据表明，花生四烯酸（AA）作为内源性激活剂的TRPV 4在FMD的HCAs，可能通过一个潜在的新的花生四烯酸识别序列（ARS）的TRPV 4蛋白。我们还将测试创新的假设，即NO和H2 O2介导的TRPV通道的变构调节加强了两个不同的途径，血管扩张剂释放在非CAD与CAD。最后，目的3将确定TRPV 4是否作为分子开关促进内皮扩张因子和氧化还原平衡的变化。将使用多方面方法对新鲜分离的人冠状血管和内皮细胞进行研究，该方法包括分离的血管反应性、Ca 2+成像、TRP通道活性的膜片钳记录、生物化学方法（如配体结合试验）和分子技术（包括基因过表达和蛋白质诱变）。基因工程小鼠也将用于证实人类数据，并支持因果关系和体内意义。我们期望，所提出的工作的成功将提供新的翻译和机制的见解负责的扩张因子和血管氧化应激在CAD的过渡的途径，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.