Enhanced vascularization of cultured skin substitutes genetically modifiedto overexpress vascular endothelial growth factor

Cultured skin substitutes have been used as adjunctive therapies in the tre atment of burns and chronic wounds, but they are limited by lack of a vascu lar plexus. This deficiency leads to greater time for vascularization compa red with native skin autografts and contributes to graft failure. Genetic m odification of cultured skin substitutes to enhance vascularization could h ypothetically lead to improved wound healing. To address this hypothesis, h uman keratinocytes were genetically modified by transduction with a replica tion incompetent retrovirus to overexpress vascular endothelial growth fact or, a specific and potent mitogen for endothelial cells. Cultured skin subs titutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified o r control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endoth elial growth factor mRNA in genetically modified keratinocytes and in cultu red skin substitutes prepared with modified cells. Furthermore, the vascula r endothelial growth factor-modified cultured skin substitutes secreted gre atly elevated levels of vascular endothelial growth factor protein througho ut the entire culture period. The bioactivity of vascular endothelial growt h factor protein secreted by the genetically modified cultured skin substit utes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin subst itutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modi fied grafts for at least 2 wk after surgery. Vascular endothelial growth fa ctor-modified grafts exhibited increased numbers of dermal blood vessels an d decreased time to vascularization compared with controls. These results i ndicate that genetic modification of keratinocytes in cultured skin substit utes can lead to increased vascular endothelial growth factor expression, w hich could prospectively improve vascularization of cultured skin substitut es for wound healing applications.