Cu transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2

Copper (Cu), an essential micronutrient, is involved in angiogenesis with unknown mechanism. Since excess Cu is toxic, intracellular Cu levels are tightly controlled by Cu transporting ATPase (ATP7A) which transports Cu to secretory Cu enzymes such as lysyl oxidase (LOX) at TGN or export excess Cu. Autophagy is a lysosome or vacuoles-mediated intracellular degradation pathway, and is shown to be involved in VEGF-induced angiogenesis in endothelial cells (ECs). However, role and mechanism of ATP7A and its linkage with autophagy in VEGFR2 signaling and postnatal neovascularization remain unclear. Our preliminary data show that ATP7A expression is dramatically increased in the angiogenic ECs in mice hindlimb ischemia model while ATP7A mutant (ATP7Amut) mice with recued Cu transporter function show impaired post-ischemic angiogenesis. In cultured ECs, VEGF stimulation promoted ATP7A binding to VEGFR2. Together with other preliminary data, we will test the novel hypothesis that ATP7A promotes VEGFR2 signaling and angiogenesis via preventing selective autophagic cargo/adaptor p62/SQSTM1-mediated autophagic VEGFR2 degradation as well as activating Cu-dependent LOX activity in ECs, which is required for restoring neovascularization in ischemic disease. Aim 1 will determine the role and mechanism of ATP7A in promoting VEGF-induced angiogenesis via preventing p62-mediated autophagic degradation of VEGFR2 in cultured ECs. We will use gain- and loss of ATP7A function approach, LC3-GFP-RFP fluorescence imaging, and identify the ATP7A-p62 binding sites. Aim 2 will determine if ATP7A in ECs promotes angiogenesis by preventing autophagic VEGFR2 degradation. We will use hindlimb ischemia model and inducible EC-specific ATP7A knockout (iEC-ATP7A KO) mice crossed with GFP-LC3B transgenic mice. Innovative Cu imaging such as X-ray fluorescence microscopy will be used. Our studies should provide new insights into Cu transport proteins as potential therapeutic targets for treatment of ischemic cardiovascular diseases. (AHA Program: Postdoctoral Fellowship)