Regulation of skin microvasculature angiogenesis, cell migration, and permeability by a specific inhibitor of PKCalpha.

Activation of protein kinase C (PKC) induces phenotypic changes in the morphology of microvascular endothelial cells that affect major functions of the microvasculature. These functions include the first stages of sprouting in angiogenesis, cell migration following wounding, and vascular permeability. The specific isoform(s) of PKC responsible for each of these changes has not been previously identified. In this study, we used two inflammatory agents, IL-1beta and phorbol myristic acetate, to activate PKC isozymes and specific inhibitors of PKCalpha (Gö6976) and PKCbeta (hispidin) to distinguish how each of these isoform(s) controls angiogenesis, wound healing, and permeability. In all cases, only inhibition of PKCalpha inhibited each of these functions when compared to the inhibition of PKCbeta. Additional analysis of the mechanism of action of Gö6976 (RT-PCR, Western blots, and immunohistochemistry) of the changes in the phosphorylated and nonphosphorylated forms of PKCalpha in the cell membrane and cytoplasm confirmed the specificity of PKCalpha inhibition by Gö6976. These studies therefore indicate a specific and a regulatory role of the PKCalpha isoform in three major endothelial cell functions that are important in the maintenance of microvascular homeostasis.