Ethanol Prevents Microvascular Dysfunction

描述(由申请人提供)：低到中等水平的酒精消费保护心脏和血管系统免受缺血和再灌注(I/R)的有害影响。我们最近的工作表明，预先摄入乙醇会刺激毛细血管后小静脉形成抗炎表型，导致这些微血管不能支持I/R期间白细胞/内皮细胞的黏附作用。令人惊讶的是，AMP激活的激酶(AMPK)在启动这种适应性转化为毛细血管后小静脉保护表型的过程中发挥了必要的作用。然而，目前还不清楚这种以代谢主开关的作用而闻名的酶是如何参与转导乙醇诱导的信号，从而在微循环中影响对I/R耐受性的获得。我们的总体假设是，乙醇诱导的依赖AMPK的eNOS激活是触发一系列信号事件的重要启动因素，这些信号事件最终促进细胞色素P450环氧合酶(CYP)的表达，CYP通过催化反应产物的形成而成为乙醇有益的微血管作用的重要效应者，反应产物具有强大的抗粘连和抗氧化特性。为了解决这一假设，我们建议确定：(1)乙醇诱导的AMPK激活是否通过刺激eNOS来源的NO的形成来启动抗炎表型的形成；(2)NO诱导的降钙素基因相关肽(CGRP)的释放在获得对缺血的耐受中起重要作用；(3)囊性纤维化跨膜调节因子(CFTR)作为必需的下游信号元件参与了乙醇诱导的AMPK激活所沉淀的受保护表型的形成；(4)乙醇诱导的AMPK激活的CYP活性在I/R期间通过抑制P-选择素的表达来预防缺血后白细胞在毛细血管后小静脉的黏附。活体显微镜方法将被用来量化野生型对照小鼠和缺乏AMPK、eNOS、CGRP或CFTR的突变小鼠的白细胞滚动和黏附。此外，还将观察乙醇对L/R诱导的黏附分子表达、AMPK和eNOS活性、CGRP释放以及CYP蛋白表达和活性的影响。总而言之，这些目标解决了先天摄入乙醇诱导毛细血管后小静脉中抗炎表型发展的新机制。这项工作将确定乙醇诱导的、AMPK依赖的、eNOS来源的NO形成作为基本触发元件、CGRP释放和CFTR作为必需的下游调节因子以及CYP活性增加作为预先摄入乙醇获得I/R耐受性的主要影响因素之间的新联系。

DESCRIPTION (provided by applicant): Ethanol consumption at low to moderate levels protects the heart and vasculature from the deleterious effects of ischemia and reperfusion (I/R). Our recent work indicates that antecedent ethanol ingestion provokes the development of an anti-inflammatory phenotype in postcapillary venules such that these microvessels fail to support leukocyte/endothelial cell adhesive interactions during I/R. Surprisingly, AMP-activated kinase (AMPK) plays a necessary role in initiating this adaptive transformation to a protected phenotype in postcapillary venules. However, it is not clear how this enzyme, best known for its role as a metabolic master switch, is involved in transducing signals elicited by ethanol to effect the acquisition of tolerance to I/R in the microcirculation. Our overall hypothesis is that ethanol-induced, AMPK-dependent eNOS activation serves as an important initiating factor to trigger a cascade of signaling events that ultimately act to promote the expression of cytochrome P450 epoxygenases (CYP), which serve as an important effector of the beneficial microvascular actions of ethanol by catalyzing the formation of reaction products which exhibit powerful anti-adhesive and antioxidant properties. To address this postulate, we propose to determine whether: (1) ethanol-induced AMPK activation initiates the development of an anti-inflammatory phenotype by stimulating the formation of eNOS-derived NO; (2) NO-induced release of calcitonin gene-related peptide (CGRP) plays an essential role in the acquisition of tolerance to ischemia that is evoked by antecedent ethanol consumption; (3) the cystic fibrosis transmembrane regulator (CFTR) serves as an obligatory downstream signaling element that participates in inaugurating the development of the protected phenotype precipitated by ethanol-induced AMPK activation; and (4) ethanol-induced, AMPK-triggered increases in CYP activity during I/R prevents postischemic leukocyte adhesion in postcapillary venules by abrogating P-selectin expression. Intravital microscopic approaches will be used to quantify leukocyte rolling and adhesion in wild-type control mice and in mutant mice lacking AMPK, eNOS, CGRP, or CFTR. The influence of ethanol on l/R-induced adhesion molecule expression, AMPK and eNOS activity, CGRP release, and CYP protein expression and activity will also be investigated. Collectively, these aims address novel mechanisms whereby antecedent ethanol ingestion induces the development of an anti-inflammatory phenotype in postcapillary venules. This work will identify new links between ethanol-induced, AMPK-dependent, eNOS-derived NO formation as essential triggering elements, CGRP release and CFTR function as obligatory downstream mediators, and increased CYP activity as a major effector in the acquisition of tolerance to I/R by antecedent ethanol ingestion.