Protein Kinase C Inhibitors Prevent Cultured Human Endothelial Cell Stress Fiber Formation, but not Heightened Endocytosis

In order to study the signal transduction mechanism of endothelial perturbation, the effects of phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), both protein kinase C (PKC) activators, on cultured human endothelial cell (EC) hydrogen peroxide (H2O2) generation, endocytotic activity, and cytoskeletal structure have been investigated. EC were incubated with 1-100 nM PMA, or PDBu, and cellular H2O2 generation and endocytotic activity measured. PMA and PDBu exposure caused dose-dependent rises in EC H2O2 production. Likewise, EC incubated with PMA and PDBu had dose-related endocytosis increases. Cytoskeletal inspection of 10 nM PMA-perturbed EC revealed structural remodeling with stress fiber formation. Similar cellular functional changes occur in EC exposed to high low-density lipoprotein (LDL) concentrations. Protein kinase C (PKC) inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) prevented cytoskeletal remodeling in PMA-stimulated EC. In difference to this inhibitory effect, H-7 and calphostin C, another PKC inhibitor, augmented EC H2O2 generation and endocytotic activity. Similar cellular responses to H-7 and calphostin C occurred in high LDL-perturbed EC. These findings suggest that PKC activation contributes to the stress fiber formation mechanism, whereas H2O2 generation and heightened endocytosis can occur by both PKC-dependent and PKC-independent mechanisms. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in atherogenesis.