Angiopoietin-1 gene transfer improves impaired wound healing in genetically diabetic mice without increasing VEGF
Alessandra Bitto1, Letteria Minutoli, Maria Rosaria Galeano
1Section of Pharmacology, Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy.
Abstract:
Ang-1 (angiopoietin-1) improves the ineffective angiogenesis and impaired wound healing in diabetes; however, the mechanism underlying this positive effect is still far from being completely understood. In the present study, we investigated whether rAAV (recombinant adeno-associated virus)-Ang-1 gene transfer could improve wound repair in genetically diabetic mice (db+/db+) and the mechanism(s) by which it causes new vessel formation. An incisional skin-wound model in diabetic and normoglycaemic mice was used. After the incision, animals received rAAV-LacZ or rAAV-Ang-1 in the wound edge. After 7 and 14 days, wounds were used to (i) confirm Ang-1 gene transfer, (ii) assess histologically the healing process, (iii) evaluate wound-breaking strength, and (iv) study new vessel formation by PECAM-1 (platelet/endothelial cell adhesion molecule-1) immunostaining. Finally, we investigated VEGF (vascular endothelial growth factor) mRNA and protein levels, eNOS (endothelial NO synthase) expression and VEGFR-1 and VEGFR-2 (VEGF receptor-1 and -2 respectively) immunostaining. The efficiency of Ang-1 gene transfer was confirmed by increased mRNA and protein expression of the protein. rAAV-Ang-1 significantly improved the healing process, stimulating re-epithelization and collagen maturation, increasing breaking strength and significantly augmenting the number of new vessels, as indicated by PECAM-1 immunostaining. However, Ang-1 gene transfer did not modify the decrease in VEGF mRNA and protein expression in diabetic mice; in contrast, Ang-1 increased eNOS expression and augmented nitrate wound content and VEGFR-2 immunostaining and protein expression. Ang-1 gene transfer did not change vascular permeability. Similar results were obtained in normoglycaemic animals. In conclusion, our results provide strong evidence that Ang-1 gene transfer improves the delayed wound repair in diabetes by inducing angiogenesis in a VEGF-independent manner.
