Abstract 5329: ROS-dependent Cysteine Sulfenic Acid Formation of IQGAP1 and ERK1/2 in VEGF-induced Endothelial Cell Proliferation and Migration

Abstract

VEGF receptor type 2 (VEGFR2) activation induces endothelial cell (EC) proliferation and migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, role of Cys-SOH formation in VEGF signaling linked to angiogenesis is unknown. We previously identified IQGAP1, as a novel VEGFR2 binding protein, involved in regulating ROS-dependent EC migration and proliferation by recruiting Nox2 NADPH oxidase to activated VEGFR2 signaling complex. By using a newly-developed Cys-SOH trapping reagent we show that VEGF stimulation significantly increases protein-Cys-SOH formation in HUVECs. Immunofluorescence reveals that Cys-SOH modified proteins accumulate at the leading edge where they colocalize with NADPH oxidase, F-actin and IQGAP1 in actively migrating ECs. VEGF stimulation increases IQGAP1-Cys-SOH formation within 5min (60%) with a maximum increase at 15min (90%). VEGF-induced Cys-SOH formation of IQGAP1 is prevented by pretreatment with a GSH donor antioxidant, N-acetylcysteine. Knockdown of IQGAP1 by siRNA or trapping of Cys-SOH with dimedone, blocks wound-induced Cys-SOH formation at the leading edge and EC migration toward the injured site (80%), EC chemotaxis (90%) and matrigel angiogenesis (80%). These suggest linkage between IQGAP1-Cys-SOH formation and IQGAP1-mediated EC migration. In addition, dimedone pretreatment inhibits VEGF-stimulated EC proliferation. VEGF stimulation also induces oxidation of ERK1/2 upon 5 min (50%) and 15min (80%) stimulation. Trapping Cys-SOH by dimedone inhibits ERK phosphorylation (95%), which is associated with increasing IQGAP1/ERK complex (50%) after VEGF stimulation. Hind limb ischemia increases oxidation of tissue proteins including IQGAP1 (80%) and ERK1/2 (60%) at day 1 after injury, which is prevented in mice lacking Nox2. Thus, oxidation of IQGAP1 and ERK contributes to directional migration and proliferation of ECs and to neo-vascularization in ischemic tissue. The present study should provide insights into identifying and visualizing Cys-SOH formed proteins as important strategy for understanding mechanisms by which ROS regulate EC function such as angiogenesis.