Endothelium-Myocardium Interaction in Netrin-1 Induced Cardioprotection

描述（由申请人提供）：本申请的中心重点是研究心脏缺血再灌注（I/R）损伤期间心脏微血管内皮在介导心脏保护中的关键信号作用。我们提出了一个创新的假设，即在netrin-1再灌注等条件下，心脏微血管内皮细胞向邻近的心肌细胞发出信号以诱导心脏保护。这与血管平滑肌松弛和导管动脉信号的内皮调节有一定的相似性。心脏I/R损伤是一个主要的临床问题，分子机制尚不清楚，因此缺乏新药。我们最近的工作已经确定了netrin-1的极其有效的心脏保护作用及其信号机制，使用体外Langendorff灌注系统治疗心脏I/R损伤。为了检验netrin-1在体内诱导心脏保护是否同样强大，以及这种保护是否通过在netrin-1灌注后应立即激活的心脏微血管内皮细胞中eNOS /一氧化氮（NO”）的dcc - erk1 /2深度激活介导，我们将采用小鼠左冠状动脉结扎和再灌注模型。目标1：确定netrin-1诱导的体内心脏保护的分子机制：一氧化氮（NO）途径的作用。分析梗死面积和心肌肌钙蛋白I释放，以及心功能超声心动图分析，将用于检测netrin-1在体内的心脏保护作用。eNOS在体内的激活机制以及netrin-1刺激的心脏保护对DCC、ERK1/2和NO的依赖性将被充分描述。目的2：确定netrin-1激活心肌微血管内皮细胞（CMECs） eNOS是否能保护心肌细胞免于凋亡。eNOS的激活机制和基于蛋白酶体降解抑制的机制介导NO前馈上调DCC，以及cmec来源的NO对心肌细胞凋亡和自噬的影响将被深入探讨。目的3：确定netrin-1抑制NADPH氧化酶4 （NOX4）和氧化应激是否由cmec衍生的NO介导。我们将详细研究netrin-1对总氧化应激的影响，以及不同NOX亚型的表达和活性，以及NO“对心肌细胞和cmec中NOX4抑制的影响。目的4：确定netrin-1抑制NOX4是否能阻止I/R期间eNOS解偶联和线粒体功能障碍，从而促进心脏保护。我们将通过RNAi敲低NOX4和eNOS的鞘叶蝶呤重偶联来检测NOX4/eNOS解偶联衰减对梗死面积、线粒体功能和线粒体活性氧产生的影响，这也可以通过netrin-1灌注来实现。NOX4 RNAi对I/R过程中eNOS解耦状态的影响也将被研究。这些明确的、高度重要的、可转化的目标和亚目标的实现将最终促进基于netrin-1的心脏I/R损伤治疗新方法的发展。cmec -心肌细胞信号轴的表征可能为潜在的基于细胞的治疗提供新的见解。

DESCRIPTION (provided by applicant): The central focus of this application is to examine a previously uncharacterized, critical signaling role of cardiac microvascular endothelium in mediating cardioprotection during ischemia reperfusion (I/R) injury of the heart. We propose an innovative hypothesis that cardiac microvascular endothelial cells signal to adjacent cardiomyocytes to induce cardioprotection, under conditions such as netrin-1 reperfusion. This shares some similarity with endothelium regulation of vascular smooth muscle relaxation and signaling in conduit arteries. Cardiac I/R injury is a major clinical problem with unclear molecula mechanisms, and therefore lack of new medicines. Our recent work has identified an extremely potent cardioprotective effect of netrin-1 and the signaling mechanisms involved, using an ex vivo Langendorff perfusion system for cardiac I/R injury. To examine whether netrin-1 is equally robust in inducing cardioprotection in vivo and whether the protection is mediated by a DCC-ERK1/2-deepdent activation of eNOS /nitric oxide (NO") in cardiac microvascular endothelial cells that should be immediately activated upon netrin-1 perfusion, a murine model of left coronary artery ligation and reperfusion will be employed. Four specific aims will be addressed: Aim 1: To identify molecular mechanisms underlying netrin-1 induced cardioprotection in vivo: the role of nitric oxide (NO") pathway. Analyses of infarct size and cardiac Troponin I release, and echocardiography analysis of cardiac function, will be used to examine the cardioprotective effect of netrin-1 in vivo. The eNOS activation mechanisms in vivo and the dependency on DCC, ERK1/2, and NO" of netrin-1 stimulated cardioprotection will be fully delineated. Aim 2: To determine whether netrin-1 activation of eNOS in cardiac microvascular endothelial cells (CMECs) protects cardiomyocytes from apoptosis. The eNOS activation mechanisms and the proteasome-degradation inhibition based mechanisms mediating feed-forward upregulation of DCC by NO", as well as the effects of CMEC-derived NO" on cardiomyocyte apoptosis and autophagy will be characterized in depth. Aim 3: To determine whether netrin-1 inhibition of NADPH oxidase 4 (NOX4) and oxidative stress is mediated by CMEC-derived NO". Effects of netrin-1 on total oxidative stress, and expression and activity of different NOX isoforms, as well as effects of NO" on NOX4 inhibition in both cardiomyocytes and CMECs, will be examined in details. Aim 4: To determine whether netrin-1 inhibition of NOX4 prevents eNOS uncoupling and mitochondrial dysfunction during I/R, therefore promoting cardioprotection. RNAi knockdown of NOX4 and sepiapterin recoupling of eNOS will be employed to examine the effects on infarct size, mitochondrial function and mitochondrial reactive oxygen species production of NOX4/eNOS uncoupling attenuation, which can also be achieved by netrin-1 perfusion. Effects of NOX4 RNAi on eNOS uncoupling status during I/R will also be examined. Accomplishment of these well-defined, highly significant and translational aims and subaims would ultimately promote novel netrin-1 based therapeutics for cardiac I/R injury. Characterization of the CMEC-cardiomyocyte signaling axis may additionally provide novel insights into potential cell-based therapies.