TRPC Channels in the Metabolic Syndrome

描述（由申请人提供）：典型TRP通道（TRPC）在血管平滑肌细胞的质膜中形成储存和受体操作的Ca2+渗透通道。TRPCs参与调节血管张力和平滑肌细胞增殖。该提案将确定TRPC通道在与代谢综合征（MetS）相关的冠状动脉重构中的分子作用。MetS的特征是中枢性肥胖、血浆胆固醇和空腹血糖升高、高血压、胰岛素抵抗和动脉粥样硬化。在美国成年人中，met的患病率为24%。在本研究项目中，我们将利用具有MetS所有特征的Ossabaw猪模型，包括过度活跃的肾素-血管紧张素-醛固酮系统（RAAS）。我们证明TRPC1和TRPC6通道的表达水平在MetS的Ossabaw猪冠状动脉中显着升高，表现出动脉粥样硬化和过度收缩。一致地，新分离的MetS冠状动脉平滑肌细胞具有升高的储存/受体操作的Ca2+内流和大的储存/受体操作的trpc样电流。由于血管紧张素II和醛固酮是TRPC表达的正调节因子，我们假设，在MetS中，过度活跃的RAAS上调TRPC1和TRPC6的表达，从而导致冠状动脉平滑肌细胞增殖增加和冠状动脉过度收缩。本研究的具体目的如下：1)确定冠状动脉平滑肌细胞在met相关重构过程中TRPCs的分子表达是如何改变的；2)确定trpc对内源性储存和受体激活的Ca2+内流/电流在对照和MetS冠状动脉平滑肌细胞中的贡献；3)确定内源性TRPC通道的功能表达是否直接受冠状动脉平滑肌细胞中RAAS组分、血管紧张素II和Aldo的调控；4)确定在体内，冠状动脉靶向下调TRPCs是否减缓了MetS猪动脉粥样硬化的进展并降低了冠状动脉的过度收缩。在这个研究项目中，我们将使用分子生物学、生物化学、电生理学和荧光成像方法以及血管内超声、等长张力和冠状动脉环管腔面积测量。此外，将采用冠状动脉靶向递送方法在体内将shRNAs和cDNA构建物部署到冠状动脉壁中。重要的是，我们将确定TRPC1和TRPC6在met相关的原生动脉粥样硬化进展中的独特作用。

DESCRIPTION (provided by applicant): Canonical TRP channels (TRPC) form store- and receptor-operated Ca2+-permeable channels in the plasma membrane of vascular smooth muscle cells. TRPCs have been implicated in regulating vascular tone and smooth muscle cell proliferation. This proposal will define the molecular roles of TRPC channels during coronary artery remodeling associated with metabolic syndrome (MetS). MetS is characterized by central obesity, elevated plasma cholesterol and fasting glucose, hypertension, insulin resistance, and atherosclerosis. The prevalence of MetS among adults in the USA is 24%. In this research program, we will utilize the MetS Ossabaw pig model that exhibits all of the characteristics of MetS, including the overactive renin-angiotensin- aldosterone system (RAAS). We demonstrated that the expression levels of TRPC1 and TRPC6 channels are markedly elevated in MetS Ossabaw pig coronary arteries exhibiting atherosclerosis and hypercontractility. Consistently, freshly isolated MetS coronary artery smooth muscle cells had elevated store-/receptor-operated Ca2+ influx and large store-/receptor-operated TRPC-like currents. Since angiotensin II and aldosterone are positive regulators of TRPC expression, we hypothesize that, in MetS, the overactive RAAS upregulates TRPC1 and TRPC6 expression, which drives increased coronary smooth muscle cell proliferation and coronary artery hypercontractility. The following Specific Aims will be pursued: 1) To determine how the molecular expression of TRPCs is altered during the MetS-associated remodeling of coronary artery smooth muscle cells; 2) To define the contribution of TRPCs to endogenous store- and receptor-activated Ca2+ influx/currents in control and MetS coronary artery smooth muscle cells; 3) To determine whether the functional expression of endogenous TRPC channels is directly regulated by RAAS components, angiotensin II and Aldo, in coronary artery smooth muscle cells; 4) To determine whether the in vivo, coronary artery targeted down- regulation of TRPCs slows atheroma progression and decreases coronary artery hypercontractility in MetS pigs. During this research program, we will use molecular biological, biochemical, electrophysiological, and fluorescence imaging approaches as well as intravascular ultrasound, isometric tension and coronary artery ring lumen area measurements. Additionally, a coronary artery targeted delivery approach will be employed to deploy shRNAs and cDNA constructs into the coronary artery wall in vivo. Importantly, we will determine the distinct roles of TRPC1 and TRPC6 during MetS-associated NATIVE atherosclerosis progression.