investigates whether overexpression of Ang-1 promotes recruit- ment of hematopoietic progenitor cells into ischemic sites and whether this leads to attenuation of myocardial ischemic injury through SDF-1a/CXCR-4 signaling. Our data suggest that Ang- 1/Tie-2 plays a crucial role in regulation of hematopoietic progenitor cell recruitment and cardiac repair in the diabetic infarcted heart. Methods Ethics Statement All procedures conformed to the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee (Protocol ID: #1280). Diabetic mouse myocardial ischemia model db/db mice (12–14 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, Maine). Experimental mice were anesthetized with ketamine (100 mg/kg) plus xylazine (15 mg/ kg), intubated and artificially ventilated with room air. Adequate anesthesia was monitored by toe pinch. A left thoracotomy was performed and myocardial ischemia was achieved by ligation of the left anterior descending coronary artery (LAD) [12,15]. Sham controls underwent the surgery without LAD ligation. Experi- mental mice were divided into three groups: (1) sham control (n = 12 mice); (2) Ad-b-gal + ischemia 24 hours (n = 22 mice) and 14 days (n = 13 mice) and (3) Ad-Ang-1 + ischemia 24 hours (n = 16 mice) and 14 days (n = 19 mice). Experimental mice were sacrificed under anesthesia with isoflurane at 24 hours and 14 days after LAD ligation for the analyses described below. Systemic delivery of Ang-1 to experimental mice After surgery, db/db mice received an intravenous tail vein injection of either Ad-Ang-1 (16109 PFU) or Ad-b-gal (16109 PFU) [4,12]. Western analysis of Ang-1, SDF-1a, VEGF, apelin, APLNR (APJ), Jagged 1 and Notch3 expression After 24 hours of myocardial ischemia, the hearts and BM were harvested and homogenized in lysis buffer. The membranes were immunoblotted with SDF-1a, VEGF, apelin, APNLR, Jagged1 and Notch3 (1:1000, Santa Cruz, CA) or Ang-1 (1:1000, Sigma, MO) antibodies. The membranes were washed and incubated with a secondary antibody coupled to horseradish peroxidase and densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0). Myocardial Ang-1, Tie-2, CD133, CD45, C-kit, CXCR-4, SDF-1a, SMA and Notch3 expression Heart tissue sections (8 mm thick) were incubated with Ang-1, CD133, Tie-2 and Notch3, CXCR-4, CD45, c-kit and SDF-1a (1:200 Santa Cruz, CA) antibodies overnight. Ang-1, CD133, Tie- 2 and Notch3 were visualized using FITC labeled goat anti-mouse IgG antibodies; CXCR-4, CD45 and SDF-1a were visualized with FluorolinkTM CyTM3 labeled goat anti-mouse IgG antibodies (1:200). Cy3-conjugated anti-a smooth muscle actin (SMA, 1:100; Sigma, MO) was used to identify smooth muscle cells. Myocardial CD133, CD45 and CXCR-4 expression was assessed by counting the number of positive cells per square millimeter (mm2) of tissue. Co-localization of CD133 with either CXCR-4 or SDF-1a, or Ang-1 Reduces Myocardial Infarction in db/db Mice Ang-1 with SMA was visualized with a fluorescence microscope (Nikon TE 2000, Japan). Sections were counterstained with DAPI. Bone marrow (BM) cell differentiation into CD31+ and SMA+ cells At 24 hours of myocardial ischemia, BM cells were obtained by flushing the tibias and femurs with 10% FBS EGM. Immediately after isolation, 105 bone marrow–derived mononuclear cells were plated on 6-well culture plates coated with fibronectin (50 mg/ml, Sigma). After 4 days of culture, the nonadherent cells were removed and the adherent cells were incubated with -FITC- labeled CD31 antibody (1:100, BD Biosciences) and -Cy3- conjugated anti-a smooth muscle actin (SMA, 1:100; Sigma, MO). Positively-stained cells were counted per 105 BM cells. Analysis of myocardial capillary density After 14 days of myocardial ischemia, the hearts were harvested and immediately flash frozen. Five mm sections were incubated with Isolectin B4 (1:200, IB4, Molecular Probe, Invitrogen) to identify capillaries. The number of capillaries was counted and expressed as capillary density per square millimeter (mm2) [12,16]. Analysis of VSMC and arteriole formation To assess VSMC in the border zone of the ischemic area after 24 hours of MI, VSMC were stained with Cy3-conjugated anti- a smooth muscle actin (SMA, 1:100; Sigma, MO). Arterioles (Ang- 1+/SMA+) was examined at 14 days of MI. Myocardial cell/endothelial apoptosis Heart tissue sections underwent transferase deoxyuridine nick end labeling (TUNEL) following the manufacturer’s instructions (Promega, WI). Sections were counterstained with DAPI. Apoptosis was indexed by counting TUNEL positive cells per square millimeter (mm2). To determine endothelial or SMC apoptosis, sections of myocardium were incubated with TUNEL and vWF (1:200, Santa Cruz, CA) or Cy3-conjugated anti- a smooth muscle actin (SMA, 1:100; Sigma, MO) overnight, followed by incubation with FluorolinkTM CyTM3 for vWF. Co- localization of TUNEL with vWF or SMA was visualized with a fluorescence microscope (Nikon TE 2000, Japan). Sections were counterstained with DAPI. Endothelial cell/SMC apoptosis was indexed by counting TUNEL/vWF or TUNEL/SMA positive 2 cells per square millimeter (mm ). Assessment of cardiac function Briefly, experimental mice were anesthetized with ketamine (100 mg/kg) plus xylazine (15 mg/kg), intubated and artificially ventilated with room air. A 1.4-Fr pressure–conductance catheter (SPR-839, Millar Instrument, Houston, TX) was insert into left ventricle (LV) to record baseline hemodynamics of hearts. Data were digitized with a sampling rate of 1000 Hz. All data were analyzed with LabChart software package from AD instruments. Myocardial fibrosis and heart weight to body weight ratio (HW/BW) To determine cardiac fibrosis formation, 8 mm sections from hearts after 14 days of myocardial ischemia were stained with Masson’s trichrome (MT, Sigma, MO). Myocardial fibrosis was quantified by measuring the MT staining area (blue) in the infarcted area using NIH Image analysis software [4]. Cardiac hypertrophy was assessed by measuring heart-to-body weight ratio at 14 days post-myocardial ischemia. Each heart weight was PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35905