DERMAL-EPIDERMAL INTERACTIONS IN DIABETIC WOUND HEALING

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Our long-term goal is to develop gene transfer based treatment strategies to completely correct the wound healing impairment in diabetes. The overall objective of this proposal is to understand the mechanisms by which dermal-epidermal interactions normally induce the process of re- epithelialization in wound healing, how this process is disregulated in diabetic wound healing, and how over-expression of PDGF-B can "jump start" dermal-epidermal interactions restoring the wound healing process. Based on preliminary work at our laboratory we have developed the overall working hypothesis that: diabetic wounds have disordered dermal-epidermal interactions lacking the normal level and sequence of growth necessary for the orderly progression of wound healing and this disordered dermal-epidermal interaction is corrected by PDGF-B over- expression-induced growth factors made by fibroblasts which accelerate keratinocyte proliferation and migration. To investigate this hypothesis we plan experiments with the following specific aims: I. To test the hypothesis that the mechanisms by which PDGF over-expression induces dermal signaling to the epidermis is mediated by induction of PDGF-A in dermal fibroblasts; II. To test the hypothesis that one mechanism by which PDGF over-expression induces dermal signaling to the epidermis is mediated by induction of PDGF-A in dermal fibroblasts; II. To test the hypothesis that one mechanism by which PDGF-B over-expression corrects diabetic impaired keratinocyte migration and proliferation is the induction of TGF-alpha expression in dermal fibroblasts; III. To test the hypothesis that another mechanism by which PDGF-B over-expression corrects-epidermal interactions in diabetes impaired re-epithelialization is by inducing production of fibronectin by dermal fibroblasts and alpha5bneta1 integrin expression in keratinocytes. We will use a combination in vivo models of diabetic impaired wound healing including the db/db mouse model, non-obese diabetic mice, and streptotozocin-inducing diabetes in TGF-alpha knock out mice. In addition, in vitro techniques using transwell, co-culture and organotypic skin reconstruct models will be used. We will analyze the effects of growth factor over-expression by standard history, immunocytochemistry, in situ hybridization, immunofluorescent staining and confocal microscopy, PCR, RT-PCR, Western blot, and ELISA. The results of these studies will have direct bearing on the translation of adenoviral mediated gene transfer strategies to the treatment of non- healing diabetic foot ulcers with the potential to ameliorate one of the most common complications of this disease.