Mechanisms for Redox Regulation of Selective VEGF Signaling Pathways in Endothelial Cells

Project: Research

Investigators

  • Ruhul Abid (PI)

Description

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular physiology and pathophysiology including hypertension, angiogenesis, and vascular complications in arthritis and diabetes. Traditionally, ROS have been considered harmful for the endothelium. However, recent findings suggest that ROS play critical roles in signal transduction in endothelial cells (ECs). We and others have identified Rac-1-dependent NADPH oxidase as the major source of VEGF-inducible and ambient levels of ROS in endothelial cells (ECs), and is essential for proliferation and migration in ECs, and angiogenesis. Recently, we have demonstrated that NADPH oxidase is not essential for global VEGF signaling, instead ROS modulate activities of selective signaling intermediates (Src, FAK, PI3K-Akt-eNOS but not ERK1/2) downstream of VEGF receptor 2 (VEGFR2). Here, we demonstrate that NADPH oxidase-derived ROS are essential for recruitment and activation of SHP2 by Shb-VEGFR2, followed by activation of Src and inhibition of PLCgammma-1. We will test the HYPOTHESIS that transient oxidation of SHP2 by NADPH oxidase-derived ROS is essential for binding and activation of SHP2 by Shb-VEGFR2 complex, and the activated SHP2 activates PI3K-Akt signaling pathway by two independent mechanisms: one, by activating Src through PAG/CSK inhibition, and second, by competing with PLCgamma-1 for a common binding site on VEGFR2. Using primary ECs from human coronary, siRNA against p47phox and cysteinyl labeling assay, we will elucidate the molecular mechanisms by which NADPH oxidase-derived ROS induce recruitment of SHP2 into a complex of Shb and VEGFR2 to activate Src (Specific Aim#1). We will also employ phoshpholipase assay, PAE-Y1175F VEGFR2 mutant, and binding competition assay between PLCgamma and SHP2 for VEGFR2-Shb to delineate the molecular mechanisms by which NADPH oxidase prevent hyperactivation of PLCgamma to increase availability of the common substrate, PIP2, to PI3K (Specific Aim#2). Finally, using NADPH oxidase knockout mice for coronary vessel relaxation and wound healing assay, we will determine the molecular mechanisms that link NADPH oxidase activity to selective VEGF functions in the endothelium such as microvascular reactivity and angiogenesis (Specific Aim#3). This study should provide a framework for tailoring and fine tuning therapeutic modalities in vascular disease states by selectively manipulating redox-sensitive endothelial functions
Award amount$198,000.00
Award date07/01/2010
Program typeGrant-in-Aid
Award ID10GRNT3640011
Effective start/end date07/01/201006/30/2013
StatusFinished