Abstract 17428: Cu Transporter ATP7A Promotes Angiogenesis by Limiting Autophagic Degradation of VEGFR2

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Abstract

Background: VEGF stimulates angiogenesis through VEGFR2 in endothelial cells (ECs). Copper (Cu), an essential micronutrient, is involved in angiogenesis. Since excess Cu is toxic, intracellular Cu levels are tightly controlled by Cu transporting ATPase (ATP7A) which transports Cu to secretory Cu enzymes at TGN or export excess Cu. However, the role of ATP7A in VEGFR2 signaling and angiogenesis remains unknown.

Results: Here we show that ATP7A expression is dramatically increased in the angiogenic endothelial cells (ECs) in mice hindlimb ischemia model while ATP7A mutant mice with reduced Cu transporter function showed impaired ischemia-induced blood flow recovery (60±6.7% inhibition, p<0.05) and decreased capillary density and VEGFR2 expression. In human cultured ECs, ATP7A knockdown with siRNA significantly inhibited VEGF-induced migration (66±5.5% inhibition, p<0.05), capillary network formation on Matrigel (46±3.0% inhibition, p<0.05) and Cu enzyme LOX activation (62%±9.0 inhibition). Mechanistically, ATP7A depletion promoted VEGF-induced VEGFR2 protein degradation (55±5.2% decrease, p<0.05) and ubiquitination (2.0±0.3-fold increase). This was associated with reduced cell surface VEGFR2 expression, increased VEGFR2 binding to selective autophagic cargo/adaptor p62/SQSTM1 (60±12% increase, p<0.05) and LC3-GFP+autophagic puncta (34±7% increase) and autolysosome formation. Role of autophagy was further confirmed as inhibition of autophagy by bafilomycin A1 or chloroquine prevented enhanced VEGFR2 degradation in ATP7A-depleted ECs. Furthermore, overexpression of p62 or autophagy inducer rapamycin treatment increased VEGFR2 degradation (26±3.5% and 87±1.0% decrease, respectively, p<0.05) and p62 overexpression inhibited ECs migration (29±1.9% inhibition, p<0.05).

Conclusion: Our study demonstrates the novel linkage between Cu transport proteins and VEGFR2 signaling, and provides the evidence that ATP7A functions to promote VEGF-induced angiogenesis via preventing p62/SQSTM1-mediated autophagic VEGFR2 degradation as well as activating Cu-dependent LOX activity, which is required for enhancing therapeutic neovascularization in ischemic disease.

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