Ang-1 Reduces Myocardial Infarction in db/db Mice Figure 6. Ang-1 increases number of SMA+ cells in the db/db mouse BM and hearts. A. Representative images showing that BM cells differentiate into vascular smooth muscle-like (SMA+) cells in Ad-b-gal treated db/db mice subjected to MI for 24 hours (left panel) and in the Ad-Ang- 1treated db/db mice (right panel). B. Quantitative analysis showing a significant increase in number of SMA+ cells after myocardial ischemia for 24 hours following Ad-Ang-1 compared to Ad-b-gal in db/db mice subjected to MI. All data represent mean 6 SD (n = 3 mice); *p,0.05. C. Representative images of myocardial VSMC in infarcted areas in db/db+IS+Ad-b-gal and db/db+IS+Ad-Ang-l mice stained by Ang-1+/SMA+ cells. Ang- 1 was stained with monoclonal anti-angiopoietin-1 linked to FITC (green, 40X). VSMCs were stained with smooth muscle actin (SMA, Red, 40X) and nuclei were stained with DAPI counterstaining (blue, 40x). Merged image showed that over-expressed Ang-1 co-localized with VSMC. Top and middle panel: representative images showing that systemic delivery of Ad-Ang-1 led to a dramatic increase in the number of Ang-1+/SMA+ cells in db/db mouse subjected to myocardial ischemia for 24 hours compared to Ad-b-gal in db/db mice. Bottom panel: representative images showing the newly formed arteriole (Ang-1+/ SMA+) in the Ad-Ang-1 treated db/db mouse heart after ischemia for 14 days. doi:10.1371/journal.pone.0035905.g006 Overexpression of Ang-1 upregulates Jagged1/Notch3 and apelin expression in diabetic db/db mouse hearts To explore the potential intracellular molecular mechanisms by which overexpression of Ang-1 increased angiogenesis in diabetes, myocardial Jagged 1/Notch3 and apelin/APLNR expression in db/db mice was examined. Systemic delivery of Ad-Ang-1 resulted in a significant increase in Jagged 1 and Notch3 expression 24 hours after myocardial ischemia compared to Ad-b-gal (Figure 7A and B). Immunohistochemical analysis confirmed that the increased Notch3 localized in the wall of vessels in diabetic infarcted hearts treated with Ad-Ang-1 (Figure 7C). Systemic delivery of Ad-Ang-1 also leads to a significant increase in apelin expression at 24 hours after MI compared to Ad-b-gal (Figure 7D). APLNR (APJ) expression in the db/db mice treated with Ad-Ang-1 showed a trend to increase, however, failed to achieve statistical significance (Figure 7E). Sham control had little effect on Jagged1/Notch3 and apelin/APLNR expression (Data not shown). Overexpession of Ang-1 improves diabetic cardiac function after myocardial ischemia To determine whether increased homing of hematopoietic stem cell and angiogenesis by Ang-1 promoted cardiac functional recovery, cardiac function was measured by P-V loop at 14 days of myocardial ischemia. As shown in Fig 8 A and B, exposure of diabetic hearts to ischemia for 14 days resulted in a dramatic reduction of cardiac output (CO) and ejection fraction (EF%). Overexpression of Ang-1 led to a significant improvement of CO and EF% in diabetic infarcted hearts. Discussion Our data establish, for the first time, that Ang-1 plays a critical role in the recruitment of hematopoietic stem cell CD133+/c-kit+ and CD45+ cells into ischemic areas of the diabetic infarcted heart. This is accompanied with a significant reduction in myocardial/ endothelial apoptosis and cardiac hypertrophy as well as fibrosis formation in diabetic heart. Our study also demonstrates that PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35905