Angiopoietin-1 gene transfer improves impaired wound healing in genetically diabetic mice without increasing VEGF expression.
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.
Bitto
Alessandra
A
Section of Pharmacology, Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy.
Minutoli
Letteria
L
Galeano
Maria Rosaria
MR
Altavilla
Domenica
D
Polito
Francesca
F
Fiumara
Tiziana
T
Calò
Margherita
M
Lo Cascio
Patrizia
P
Zentilin
Lorena
L
Giacca
Mauro
M
Squadrito
Francesco
F
D042683:Angiopoietin-1 / Q000235:genetics* / Q000502:physiology; D000818:Animals; D002199:Capillary Permeability; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D012867:Skin / Q000098:blood supply / Q000293:injuries* / Q000378:metabolism / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D014945:Wound Healing / Q000235:genetics*
D042683:Angiopoietin-1; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2