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研究心脏微血管内皮细胞在心脏缺血再灌注(I/R)损伤中介导心脏保护的一个以前未被表征的关键信号作用。我们提出了一个创新的假设，即心脏微血管内皮细胞向邻近的心肌细胞发出信号，在Netrin-1再灌注等条件下诱导心脏保护。这与血管内皮细胞调节血管平滑肌松弛和导管动脉的信号传导有一些相似之处。心脏I/R损伤是一大临床难题，其分子机制尚不清楚，因此缺乏新药。我们最近的工作已经确定了netrin-1的极其强大的心脏保护作用及其相关的信号机制，使用体外Langendorff灌流系统治疗心脏I/R损伤。采用小鼠左冠状动脉结扎再灌流模型，研究netrin-1在体内是否同样有效地诱导心肌保护，以及这种保护是否由dcc-ERK1/2深度激活心脏微血管内皮细胞eNOS/NO“介导的，而这种激活应被立即激活。对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，将被用来检验netrin-1在体内的心脏保护作用。目的2：探讨心脏微血管内皮细胞(CMECs)中Netrin-1激活eNOS对心肌细胞的保护作用。目的：探讨心肌微血管内皮细胞(CMECs)中Netrin-1激活eNOS是否对心肌细胞有保护作用。NO“前馈上调心肌细胞eNOS的激活机制和蛋白酶体降解抑制机制，以及CMEC来源的NO”对心肌细胞凋亡和自噬的影响。目的3：确定心肌微血管内皮细胞(CMECs)中Netrin-1抑制NADPH-4(NOX4)和氧化应激是否通过CMEC来源的NO“介导。将详细研究netrin-1对心肌细胞和CMECs总氧化应激、不同NOX亚型的表达和活性的影响，以及NO“对NOX4抑制的影响。目的4：探讨抑制网织蛋白-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，从而促进心肌保护。我们将利用NOX4的RNAi敲除和eNOS的sepiapterin重新偶联来检测NOX4/eNOS去偶联对心肌梗死面积、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灌流来实现。还将研究NOX4 RNAi对I/R期间eNOS解偶联状态的影响。这些明确的、意义重大的翻译目标和子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-心肌细胞信号轴的特征可能为潜在的基于细胞的治疗提供新的见解。