This grant will elucidate the novel protective role of ?Copper transporting ATPase (ATP7A)? against impaired reparative neovascularization in diabetic ischemic vascular diseases. Diabetic complication leads to defective neovascularization in ischemic peripheral vascular disease due to impaired angiogenesis and endothelial cell (EC) barrier dysfunction with unknown mechanisms. Copper (Cu), an essential micronutrient, is involved in angiogenesis while excess Cu contributes to inflammatory diseases such as diabetes. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by ATP7A which delivers Cu to the secretory Cu enzymes, or exports Cu to extracellular space. Our lab discovered that ATP7A in VSMC protects against hypertension. We also identified ?IQGAP1? as a VEGF receptor2 (VEGFR2) binding scaffold protein promoting VEGF signaling and post-ischemic angiogenesis. However, role of ATP7A in ECs for defective post-ischemic revascularization in diabetes is entirely unknown. Preliminary data suggest that ATP7A prevents VEGFR2 degradation through binding to IQGAP1 and maintains basal EC barrier function via regulating VE-cadherin (VE- Cad). ATP7A expression is markedly decreased in ECs from diabetic mice or microvessels of type2 diabetes patients. ATP7A mutant (ATP7Amut) mice with reduced Cu transport function or diabetic mice show impaired ischemia-induced reparative angiogenesis with excess tissue Cu and vascular permeability/tissue damage, which are rescued by overexpression of ATP7A. We thus hypothesize that ATP7A functions to promote and integrate key vascular repair programs such as angiogenesis and maintaining endothelial barrier function in a Cu-dependent manner, which is required for restoring neovascularization in diabetic ischemic vascular disease. Aim 1 will define the protective role of ATP7A against: i) impaired VEGF-induced angiogenesis by stabilizing VEGFR2 and ii) endothelial barrier dysfunction by maintaining Cu homeostasis in ECs isolated from diabetic mice and human microvessels of type2 diabetic patients. Aim 2 will determine the molecular mechanism by which ATP7A downregulation in diabetes impairs VEGFR2 signaling and endothelial barrier integrity by focusing on; i) ATP7A binding to IQGAP1 that prevents VEGFR2 ubiquitination/degradation in a Cu-independent manner, and ii) role of ATP7A in regulating miR-125b that represses VE-Cad via Cu- dependent transcription factor Atox1. Aim 3 will define the protective role of ATP7A against diabetes-induced impaired post-ischemic neovascularization and tissue repair in vivo and address underlying mechanisms using animal model of critical limb ischemia. We will use ATP7Amut and ATP7A transgenic mice; inducible EC-specific ATP7A-/- or Cu importer CTR1-/- mice or type1 and type2 diabetes mice; innovative ICP-Mass Spec, X-ray fluorescence microscopy to analyze [Cu]i in cells and tissues; FRET or BiFC-based protein-protein interaction; and intravital microscopy. Our study will uncover Cu transporter ATP7A as a novel potential therapeutic target to enhance integrated vascular repair program in patients with diabetic vascular complications.
- National Heart, Lung, and Blood Institute
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