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 CβII 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<0.001). The newly developed wound healing assay revealed that re-endothelialisation occurred at a greater rate (p<0.001) than endothelialisation. Inhibition of p42/44MAPK significantly reduced endothelial cell migration at both the intact and the wounded edge in 20 mmol/l glucose but not 5 mmol/l glucose. Inhibition of Pi3 kinase significantly (p<0.001) reduced migration in all test conditions, while inhibition of PKCβ restored glucose mediated impaired migration (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 PKCβII pathway which correlated with an increase in the level of HIF1α protein.