The Regulation of Vascular Endothelial Growth Factor-induced Microvascular Permeability Requires Rac and Reactive Oxygen Species

Elizabeth Monaghan-Benson,Keith Burridge

doi:10.1074/jbc.m109.009894

Abstract

Vascular permeability is a complex process involving the coordinated regulation of multiple signaling pathways in the endothelial cell. It has long been documented that vascular endothelial growth factor (VEGF) greatly enhances microvascular permeability; however, the molecular mechanisms controlling VEGF-induced permeability remain unknown. Treatment of microvascular endothelial cells with VEGF led to an increase in reactive oxygen species (ROS) production. ROS are required for VEGF-induced permeability as treatment with the free radical scavenger, N-acetylcysteine, inhibited this effect. Additionally, treatment with VEGF caused ROS-dependent tyrosine phosphorylation of both vascular-endothelial (VE)-cadherin and beta-catenin. Rac1 was required for the VEGF-induced increase in permeability and adherens junction protein phosphorylation. Knockdown of Rac1 inhibited VEGF-induced ROS production consistent with Rac lying upstream of ROS in this pathway. Collectively, these data suggest that VEGF leads to a Rac-mediated generation of ROS, which, in turn, elevates the tyrosine phosphorylation of VE-cadherin and beta-catenin, ultimately regulating adherens junction integrity.

Highlights

VEGF2 was first discovered as a potent vascular permeability factor that stimulated a rapid and reversible increase in micro
Our results show that vascular endothelial growth factor (VEGF) treatment of human microvascular endothelial cells results in the Rac-dependent producboxy-2Ј,7Ј-dichlorofluorescein diacetate di(acetoxymethyl ester); HMVEC, human pulmonary microvessel endothelial cell; WT, wild type; siRNA, small interference RNA; FITC, fluorescence isothiocyanate; NAC, N-acetylcysteine; DPI, diphenyleneiodonium chloride; PAK, p21-activating kinase
If reactive oxygen species (ROS) are scavenged with NAC we found that the VEGF-induced changes in VE-cadherin staining are attenuated (Fig. 2C, panel d)

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Unless otherwise stated all chemicals were obtained from Sigma. Cells were loaded with 5 ␮M DCF in serum-free medium for 30 min at 37 °C. Cells were stimulated as described in the figure legends. Cells were serum-starved for 2 h before treatment with VEGF. To visualize VE-cadherin cells were stained with a monoclonal antibody against VE-cadherin (Santa Cruz Biotechnology) followed by goat anti-mouse Alexa 488 (Molecular Probes). After stimulation cells were kept on ice, washed with ice-cold phosphate-buffered saline, and assayed for Rac activation with glutathione S-transferase-p21-activating kinase, as described by Sander et al [62]. The beads were resuspended in sample buffer, boiled for 10 min, and analyzed by Western blotting. Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell Bioscience) and processed for Western analysis using the antibodies described in the figure legends. For quantification of Western blots, intensity values of bands were measured from three different repeats for each experiment using ImageJ (National Institutes of Health)

RESULTS

ROS formation we asked whether

DISCUSSION