Ang-1 Reduces Myocardial Infarction in db/db Mice sections from sham control db/db mouse, db/db mouse treated with Ad-b-gal or Ad-Ang-1 at 24 hours and 14 days of MI. Myocardial and SMCs apoptotic cells in the infarcted area of the ischemic hearts were identified by TUNEL (green) and SMA (red) positive staining, and total nuclei by DAPI counterstaining (blue). F. Representative images of cardiac fibrosis formation and quantitative analysis of cardiac fibrosis area in db/db mice treated with Ad-b-gal or Ad-Ang-1 stained by Masson’s trichrome. Ad-Ang-1 significantly attenuated area of cardiac fibrosis in db/db mice compared to Ad-b- gal mice. All data represent mean 6 SD (n = 5 mice); *p,0.05. G. Heart weight/body weight (HW/BW) ratio in db/db and Ad-Ang-1-treated db/db mouse hearts at 14 days after MI. Treatment with Ad-Ang-1 significantly attenuated cardiac hypertrophy in db/db mouse hearts (n = 5) compared to Ad-b-gal mice (n = 4). All data represent mean 6 SD; *p,0.05. doi:10.1371/journal.pone.0035905.g004 24 hours of myocardial ischemia. The number of BM-CD31+ was significantly increased in db/db mice treated with Ad-Ang-1 compared to db/db mice treated with Ad-b-gal (Figure 5A and B). Overexpression of Ang-1 in BM also resulted in a significant increase in VEGF expression (Figure 5C). Further, overexpres- sion of Ang-1 in db/db mouse hearts resulted in a significant increase in myocardial capillary density at 14 days in the border zone of infarcted myocardium compared to db/db mice treated with Ad-b-gal (Figure 5D and E). The sham control had little effect on BM-CD31+ cells and myocardial capillary density (data not shown). Overexpression of Ang-1 increases BM-SMA+ cells and number of VSMC in the db/db mouse hearts The number of BM-SMA+ cells was significantly increased in db/db mice treated with Ad-Ang-1 compared to db/db mice treated with Ad-b-gal after MI (Figure 6A & B). Next, the number of VSMC in the ischemic area was examined after 24 hours of ischemia by the co-localization of Ang-1 with SMA. Ang-1 expression co-localized with SMA (middle panel). After 2 weeks of MI, Ang-1-positive arteriole with large diameter was also found in the border zone of infarcted myocardial area (Figure 6C). Sham control had little effect on BM-SMA+ and VSMC cell number (Data not shown). Figure 5. Ang-1 increases CD31+ cells and VEGF expression in the BM and promotes capillary formation in db/db mouse hearts. A. Representative images showing that BM cells differentiate into CD31+ cells in Ad-b-gal treated db/db mice subjected to myocardial ischemia for 24 hours (left panel) and in the Ad-Ang-1treated db/db mice (right panel). B. Quantitative analysis of BM differentiation into CD31+ after myocardial ischemia. The number of BM cells that differentiate into CD31+ was significantly increased in the Ad-Ang-1treated db/db mice compared to the Ad-b- gal treated db/db mice. There was not difference between sham control and ischemic group. All data represent mean 6 SD (n = 3 mice); *p,0.05. C. Western blot densitometric analysis of VEGF expression revealed that systemic delivery of Ad-Ang-1 resulted in a significant increase in VEGF expression in the BM of db/db mouse compared to the Ad-b-gal db/db treated mice. All data represent mean 6 SD (n = 3 mice); *p,0.05. D and E. Representative images and quantitative analysis showing that treatment with Ad-Ang-1 significantly increased capillary formation in db/db mice subjected to ischemia compared to Ad-aˆ-gal-treated db/db mice. All data represent mean 6 SD (n = 7 mice); *p,0.05. doi:10.1371/journal.pone.0035905.g005 PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35905