Endothelium-Myocardium Interaction in Netrin-1 Induced Cardioprotection

描述（由申请人提供）：本申请的中心焦点是检查心脏微血管内皮在心脏缺血再灌注（I/R）损伤期间介导心脏保护中的先前未表征的关键信号传导作用。我们提出了一个创新的假设，即心脏微血管内皮细胞向相邻的心肌细胞发出信号，以诱导心脏保护，在netrin-1再灌注的条件下。这与导管动脉中血管平滑肌松弛和信号传导的内皮调节有一些相似之处。心脏I/R损伤是临床上的一个主要问题，其分子机制尚不清楚，因此缺乏新的药物。我们最近的工作使用离体Langendoff灌注系统治疗心脏I/R损伤，确定了netrin-1的极其有效的心脏保护作用及其相关的信号传导机制。为了检查netrin-1在体内诱导心脏保护中是否同样稳健，以及该保护是否由心脏微血管内皮细胞中eNOS /一氧化氮（NO-）的DCC-ERK 1/2-深度激活介导，所述eNOS/NO-在netrin-1灌注后应立即激活，将采用左冠状动脉结扎和再灌注的鼠模型。目的1：确定netrin-1诱导的体内心脏保护的分子机制：一氧化氮（NO-1）通路的作用。梗死面积和心肌肌钙蛋白I释放的分析以及心脏功能的超声心动图分析将用于检查netrin-1在体内的心脏保护作用。将充分描述体内eNOS激活机制和netrin-1刺激的心脏保护对DCC、ERK 1/2和NO-1的依赖性。目的2：探讨心肌微血管内皮细胞（CMECs）中netrin-1激活eNOS是否具有保护心肌细胞凋亡的作用。eNOS激活机制和基于蛋白酶体降解抑制的机制介导通过NO-前馈上调DCC，以及CMEC衍生的NO-对心肌细胞凋亡和自噬的影响将被深入表征。目的3：确定netrin-1对NADPH氧化酶4（NOX 4）和氧化应激的抑制是否由CMEC衍生的NO-介导。将详细检查netrin-1对心肌细胞和CMEC中总氧化应激和不同NOX同工型的表达和活性的影响，以及NO-1对NOX 4抑制的影响。目标4：确定netrin-1抑制NOX 4是否可防止I/R期间eNOS解偶联和线粒体功能障碍，从而促进心脏保护。将采用N 0X 4的RNAi敲低和eNOS的sepiapterin再偶联来检查N 0X 4/eNOS解偶联衰减对梗塞大小、线粒体功能和线粒体活性氧物质产生的影响，这也可以通过netrin-1灌注来实现。还将检查I/R期间NOX 4 RNAi对eNOS解偶联状态的影响。 这些明确定义的、高度重要的和转化的目标和子目标的实现将最终促进用于心脏I/R损伤的基于netrin-1的新疗法。CMEC-心肌细胞信号传导轴的表征可以额外地为潜在的基于细胞的疗法提供新的见解。

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.