VEGF111b Gene Therapy On Cutaneous Wound Healing:An In Vivo Study

Background:Vascular endothelial growth factor A (VEGF-A, hereafter referred to as VEGF) plays key roles in both physiological and pathological angiogenesis. There are two subfamilies of VEGF isoforms with opposite effects on angiogenesis which were generated by alternate splice-site selection in the terminal exon. The pro-angiogenic VEGFxxx isoforms are generated from proximal splice-site selectionin exon 8, whereas the anti-angiogenic VEGFxxxb isoforms are produced as the result of distal splice-site selection. It is well-known that VEGFxxx subfamily members, such as VEGF 165, VEGF 189 and VEGF121, have been proved to be potent promoters for repairing many different types of wounds by enhancing angiogenesis. However, whether VEGFxxxb subfamily members are involved in wound healing process remains unknown. Recently, two novel VEGF isoforms, VEGF111 and VEGF111b, had been identified and found to be resistant to cleavage by plasmin or matrix metalloproteinases. Moreover, paradoxical effects of VEGF111b on angiogenesis had been reported by different researchers. Objective:In order to elucidate the effects of VEGFxxxb on wound repair, we evaluated the wound healing efficacy of VEGF111b gene therapy by using VEGF 111 as a control. Methods and Results:Total RNA were extracted from human MCF7 breast cancer cells and were used as templates for VEGF111 and VEGF111b cDNAs by RT-PCR. Then VEGF111 and VEGF111b eukaryotic expression vectors were constructed by subcloning VEGF111 and VEGF111b cDNAs into plasmid pIRES2-EGFP, respectively. Large-scale extraction of plasmids were carried out by a strategy of silicon dioxide absorption followed by a endotoxin removing step by a Triton X-114 isothermal extraction method. Mouse models of full-thickness skin defect wounds were made by creating two 6 mm diameter full-thickness wounds on the dorsal surface by using a standard 6 mm skin biopsy punch. The polyethylenimine (PEI)-plasmid DNA complexes encapsulated in Pluronic F127 hydrogel were injected into the skin defect region immediately after wounding. And all the wounds were dressed with self-adhering elastic bandages. At all the observation time point, both VEGF111-and VEGF111b-treated groups showed less unhealed area, compared to pIRES2-EGFP control. And both the experimental groups were almost completely healed at day 7. After the mice were euthanized, the wounded tissues were harvested and processed for histological analysis. The number of capillary vessels around wounded tissues was higher in the experimental groups when compared with the control groups. Complete re-epithelialization was observed in both VEGF111-and VEGF111b-treated groups but not in any of the control groups. Masson's trichrome staining demonstrated that blue-stained collagen deposits was significant in all the control groups but not in the experimental groups. It is worth noting that newly formed epithelial layer that protruded into dermal layer and formed a hair follicle-like structure was observed only in VEGF111b-treated group as early as day 5 postoperative, which had been previously described as hair follicle neogenesis and an indicator of regenerative healing. Conclusion:In this study, we found that VEGF111b, the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b, was a potent stimulator for in vivo angiogensis. In addition, both VEGF111 and VEGF111b significantly accelerate the wound repair process through enhanced angiogenesis and re-epithelialization. Moreover, VEGF111b has the potential of healing skin wounds in a regenerative manner.