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.

Public Health Relevance

Ischemia reperfusion (I/R) injury of the heart is a major clinical problem. The current project investigates a previously uncharacterized, critical signaling role of cardiac microvascular endothelium in mediating cardioprotection during I/R injury of the heart, under conditions such as netrin-1 perfusion. Accomplishment of the proposed aims and subaims will ultimately promote novel therapeutics for cardiac I/R injury that are netrin-1 based and/or cell- based.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Schwartz, Lisa
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Los Angeles
Schools of Medicine
Los Angeles
United States
Zip Code
Bouhidel, Jalaleddinne Omar; Wang, Ping; Siu, Kin Lung et al. (2015) Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy. Biochim Biophys Acta 1852:277-89
Siu, Kin Lung; Lotz, Christopher; Ping, Peipei et al. (2015) Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS. J Mol Cell Cardiol 78:174-85
Youn, Ji-Youn; Siu, Kin Lung; Lob, Heinrich E et al. (2014) Role of vascular oxidative stress in obesity and metabolic syndrome. Diabetes 63:2344-55
Bouhidel, Jalaleddinne Omar; Wang, Ping; Li, Qiang et al. (2014) Pharmacological postconditioning treatment of myocardial infarction with netrin-1. Front Biosci (Landmark Ed) 19:566-70
Zhang, Yixuan; Shimizu, Hirohito; Siu, Kin Lung et al. (2014) NADPH oxidase 4 induces cardiac arrhythmic phenotype in zebrafish. J Biol Chem 289:23200-8
Siu, Kin Lung; Miao, Xiao Niu; Cai, Hua (2014) Recoupling of eNOS with folic acid prevents abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E null mice. PLoS One 9:e88899