application) One of the stated goals of Healthy People 2000 is to reduce the incidence of lower extremity amputation in diabetic patients from the 1988 baseline rate of 8.2/1000 to a target incidence of 4.9/1000 in the year 2000. Despite efforts directed at prevention, the rate of amputation has continued to rise in patients with DM. A better understanding of the pathogenesis of diabetic ulcers and novel treatment strategies are required to reverse the rising trend in the rate of amputation. The goal of this application is to use new molecular technologies to enhance healing of diabetic ulcers by increasing the migration of cells (keratinocytes) at the ulcer margin. The laminin 5 (Lam 5) cell-signaling pathway is the focus of this investigation. An interdisciplinary team will conduct the proposed experiments in patients and mice with DM. This application will test three general HYPOTHESES: 1. The principle deficit in closure of diabetic ulcers is failure of keratinocyte migration despite marked keratinocyte proliferation at the ulcer margin. 2. Lam 5 mediated signaling, essential for keratinocyte migration, is impaired in diabetes mellitus. 3. Clarification of Lam 5 cellsignaling pathways will lead to novel approaches to stimulate keratinocyte migration and speed healing of diabetic ulcers. In order to test these hypotheses we will carry out the following Specific Aims:
Specific Aim 1. Characterize chronic ulcers from patients with diabetes and acute wounds from diabetic and normal human subjects/mice regarding Lam 5 isoforms and Lam 5 mRNA, the Lam 5 associated integrins alpha6-beta4 alpha3-beta1 and the proliferation marker Ki67.
Specific Aim 2. Evaluate functions for Lam 5 sub-domains as well as design and produce recombinant isoforms of Lam 5.
Specific Aim 3. Evaluate mechanisms by which keratinocytes regulate synthesis and deposition of Lam 5, and evaluate the role of Lam 5 in the migration of normal and diabetic cells.
Specific Aim 4. Assess the effects of application of recombinant Lam 5 isoforms to the wounds of db/db diabetic mice and develop bioniaterials that elicit a maximal increase in healing rate.
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