Abstract 16809: Novel Role of Copper Transporter ATP7A in VEGF Signaling and Post-Ischemic Neovascularization
VEGF stimulates angiogenesis through activating VEGF receptor2 (VEGFR2) on cell surface in endothelial cells (ECs). Copper (Cu), an essential micronutrient, is involved in angiogenesis and wound repair. Since excess Cu is toxic, intracellular Cu levels are tightly controlled by Cu transporter ATP7A. However, the role of ATP7A in VEGFR2 signaling and post-ischemic neovascularization remains unknown. Here, we show that ATP7A expression is dramatically increased in the angiogenic ECs in a mouse hindlimb ischemia model. ATP7A knockdown by siRNA in ECs inhibits VEGF-induced endothelial migration in a Cu-dependent manner, as measured by modified Boyden chamber assay (78%) and capillary formation on Matrigel. Mechanistically, ATP7A siRNA promotes VEGF-induced VEGFR2 degradation (>=15min; 60%) without affecting mRNA, which is associated with decreased membrane VEGFR2 as visualized by immunofluorescence staining and biotinylation assay. Further, ATP7A siRNA increases VEGFR2 ubiquitination (2 fold) through increasing c-Cbl mediated degradation. Proteosomal inhibitor, MG132 prevents VEGFR2 degradation in ATP7A depleted ECs, suggesting that ATP7A is involved in stabilizing VEGFR2 protein on cell surface. Moreover, VEGF increases ATP7A translocation to the plasma membrane and association with VEGFR2 within 15 min through direct binding to IQGAP1, a VEGFR2 binding scaffold protein that stabilizes VEGFR2. As consequence, ATP7A siRNA inhibits autophosphorylation of VEGFR2 (60%) and its downstream angiogenic signaling such as phosphorylation of Akt (71%), p38 MAPK (68%) and H2O2 production (62%) (>=15min). In vivo studies using hindlimb ischemia model reveal that ischemia-induced blood flow recovery (60% inhibition) and CD 31+ capillary density and α-actin+ arterioles are inhibited (P<0.05) in ATP7A mutant mice with reduced Cu transporter function than wild type mice. Impaired perfusion in ATP7A mutant mice is rescued by adenovirus mediated gene transfer of ATP7A. In summary, this study uncovers a novel role of ATP7A to promote VEGF signaling and angiogenesis by stabilizing VEGFR2 on cell surface through binding to IQGAP1. Thus, ATP7A is an important therapeutic target for promoting neovascularization in ischemic cardiovascular diseases.
- © 2013 by American Heart Association, Inc.