OBJECTIVE: Diabetic impaired angiogenesis is associated with impairment of hypoxia-inducible factor-1alpha (HIF-1alpha) as well as vasculature maturation. We investigated the potential roles and intracellular mechanisms of angiopoietin-1 (Ang-1) gene therapy on myocardial HIF-1alpha stabilization and vascular maturation in db/db mice. RESEARCH DESIGN AND METHODS: db/db mice were systemically administrated adenovirus Ang-1 (Ad-CMV-Ang-1). Myocardial HIF-1alpha, vascular endothelial growth factor (VEGF), hemeoxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), Akt, and HIF-1alpha-prolyl-4-hydroxylase-2 (PHD)2 expression were measured. Vasculature maturation, capillary and arteriole densities, and cardiac interstitial fibrosis were analyzed in the border zone of infarcted myocardium. RESULTS: Systemic administration of Ad-CMV-Ang-1 results in overexpression of Ang-1 in db/db mice hearts. Ang-1 gene therapy causes a significant increase in Akt and eNOS expression and HIF-1alpha stabilization. This is accompanied by a significant upregulation of VEGF and HO-1 expression. Intriguingly, Ang-1 gene therapy also leads to a significant inhibition of PHD2 expression. Smooth muscle recruitment and smooth muscle coverage in the neovessels of the border zone of infarcted myocardium are severely impaired in db/db mice compared with wild-type mice. Ang-1 gene therapy rescues these abnormalities, which leads to a dramatic increase in capillary and arteriole densities and a significant reduction of cardiac hypertrophy and interstitial fibrosis at 14 days after ischemia. Taken together, our data show that Ang-1 increases myocardial vascular maturation and angiogenesis together with suppression of PHD2 and the upregulation of HIF-1alpha signaling. CONCLUSIONS: Normalization of immature vasculature by Ang-1 gene therapy may represent a novel therapeutic strategy for treatment of the diabetes-associated impairment of myocardial angiogenesis.