Substance P (SP), a sensory nerve derived neuropeptide, has been implicated in wound repair. Our hypothesis was that oxidative effects of elevated glucose and fatty acid levels as seen with diabetes mellitus inhibit SP-mediated endothelial cell directional migration and proliferation. Using a 2% agarose gel, immortalized human microvascular endothelial cells (HMEC-1) were plated into a 1.5-mm well, and agonist (SP; 10(-4) mol/L) was loaded into a 3-mm well; controls included NaCl, albumin (bovine serum albumin), and vascular endothelial cell growth factor. The SP receptor antagonist spantide 1 was used to confirm SP specificity. Elevated glucose (40 mmol/L) and fatty acids (40 micromol/L) were added to the medium with and without vitamin E and vitamin C treatment to determine whether endothelial cell responses to SP were altered by metabolic perturbations and whether they could be recovered with antioxidant treatment. Using computer-assisted image analysis, migration distance was measured. Cells were counted using a hemocytometer. Human microvascular endothelial cell 1 migration toward the SP exceeded NaCl or bovine serum albumin; vascular endothelial cell growth factor had similar effects. The SP receptor antagonist, spantide, inhibited SP-induced HMEC-1 migration. Substance P treatment was associated with increased cell number. Ki-67 staining was increased in SP-treated cells compared with controls. Elevated glucose and fatty acid levels diminished cell migration toward SP. The antioxidants vitamins C and E significantly improved proliferation but only marginally improved migration. Our data suggest that glucose and fatty acids perturb SP-induced HMEC-1 migration and proliferation in an agarose gel migration model.