Mineralocorticoid Receptor-Mediated Vascular Insulin Resistance

描述(由申请人提供)： 冠状动脉微血管功能障碍是胰岛素抵抗(IR)状态下的一种早期和持续性的缺陷，与肾素-血管紧张素-醛固酮系统(RAAS)的不适当激活密切相关。因此，RAAS激活是IR相关血管功能障碍的主要因素，其作用机制包括氧化应激、纤维化、内皮功能障碍和丝氨酸激酶的激活，如Rho激酶(ROK)。许多现有证据表明血管紧张素II参与了这些不良反应；然而，越来越多的证据表明，醛固酮通过血管盐皮质激素受体(MR)发挥显著作用，可能是IR状态下血管功能障碍的介质。在临床试验中，MR拮抗剂通过未知的机制降低心血管死亡率。此外，最近的证据表明，MR拮抗对改善糖尿病患者的冠状动脉血流储备(CFR)有显著效果。MR介导的冠状动脉血管功能障碍的确切机制(S)；然而，尚不清楚。越来越多的证据表明，MR信号是细胞内钙稳态和收缩的主要调节因子。具体地说，MR信号似乎是心肌细胞L型钙通道(CaV1.2)功能/表达的紧张性调节因子。我们最近的工作表明，醛固酮增加冠状动脉血管MR的激活导致激动剂和CaV1.2介导的冠状动脉血管收缩反应性增加，这与IR状态下的结果相似。此外，我们的数据表明，在血管平滑肌细胞(VSMC)中，通过MR信号直接激活了钙敏感性的主要调节剂ROK。因此，本研究试图探讨VSMC MR信号促进VSMC过度收缩、增加冠脉收缩和减少与IR相关的CFR的新的分子机制。我们推测，血管的不适当激活，特别是VSMC、MRS的不适当激活，通过调节钙内流和IR敏感性，在VSMC过度收缩的发生发展中起核心作用。这一假设的一个推论是，VSMC中的MR依赖信号直接介导了IR状态下的冠状动脉功能障碍和冠状动脉血流控制的损害。在我们的方案中，我们将利用一种新颖的、创新的VSMC特异性MR基因敲除(VSMC MR KO)小鼠模型，结合最先进的微血管成像和功能技术，评估MR信号(由外源性醛固酮或饮食诱导的IR激活)在调节VSMC钙处理、体外冠状动脉小动脉功能和体内CFR方面的作用。为了达到特定的目标1，我们将研究在原代培养的VSMC、新鲜分散的小鼠冠状动脉VSMC和完整的冠状动脉小动脉中MR激活与CaV1.2通道功能/表达和韩国激活的关系。这些参数将与包括小动脉血管收缩、血压和CFR在内的功能指标相关。具体目标2将扩展这些发现，并解决IR中VSMC中MR激活作为IR相关冠状动脉过度收缩/功能障碍的主要介体的作用及其与小动脉功能和CFR的关系。IR将在野生型和VSMC MR KO小鼠中通过高脂肪、高蔗糖饮食诱导，并将通过葡萄糖耐量测试进行评估。

DESCRIPTION (provided by applicant): Coronary microvascular dysfunction is an early and persistent defect in states of insulin resistance (IR) that are closely associated with inappropriate activation of the renin-angiotensin-aldosterone system (RAAS). Thus, RAAS activation is a major factor in IR-associated vascular dysfunction, acting via several mechanisms including oxidative stress, fibrosis, endothelial dysfunction and activation of serine kinases such as Rho kinase (ROK). Much existing evidence implicates angiotensin II involvement in these adverse effects; however, accumulating evidence demonstrates pronounced effects of aldosterone acting through vascular mineralocorticoid receptors (MR) as a putative mediator of vascular dysfunction in states of IR. In clinical trials, MR antagonism reduced cardiovascular mortality via unknown mechanisms. Furthermore, recent evidence demonstrates a pronounced effect of MR antagonism to improve coronary flow reserve (CFR) in diabetic patients. The exact mechanism(s) underlying MR-mediated coronary vascular dysfunction; however, remain unclear. Mounting evidence suggests that MR signaling is a primary regulator of cellular calcium homeostasis and contractility. Specifically, MR signaling appears to be a tonic regulator of L-type calcium channel (CaV1.2) function/expression in cardiomyocytes. Our recent work demonstrates that increased activation of coronary vascular MR by aldosterone leads to increased agonist- and CaV1.2-mediated coronary vasoconstrictor responsiveness, similar to that noted in states of IR. Furthermore, our data demonstrate direct activation of ROK, a primary modulator of calcium sensitivity, by MR signaling in vascular smooth muscle cells (VSMC). Therefore, this proposal seeks to investigate novel molecular mechanisms by which VSMC MR signaling promotes VSMC hypercontractility, increased coronary vasoconstriction and reductions in CFR associated with IR. We hypothesize that inappropriate activation of vascular, specifically VSMC, MRs plays a central role in the development of VSMC hypercontractility via the modulation of calcium influx and sensitivity in IR. A corollary to this hypothesis is that MR-dependent signaling in VSMC directly mediates coronary dysfunction and the impairment of coronary blood flow control in states of IR. In our proposal, we will utilize a novel, innovative VSMC-specific MR knockout (VSMC MR KO) mouse model coupled with state-of-the-art microvascular imaging and functional techniques, to evaluate the effect of MR signaling (activated by exogenous aldosterone or diet-induced IR) to modulate VSMC calcium handling, coronary arteriolar function ex vivo and CFR in vivo. To address Specific Aim 1, we will examine the relationship between MR activation and CaV1.2 channel function/expression and ROK activation in primary cultured VSMC, freshly dispersed murine coronary VSMC and intact coronary arterioles. These parameters will be correlated to functional measures including arteriolar vasoconstriction, blood pressure and CFR. Specific Aim 2 will extend these findings and address the role of MR activation in VSMC in IR as a primary mediator of IR-associated coronary hypercontractility/dysfunction and its relationship to arteriolar function and CFR. IR will be induced in wild-type and VSMC MR KO mice with a high-fat, high-sucrose diet and will be assessed by glucose tolerance testing.