Abstract 230: Protein Kinase D1 Mediates Vegf-induced Vascular Permeability In Vitro And In Vivo By Promoting Endothelial Cell Stress Fiber Formation

Abstract

Background Vascular hyperpermeability is critical components of many human diseases, including inflammation, cancer and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor. Better understanding of the molecular mechanisms by which VEGF induces vascular permeability may provide novel therapeutic targets for these diseases. We and others have recently demonstrated that protein kinase D1 (PKD1), a newly discovered serine/threonine protein kinase, is implicated in VEGF signaling. We hypothesized that PKD1 plays a crucial role in VEGF-induced vascular permeability in vivo and vitro by promoting endothelial cell stress fiber formation. Methods and results Using Tie2-Cre transgenic mice and PKD1 flox mice, we generated endothelium-specific PKD1 knockout (PKD1-ecKO) mice. PKD1-ecKO mice were viable and born at the expected Mendelian ratio. Comparing with wild type mice, PKD1-ecKO mice exhibited the dramatic reduction of VEGF-induced vascular permeability in lung and ear with Miles assay, expressed as Evans blue leakage. Selective pharmacological PKD inhibitor also significantly attenuated in vivo vascular permeability induced by VEGF intravenously injection in mice. In cultured endothelial monolayer, down-regulation of PKD1 by siRNA, GFP-PKD1 dominant negative mutant plasmid or PKD inhibitor significantly reduced VEGF-induced endothelial permeability, measured by bovine serum albumin leakage through endothelial monolayer cultured on transwell system. Moreover, we found that the inhibition of PKD1 diminished F-actin stress fiber formation and the dismantlement of endothelial adherens junctions that lead to intercellular gap formation and leakage in response to VEGF. We further observed that PKD1 acted as a downstream mediator of Rho activation in VEGF-induced stress fiber formation. Specifically, Rho inhibitor Y27632 blocked VEGF-induced PKD1 phosphorylation, while down-regulation of PKD1 expression and activation did not affect VEGF-stimulated Rho activation in endothelial cells. Conclusion PKD1 is required for VEGF-induced vascular permeability in vivo and in vitro through promoting F-actin stress fiber formation and disrupting endothelial adherens junctions.