Role of HIF1α and PKCβ in mediating the effect of oxygen and glucose in a novel wound assay.

Abstract Delayed wound healing is characteristic of those affected by both Type 1 and Type 2 diabetes. We have developed a novel assay to investigate endothelial cell migration using primary microvascular endothelial cells of dermal origin. Endothelial cell migration was determined using defined monolayers of cells. Net migration or migration at a wounded edge was recorded after 24 or 48 h following incubation in either 20% or 5% oxygen in combination with either 5 mmol/l or 20 mmol/l glucose. Specific intracellular inhibitors of p42/44 MAPK, Pi3 kinase and protein kinase C βII were used. Hypoxia inducible factor type 1 alpha protein was detected using immunocytochemical staining. Cell migration was increased in the presence of hypoxia and decreased with high glucose concentration ( p p p p  > 0.05). HIF-1α protein levels did not significantly reduce in the presence of a PKCβ inhibitor at the wounded edge of cells in 20 mmol/l glucose. In conclusion, we have established a novel assay to determine endothelial cell migration that is robust and reproducible. Impaired cell migration mediated by high glucose concentration was restored using an inhibitor of the PKC βII pathway which correlated with an increase in the level of HIF1α protein.