Reduction of high glucose and phorbol-myristate-acetate-induced endothelial cell permeability by protein kinase C inhibitors LY379196 and hypocrellin A

Abstract

Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. Although recent studies have established a link between protein kinase C (PKC) pathway and hyperglycaemic-induced vascular permeability, it is unclear which PKC isoforms involve increased endothelial cell permeability. In the present study, we investigated whether high glucose induced endothelial hyperpermeability via distinct PKC isoforms in human umbilical vein endothelial cells (HUVECs) and whether increased endothelial permeability could be substantially reversed by PKC inhibitors LY379196 and hypocrellin A (HA). High glucose (20 mM) and phorbol-myristate-acetate (PMA)-induced endothelial hyperpermeability was almost abolished by 150 nM HA and partially reduced by 30 nM PKC β inhibitor (LY379196). LY379196 and HA inhibited the membrane fraction of PKC activity in a dose-dependent manner. Western blot analysis revealed high-glucose-induced overexpression of PKC α and PKC β2 in the membrane fraction of HUVECs. LY379196 (30 and 150 nM) selectively inhibited PKC β2 with no significant effect on PKC α expression. HA (150 nM) significantly reduced PKC α expression with no inhibitory effect on PKC β2. At higher concentrations (300 nM), both LY379196 and HA were no longer selective for PKC β or α, respectively. This study showed that both PKC α and β2 contributed to endothelial hyperpermeability. Since reduction of endothelial hyperpermeability was greater with inhibition of PKC α rather than PKC β2, we conclude that PKC α may be a major isoform involved in endothelial permeability in HUVECs, and that PKC α-mediated endothelial permeability was significantly reversed by the PKC inhibitor HA.