Ang-1 Reduces Myocardial Infarction in db/db Mice divided by the total body weight of the mouse, resulting in a ratio representative of cardiac hypertrophy. expression was gradual increased in a dose dependent fashion both in the heart and bone marrow (Figure 1D). Statistical analysis All results are expressed as mean 6 SD. Statistical analysis was performed using one-way ANOVA followed by the multiple- comparison test (Student–Newman–Keuls or Dunn’s test). A p value ,0.05 denoted significance. Results Overexpression of Ang-1 in diabetic db/db mouse hearts and bone marrow Systemic delivery of Ad-Ang-1 led to dramatic induction of Ang-1 expression in db/db mouse hearts and bone marrow after 24 hours of myocardial ischemia (Figure 1A). Fluorescent immunohistochemical analysis confirmed the overexpression of Ang-1 in the Ad-Ang-1 treated db/db mouse hearts and bone marrow, but not in the Ad-b-gal treated db/db mouse hearts and bone marrow (Figure 1B). We also examined the location of Ang-1 expression in Ad-Ang-1–treated db/db mouse hearts. Our merged images revealed that Ang-1 localized with Isolectin B4 (IB4) on ECs and SMA on vessel SMCs (Figure 1C). To examine transfection efficiency of Ad-Ang-1 in vivo, experimental mice were systemic administrated with Ad-Ang-1 at dosage of 107–109 pfu for 24 hours. Western blot analysis showed that Ang-1 protein Overexpression of Ang-1 promotes hematopoietic stem cell recruitment via SDF-1a/CXCR-4 signaling in the ischemic hearts of db/db mouse Next, we investigated whether overexpression of Ang-1 affects recruitment of hematopoietic progenitor cells into infarcted db/db mouse hearts. CD133 and c-kit cells were examined in the ischemic hearts at 24 hour and 14 days. No CD133+ cells were observed in the sham control db/db mouse hearts without ischemia. As shown in Figure 2A and 2B, CD133+ cells were recruited into diabetic mouse infarcted hearts after 24 hours ischemia as compared to Ad-b-gal diabetic mouse infarcted hearts. Overexpression of Ang-1 led to a significant increase in CD133+ cells in diabetic infarcted hearts in comparison with Ad-b-gal treated db/db mice. Furthermore, no c-kit+ cells were detected in the sham control, db/db+ ischemia and db/db + ischemia+Ad- Ang-1 at 24 hours. Intriguingly, c-kit+ cells were detected in the Ad-Ang-1 diabetic mouse infarcted hearts at day 14 of ischemia, but not in Ad-b-gal diabetic mouse. The recruited c-kit+ cells were co-localized with CD133+ cells in db/db mouse infarcted hearts treated with Ad-Ang-1 (Figure 2C). CD45+ positive cells were also dramatically increased in diabetic infarcted hearts treated with Ad-Ang-1 at 24 hours after MI as compared to Ad-b-gal diabetic mouse infarcted hearts. Co- Figure 1. Expression and localization of Ang-1 in the diabetic db/db mouse hearts and bone marrow. (A) Western blot analysis demonstrating that systemic administration of Ad-Ang-1 (16109 PFU) resulted in overexpression of Ang-1 in the db/db mouse hearts and bone marrow at day 1 compared to mice receiving Ad-b-gal. (B) Ang-1 was stained with an Ang-1 antibody-labeled with FITC (green, 40X) and nuclei were stained with DAPI (blue, 40X). Merged images show Ang-1 expression in Ad-Ang-1 treated db/db mouse hearts and bone marrow (right panel), but not in Ad-b-gal treated db/db mouse hearts and bone marrow (left panel). (C) Ang-1 protein was localized at smooth muscle cell (SMC) and endothelial cell (EC) in heart tissue. Ang-1 expression was imunostained with Texas Red (red). Endothelial cells were stained with LB4 (green). Merged image revealed that Ang-1 was localized to EC (left panel). Ang-1 was stained with Ang-1 linked to FITC (green). SMCs were stained with SMA (Red) and nuclei were stained with DAPI counterstaining (blue). Merged image showed that Ang-1was localized to SMC (right panel). (D) Western blot analysis demonstrating that systemic administration of Ad-Ang-1 (16107 –16109 PFU) resulted in overexpression of Ang-1 in the mouse hearts and bone marrow in a dose-dependent manner. doi:10.1371/journal.pone.0035905.g001 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35905