Adeno-associated virus vectors simultaneously encoding VEGF and angiopoietin-1 enhances neovascularization in ischemic rabbit hind-limbs

Aim: Angiopoietin-1 (Ang1) and vascular endothelial growth factor A (VEGF) play important roles in vascular formation and maturation, suggesting a combination of these 2 would be a promising therapy for ischemic diseases. So we constructed an adeno-associated virus (AAV) vector, simultaneously encoding human VEGF(165) and Ang1 (AAV-VEGF/Ang1), and investigated its therapeutic effect in a rabbit ischemic hind-limb model. Methods: Four experimental groups were used to prepare the rabbit ischemic hind-limb model following AAV vectors intramuscular administration as follows: PBS (phosphate buffered solution), AAV-VEGF, AAV-Ang1, AAV-VEGF/Ang1. Results: Eight weeks after administration, human VEGF(165) and Ang1 were detected by RT-PCR, Western blotting and histochemical staining methods in AAV-VEGF/Ang1 transduced muscles. Group AAV-VEGF/Ang1 showed a significantly increased blood-flow recovery in ischemic hind-limbs compared with the other groups. Histological staining for alkaline phosphatase showed that capillary density of group AAV-VEGF/Ang1 or AAV-VEGF was significantly higher than that of group PBS or AAV-Ang1. Histological immunostaining for smooth muscle alpha-actin (alpha-SMA) revealed that group AAV-VEGF/Ang1 had the highest density of alpha-SMA-positive vessels compared with the other groups. Vascular leakage, one of the major adverse effects induced by VEGF, was very severe in group AAV-VEGF, but the permeability was obviously reduced when VEGF was co-expressed with Ang1 in group AAV-VEGF/Ang1. Conclusion: A AV vectors can simultaneously encode 2 proteins which can be efficiently and stably co-expressed in transduced tissues. AAV-mediated VEGF and Ang1 gene transfer enhances neovascularization, prevents capillary leakage, and improves blood flow in a rabbit hind-limb ischemic model. These findings suggest that intramuscular administration of AAV-VEGF/Ang1 may be useful in the treatment of ischemic diseases.