Ang-1 Reduces Myocardial Infarction in db/db Mice Figure 7. Ang-1 upregulates Jagged1, Notch3 and apelin expression in db/db mouse hearts. A. Western blot densitometry analysis of myocardial Jagged 1 expression showing that systemic delivery of Ad-Ang-1 resulted in a significant increase in Jagged 1 expression in the db/db mice subjected to myocardial ischemia for 24 hours compare to the db/db mice treated with Ad-b-gal. All data represent mean 6 SD (n = 4 mice); *p,0.05. B. Western blot densitometric analysis of myocardial Notch3 expression revealing that systemic delivery of Ad-Ang-1 (n = 5) resulted in a significant increase in Notch3 expression in db/db mice subjected to myocardial ischemia for 24 hours compare to the db/db mice treated with Ad- b-gal (n = 4). All data represent mean 6 SD; *p,0.05. C. Immunofluorescence microscopy showing expression of Notch3 in the vascular wall of db/db mice infarcted hearts treated with Ad-Ang-1. Merged images showed that Notch3 expression was expressed in newly formed arteriole of the Ad-Ang- 1 treated db/db mice (right panel), but not the Ad-b-gal-treated db/db mice (left panel). Notch3 was stained with Notch antibody conjugated to FITC (green, 40X) and nuclei were stained by DAPI (blue). No specific staining was observed in the sham control group. D. Western blot densitometry analysis showing that apelin expression was significantly increased in the db/db mice treated with Ad-Ang-1 compare to the Ad-b-gal treated db/db mice at 24 hours of ischemia. All data represent mean 6 SD (n = 4 mice); *p,0.05. E. APLNR expression was increased in the Ad-Ang-1 treated db/db mice compare to the Ad-b-gal treated db/db mice at 24 hours of ischemia, but did not achieve a statistical significantly difference. All data represent mean 6 SD (n = 4 mice); p.0.05. doi:10.1371/journal.pone.0035905.g007 upregulation of SDF-1a/CXCR-4 expression in the diabetic infarcted hearts is a novel downstream signaling pathway of Ang- 1-mediated recruitment of CD133+ cells. Further, our studies reveal that overexpression of Ang-1 results in increases in capillary density and arteriole formation together with a dramatic improve- ment of cardiac functional recovery in the diabetic infarcted hearts. These studies strongly suggest that Ang-1 gene therapy protects the diabetic heart from ischemic injury by promoting PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35905