Ang-1 Reduces Myocardial Infarction in db/db Mice Figure 2. Ang-1 promotes hematopoietic stem cell recruitment into the ischemic hearts of db/db mouse. A. Recruitment of CD133+ in db/db mouse hearts subjected to myocardial ischemia at 24 hours. The hematopoietic stem cells were stained with a CD133 antibody (green, 40X). The nuclei were stained by DAPI (blue, 40X). B: Quantitative analysis of CD133+ cells demonstrating that the number of CD133+ cells was significantly increased in the Ad-Ang-1 treated db/db mice (n = 6) compared to that of Ad-b-gal-treated db/db mice (n = 9). CD133+ cells are expressed as the total number per square mm. Sham control (n = 3). All data represent mean 6 SD; *p,0.05. ND = not detected. C. Co-localization of CD133 with c-kit in db/db mouse infarcted hearts at 14 days of MI. CD133+ cells (green); c-kit+ cells (red) and nuclei were stained by DAPI (blue, 40X). C-kit+ cells were recruited into diabetic db/db mouse infarcted hearts in the Ad-Ang-1 treated db/db mice. Merged images further show that CD133+ cells were co- localized with c-kit+ cells. No c-kit+ cells were seen in the Ad-b-gal-treated db/db mice. No specific staining was observed in the sham control group. n = 3 mice each group. D. Recruitment of hematopoietic CD45+ cells in db/db mouse hearts subjected to myocardial ischemia at 24 hours. The hematopoietic cells were stained with a CD45 antibody (red, 40X). The endothelial cells were stained with a Tie-2 antibody (green). The nuclei were stained by DAPI (blue, 40X). No specific staining was observed in the sham control group. E: Quantitative analysis of CD45+ cells demonstrating that the number of CD45+ cells was significantly increased in the Ad-Ang-1 treated db/db mice (n = 6) compared to that of Ad-b-gal-treated db/db mice (n = 9). CD45+ cells are expressed as the total number per square mm. Sham control (n = 3). All data represent mean 6 SD; *p,0.05. ND = not detected. doi:10.1371/journal.pone.0035905.g002 localization studies further revealed that the increased CD45+ cells co-localized with Tie-2+ in diabetic infarcted hearts treated with Ad-Ang-1 (Figure 2D and E). To examine the roles of SDF-1a/CXCR-4 in Ang-1-induced CD133+ cell recruitment into db/db mouse hearts, we examined the number of CXCR-4+ cells and SDF-1a/CXCR-4 protein expression in the border zone of infarcted myocardium after 24 hours of MI. As shown in Figure 3A and B, overexpression of Ang-1 in db/db mice led to a significant increase in the number of CXCR-4+ cells in the border zone of infarcted hearts in comparison with Ad-b-gal treated db/db mice. No CXCR-4+ cells were found in the sham control db/db mouse hearts. Western blot analysis revealed that overexpression of Ang-1 resulted in significant increases in CXCR-4 and SDF-1a expression in diabetic infarcted hearts as compared to Ad-b-gal diabetic mouse infarcted hearts (Figure 3 C and D). Co-localization studies further revealed that the increased CD133+ co-localized with CXCR-4+ and SDF-1a + cells in diabetic infarcted hearts treated with Ad-Ang-1 (Figure 3E). Overexpression of Ang-1 attenuates myocardial apoptosis and reduces cardiac fibrosis in diabetic db/db mouse hearts We further sought to determine whether increased recruitment of CD133+/c-kit+ cells into the ischemic area by Ang-1 would minimize myocardial apoptosis. TUNEL staining and quantitative analysis revealed a significant increase in TUNEL-positive cells in the infarcted area of db/db mice after 24 hours and 14 days of MI compared to sham control db/db mice. db/db mice treated with Ad-Ang-1 had significantly fewer TUNEL-positive cells in the infarct area compared to db/db mice that received Ad-b-gal PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35905