Abstract 15199: Cysteine Oxidation of Cu Importer CTR1 Promotes VEGF Signaling and Angiogenesis
Background: Reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, play an important role in angiogenesis with unknown mechanisms. Cysteine sulfenic acid (Cys-OH) formation is initial event of Cys oxidation by ROS involved in redox signalling. The major pathway of cellular Cu entry is via Cu transporter CTR1 at plasma membrane which possesses key redox-sensitive Cys189 at C-terminus. However, the role of CTR1 in ROS-dependent VEGF-induced angiogenesis is completely unknown.
Hypothesis: To determine if CTR1 is involved in VEGFR2 redox signalling in endothelial cells (ECs) and reparative angiogenesis in vivo.
Results: Using mouse skin wound healing angiogenesis model, here we found that CTR1 protein (8 fold) and Cu levels (p<0.05) were significantly increased after wounding, and that CTR1+/- mice showed impaired wound healing (35±1.52% inhibition) and angiogenesis (CD31+ cells) at day 7. In cultured human ECs, silencing CTR1 with siRNA significantly inhibited VEGF-induced pVEGFR2 and its downstream signalling such as pERK and p-p38MAPK as well as EC migration (78.1±3.4%). Mechanistically, biotin-labelled Cys-OH trapping reagent revealed that VEGF binding to VEGFR2 rapidly increased Cys-OH formation of CTR1 within 5 min (9.5±1.2fold, p<0.05) and continued above basal for 4hrs (n=3), which was associated with CTR1-VEGFR2 internalization (30min) and recycling to the membrane (>4hrs). Overexpression of CTR-wild type (WT) increased cell surface VEGFR2 protein expression, as measured by cell surface biotinylation, via binding to VEGFR2, which in turn promoted EC proliferation (9.1±1.1fold) and migration (8.8±1.4fold) in a Cu-dependent manner. By contrast, overexpression of CTR1 Cys oxidation defective mutant CTR1-C189A prevented VEGF induced internalization of CTR1 and VEGFR2, which results in inhibiting pVEGFR2, pERK and p-p38MAPK formation as well as EC migration.
Conclusions: Cys oxidation of CTR1 by VEGF-induced ROS at cell surface promotes CTR1-VEGFR2 internalization, which is required for sustained activation of VEGFR2 signaling, thereby enhancing therapeutic angiogenesis and wound repair. This study will provide a novel linkage between Cu transport system and VEGF redox signalling involved in angiogenesis.
Author Disclosures: A. Das: None. Y. Kim: None. S. Youn: None. S. Varadarajan: None. J. Kaplan: None. T. Fukai: None. M. Ushio-Fukai: None.
- © 2016 by American Heart Association, Inc.