Peripheral neuropathy in patients with diabetes mellitus is closely associated with development of cutaneous non-healing ulcers. Twenty to thirty thousand amputations performed annually in the United States on diabetic patients with chronic non-healing ulcers significantly impact medical costs, impairment and quality of life. Determining cellular events leading to diabetic neuropathy and impaired wound healing provides an opportunity for therapeutic intervention. We hypothesize that in patients with diabetes mellitus, microvascular endothelial cells and keratinocytes 1) do not produce necessary neurotrophic factors for sensory nerve fiber growth and 2) do not respond normally to nerve derived inflammatory mediators and these abnormalities contribute to impaired wound healing. We anticipate that hyperglycemia prevents normal signaling between cutaneous cells and sensory nerve fibers. This may result from decreased substance P due to the reduced innervation. Glycosylation of substance P, cell surface receptors or matrix molecules due to prolonged hyperglycemia may inhibit normal neuroinflammation. Alternatively, proteolytic degradation of substance P by increased levels of the enzyme neutral endopeptidase may reduce neuroinflammation. We will test our hypothesis by addressing the following: Specific Aim 1: To determine whether hyperglycemia blunts the response of cutaneous cells to substance P. We will compare substance P- induced NGF production by microvascular endothelial cells and keratinocytes under normal and hyperglycemic conditions. We will evaluate the effect of hyperglycemia on substance P-induced changes in endothelial cell integrin expression and cytoskeleton organization. Specific Aim 2: To determine whether matrix molecule glycosylation interferes with response of cutaneous cells to substance P. We will determine whether matrix molecule glycation decrease substance P-induced NGF synthesis or changes in endothelial cell cytoskeletal organization and/or integrin expression. Specific Aim 3: To determine the effect of hyperglycemia and matrix molecule glycosylation on neutral endopeptidase expression and activity by cutaneous cells. We will determine whether hyperglycemia or matrix molecule glycation increases neutral endopeptidase activity by cutaneous cells. We will determine whether hyperglycemia or matrix molecule glycation increases neutral endopeptidase expression and activity by microvascular endothelial cells or keratinocytes. Specific im 4: To determine whether restoration of neuropeptides or neurotrophins improves wound repair in diabetic (db/db) mice. Using an excisional would model in hyperglycemia db/db mice, we will replace substance P, replace NGF or inhibit neutral endopeptidase activity to evaluate the roles of neuropeptides and NGF in wound repair.