Ang-1 Reduces Myocardial Infarction in db/db Mice Figure 3. Ang-1 upregulates SDF-1a and CXCR-4 expression in the ischemic hearts of db/db mouse. A: Expression of CXCR-4 in db/db mouse hearts subjected to myocardial ischemia at 24 hours. CXCR-4 positive cell (red) and nuclei were stained by DAPI (blue). B: Quantitative analysis showing that the number of CXCR-4+ 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). CXCR-4+ 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: Western blot and densitometric analysis of myocardial CXCR-4 expression showing that overexpression of Ang-1 significantly increased CXCR-4 expression in db/db mice compared to that of Ad-b-gal-treated db/db mice. All data represent mean 6 SD (n = 4 mice); *p,0.05. D. Western blot and densitometric analysis of myocardial SDF-1a expression. Overexpression of Ang-1 significantly increased SDF-1a expression in db/db mice compared to that of Ad-b-gal-treated db/db mice. All data represent mean 6 SD (n = 4 mice); *p,0.05. E. Co-localization of CD133 with CXCR-4 and CD133 with SDF-1a in db/db mouse infarcted hearts. CD133+ cells (green); CXCR-4+ or SDF-1a+ cells (red) and nuclei were stained by DAPI (blue, 40X). Merged images show that CD133+ cells co-localized with both CXCR-4 (upper panel) and SDF-1a (bottom panel) in the Ad-Ang-1 treated db/db mice, but little co-localization was seen in the Ad-b-gal-treated db/db mice. No specific staining was observed in the sham control group. doi:10.1371/journal.pone.0035905.g003 (Figure 4A and B). Co-staining of TUNEL with vWF further revealed that treatment of db/db mice with Ad-Ang-1 significantly attenuated the number of TUNEL/vWF-positive cells in the infarct area compared to db/db mice that received Ad-b-gal (Figure 4A and C). Similarly, treatment of db/db mice with Ad- Ang-1 for 24 hours significantly reduced myocardial apoptosis in the remote area of ischemia compared to db/db mice that received Ad-b-gal (Figure 4D). However, treatment with Ad- Ang-1 has little effect on the number of TUNEL/SMA positive cells (Figure 4E). Compared to db/db mice treated with Ad-b- gal, mice treated with Ad-Ang-1 showed a significant decrease in the area of myocardial fibrosis at 14 days after MI (Figure 4F). At 14 days, treatment with Ad-Ang-1 led to a significant decrease in the HW/BW ratio in db/db mice subjected to MI as compared with db/db mice treated with Ad-b-gal (Figure 4G). Overexpression of Ang-1 increases the number of BM- CD31+ cells and myocardial capillary density in db/db mouse hearts New evidence indicates that BM-CD31+ cells have angiogenic properties and implantation of BM-CD31+ cells into ischemic hind-limb promotes recovery of tissue from ischemic injury [17]. Therefore, we examined the number of BM-CD31+ cells after PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35905