Overexpression of Angiopoietin-1 Increases CD133+/c- kit+ Cells and Reduces Myocardial Apoptosis in db/db Mouse Infarcted Hearts Heng Zeng, Lanfang Li, Jian-Xiong Chen* Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America Abstract Hematopoietic progenitor CD133+/c-kit+ cells have been shown to be involved in myocardial healing following myocardial infarction (MI). Previously we demonstrated that angiopoietin-1(Ang-1) is beneficial in the repair of diabetic infarcted hearts. We now investigate whether Ang-1 affects CD133+/c-kit+ cell recruitment to the infarcted myocardium thereby mediating cardiac repair in type II (db/db) diabetic mice. db/db mice were administered either adenovirus Ang-1 (Ad-Ang-1) or Ad-b- gal systemically immediately after ligation of the left anterior descending coronary artery (LAD). Overexpression of Ang-1 resulted in a significant increase in CXCR-4/SDF-1a expression and promoted CD133+/c-kit+, CD133+/CXCR-4+ and CD133+/ SDF-1a+ cell recruitment into ischemic hearts. Overexpression of Ang-1 led to significant increases in number of CD31+ and smooth muscle-like cells and VEGF expression in bone marrow (BM). This was accompanied by significant decreases in cardiac apoptosis and fibrosis and an increase in myocardial capillary density. Ang-1 also upregulated Jagged-1, Notch3 and apelin expression followed by increases in arteriole formation in the infarcted myocardium. Furthermore, overexpression of Ang-1 resulted in a significant improvement of cardiac functional recovery after 14 days of ischemia. Our data strongly suggest that Ang-1 attenuates cardiac apoptosis and promotes cardiac repair by a mechanism involving in promoting CD133+/c-kit+ cells and angiogenesis in diabetic db/db mouse infarcted hearts. Citation: Zeng H, Li L, Chen J-X (2012) Overexpression of Angiopoietin-1 Increases CD133+/c-kit+ Cells and Reduces Myocardial Apoptosis in db/db Mouse Infarcted Hearts. PLoS ONE 7(4): e35905. doi:10.1371/journal.pone.0035905 Editor: Ferenc Gallyas, University of Pecs Medical School, Hungary Received November 7, 2011; Accepted March 23, 2012; Published April 27, 2012 Copyright: 2012 Zeng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the United States National Institutes of Health, HL102042. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: JChen3@umc.edu Introduction Ang-1 is an oligomeric-secreted glycoprotein, which binds to Tie-2 and induces Tie-2 phosphorylation. Ang-1 is recognized as a survival factor for endothelial cells (EC). Treatment with Ang-1 prevents EC apoptosis via activation of the PI3K/Akt path- way.[1,2] Ang-1 has also been shown to prevent diabetic retinopathy by attenuating retinal permeability in the streptozo- tocin (STZ)-induced rat diabetic model.[3] Our previous studies revealed that overexpression of Ang-1 in diabetic db/db mouse heart restores Tie-2 expression and significantly increases myocardial capillary formation; this is accompanied by a dramatic decrease in myocardial hypertrophy and cardiac fibrosis.[4] These data implicate Ang-1 as a potential therapeutic target in the treatment of diabetic cardiovascular complications. Endothelial progenitor cells (EPCs) home to sites of ischemia and contribute to neovascularization in ischemic tissue.[5] Experimental and clinical studies demonstrate that treatment of acute myocardial infarction with EPCs results in a reduction in infarct size.[6,7] Vascular progenitor cells have been shown to differentiate into cardiomyocytes and vascular smooth muscle cells (VSMC), which may contribute to cardiac and/or vascular regeneration following myocardial infarction [8,9]. Intriguingly, the differentiation of EPCs is impaired in both diabetic patients with coronary artery disease and in diabetic mouse models [10,11]. Previously we demonstrate that the level of EPCs is significantly decreased in STZ-induced diabetic mouse compared to non-diabetic mice [12]. Our previous studies also reveal that disruption of BM-EPC differentiation and impairment of angio- genesis after myocardial ischemia are associated with larger myocardial infarct size in the diabetic STZ mice [12]. These studies suggest that impaired vascular progenitor cell recruitment and failure of BM differentiation to EPCs after MI may contribute to insufficient angiogenesis and exacerbation of MI in diabetes. Thus, an agent that promotes vascular progenitor cell recruitment and angiogenesis will be beneficial for ischemic injury repair and cardiac remodeling after MI in diabetic hearts. This notion is supported by our previous work demonstrating that overexpres- sion of Ang-1 significantly increased myocardial angiogenesis and reduced myocardial infarction size in the STZ diabetic mouse model [12]. However, the underlying molecular mechanism by which Ang-1 attenuates myocardial ischemic injury in the diabetic heart following MI remains poorly understood. Ang-1 has been shown to have a critical role in the maintenance of hematopoietic stem cell in the bone marrow through its binding to the Tie-2 receptor.[13]The hematopoietic stem cell cytokine SDF-1a and it receptor CXCR-4 have been identified as the central signaling axis that regulates recruitment of hematopoietic stem cells into the injured area of myocardial ischemia and in improvement of cardiac function after MI [14]. Using diabetic db/ db mice subjected to myocardial ischemia, the present study PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35905