Abstract 220: Detection of Cysteine Sulfenic Acid Formation: Role in Reactive Oxygen Species-dependent VEGF signaling Linked to Endothelial Migration
NADPH oxidase Nox2-derived reactive oxygen species (ROS) have been implicated in VEGF receptor type2 (VEGFR2)-mediated signaling linked to endothelial cell (EC) migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, the role of Cys-SOH formation in VEGF signaling and EC migration remains unknown. Using a newly-developed Cys-SOH trapping reagent, here we show that VEGF stimulation significantly increases the total Cys-SOH formation of proteins in HUVECs. VEGFR2 immunoprecipitates show significant increase in Cys-SOH within 5 min (90.3%), which remained elevated at least for 30 min (30.1%). This VEGF-induced Cys-SOH formation of VEGFR2 is prevented by pretreatment with a thiol donor antioxidant, N-acetylcysteine, which is associated with inhibition of VEGFR2 autophosphorylation (80.2%). The trapping of endogenous Cys-SOH by dimedone blocks VEGFR2 autophosphorylation (50.3%), its downstream phosphorylation of PLCgamma (70.4%) and ERK1/2 (90.1%) as well as EC migration (80.3%) without affecting p38MAPK phosphorylation. Wound scratch assay and immunofluorescence analysis reveal that Cys-SOH modified proteins are accumulated at the leading edge where they colocalize with Nox2, phospho-VEGFR2, F-actin and IQGAP1, a novel VEGFR2- and actin-binding scaffold protein, in actively migrating ECs. A significant increase in Cys-SOH is also seen in IQGAP1 coprecipitates (~250 kDa) following VEGF stimulation with peak at 2 min (50.1% increase). Knockdown of IQGAP1 using siRNA, or trapping of Cys-SOH block wound-induced Cys-SOH formation at the leading edge as well as EC migration toward the injured site. In summary, these results suggest that VEGFR2 and IQGAP1-associated proteins are targets for Cys-SOH formation at the leading edge in actively migrating ECs. The present study provides a novel mechanism of redox regulation in VEGF signaling linked to EC migration, and a rationale for the importance of identification of novel proteins that form Cys-SOH during repair process after injury and angiogenesis in vivo.
This research has received full or partial funding support from the American Heart Association, AHA National Center.