Endothelium-Myocardium Interaction in Netrin-1 Induced Cardioprotection

Netrin-1 诱导的心脏保护作用中的内皮-心肌相互作用

• 批准号: 8892236
• 负责人: Hua Linda Cai
• 金额: $ 50.01万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892236
• 关键词: Address; Adult; Apoptosis; Arteries; Attenuated; Autophagocytosis; Biochemical; Biological Availability; Blood Vessels; Cardiac; Cardiac Myocytes; Cell Therapy; Cells; Clinical; Coronary artery; Data; Dependency; Echocardiography; Endothelial Cells; Endothelium; Health; Heart; Heart Hypertrophy; Heart failure; Human; Infarction; Ischemia; Left; Ligation; MAPK3 gene; Mediating; Mediator of activation protein; Medicine; Mitochondria; Mitochondrial Swelling; Modeling; Molecular; Mus; Muscle relaxation phase; Myocardial; Myocardial Ischemia; Myocardium; NADPH Oxidase; Necrosis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Pathway; Organ; Oxidative Stress; Perfusion; Prevention; Production; Protein Isoforms; RNA Interference; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reperfusion Therapy; Role; Signal Transduction; Small Interfering RNA; Superoxides; System; Therapeutic; Troponin I; Up-Regulation; Work; attenuation; base; feeding; heart dimension/size; human NTN1 protein; in vivo; innovation; insight; mimetics; mitochondrial dysfunction; multicatalytic endopeptidase complex; netrin-1; novel; novel therapeutics; prevent; research study; sepiapterin; ubiquitin-protein ligase

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.

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先前未表征的关键信号传导作用。我们提出了一个创新假设，即在 netrin-1 再灌注等条件下，心脏微血管内皮细胞向邻近心肌细胞发出信号以诱导心脏保护。这与内皮对血管平滑肌松弛和导管动脉信号传导的调节有一些相似之处。心脏I/R损伤是一个主要的临床问题，其分子机制尚不清楚，因此缺乏新药。我们最近的工作使用离体 Langendorff 灌注系统治疗心脏 I/R 损伤，确定了 netrin-1 及其相关信号传导机制的极其有效的心脏保护作用。为了检查 netrin-1 在诱导体内心脏保护方面是否同样强大，以及这种保护是否是由心脏微血管内皮细胞中 eNOS/一氧化氮 (NO") 的 DCC-ERK1/2 深度激活介导的，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小鼠模型。将采用四个具体目标： 目标：目标 1：确定 netrin-1 诱导体内心脏保护的分子机制：一氧化氮 (NO") 途径的作用。梗塞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 体内的心脏保护作用。将全面阐明体内 eNOS 激活机制以及 netrin-1 刺激的心脏保护作用对 DCC、ERK1/2 和 NO" 的依赖性。目标 2：确定心脏微血管内皮细胞 (CMEC) 中 netrin-1 激活 eNOS 是否可以保护心肌细胞免于凋亡。eNOS 激活机制和基于蛋白酶体降解抑制的研究 NO" 介导 DCC 前馈上调的机制，以及 CMEC 衍生的 NO" 对心肌细胞凋亡和自噬的影响将得到深入表征。目标 3：确定 netrin-1 对 NADPH 氧化酶 4 (NOX4) 和氧化应激的抑制是否是由 CMEC 衍生的 NO 介导的。将详细研究 netrin-1 对总氧化应激、不同 NOX 亚型的表达和活性的影响，以及 NO" 对心肌细胞和 CMEC 中 NOX4 抑制的影响。目标 4：确定 netrin-1 对 NOX4 的抑制是否会阻止 I/R 期间 eNOS 解偶联和线粒体功能障碍，从而促进心脏保护。 RNAi 敲低 NOX4 和 eNOS 的 sepiapterin 重新偶联将用于检查 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。还将检查 I/R 期间 NOX4 RNAi 对 eNOS 解偶联状态的影响。 完成这些明确的、高度 重要的转化目标和子目标最终将促进基于 netrin-1 的新型心脏 I/R 损伤疗法。 CMEC-心肌细胞信号传导轴的表征还可能为潜在的基于细胞的疗法提供新的见解。