Cu Transporting ATPase and Diabetic Vascular Complications

铜转运 ATP 酶与糖尿病血管并发症

基本信息

批准号：
9977232
负责人：
TOHRU FUKAI
金额：
$ 68.07万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9977232
关键词：
3-Dimensional ATP phosphohydrolase Address Animal Model Animals Binding Binding Sites Bioavailable Biological Assay Biological Availability Biotinylation Blood Vessels Cardiac Cardiovascular system Cells Clinical Complications of Diabetes Mellitus Copper Data Diabetes Mellitus Diabetic Angiopathies Diabetic mouse Disease Down-Regulation Endothelial Cells Endothelium Enzymes Extracellular Space Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder Gene Transfer Goals Grant Hindlimb Homeostasis Human Hypertension Impairment Inductively Coupled Plasma Mass Spectrometry Inflammation Inflammatory Injections Ischemia Knockout Mice Lead Leukocytes Measures Micronutrients Molecular Morbidity - disease rate Mus Mutate Patients Perfusion Peripheral Peripheral Vascular Diseases Permeability Play Protein-Lysine 6-Oxidase Reporting Risk Factors Roentgen Rays Role Scaffolding Protein Signal Transduction Synchrotrons Testing Tissues Transgenic Mice Transgenic Organisms Ubiquitination Vascular Diseases Vascular Endothelial Growth Factors Vascular Permeabilities angiogenesis base cadherin 5 copper-transporting ATPase critical limb Ischemia diabetic diabetic patient extracellular in vivo innovation insight intravital microscopy live cell imaging migration mortality mutant neovascularization new therapeutic target novel overexpression prevent programs protein protein interaction recruit repaired small hairpin RNA therapeutic target tissue repair transcription factor wound healing

项目摘要

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.

这笔资助将阐明“铜转运 ATP 酶 (ATP7A)”的新型保护作用糖尿病缺血性血管疾病的修复性新生血管受损。由于血管生成受损，导致缺血性周围血管疾病中的新血管形成缺陷内皮细胞 (EC) 屏障功能障碍的机制尚不清楚，铜 (Cu) 是一种必需的微量营养素。参与血管生成，而过量的铜会导致糖尿病等炎症性疾病。 Cu 是有毒的，细胞内 Cu 的生物利用度受到 ATP7A 的严格控制，ATP7A 将 Cu 输送到分泌型 Cu 我们的实验室发现 VSMC 中的 ATP7A 可以预防。我们还鉴定出“IQGAP1”是一种促进 VEGF 受体 2 (VEGFR2) 结合的支架蛋白。 VEGF 信号传导和缺血后血管生成然而，ATP7A 在 EC 中对缺血后缺陷的作用。糖尿病的血运重建尚不清楚，初步数据表明 ATP7A 可以预防 VEGFR2。通过与 IQGAP1 结合而降解，并通过调节 VE-钙粘蛋白 (VE- 糖尿病小鼠 EC 或 2 型糖尿病微血管中 ATP7A 表达显着降低铜转运功能降低的 ATP7A 突变 (ATP7Amut) 小鼠或糖尿病小鼠表现出受损。缺血诱导的修复性血管生成与过量的组织铜和血管通透性/组织损伤， ATP7A 的过度表达可挽救这些细胞，因此我们追寻 ATP7A 的功能是促进和恢复。整合关键的血管修复程序，例如血管生成和维持内皮屏障以铜依赖性方式发挥功能，这是恢复糖尿病患者新血管形成所必需的目标 1 将定义 ATP7A 对以下疾病的保护作用： i) VEGF 诱导的受损。通过稳定 VEGFR2 来调节血管生成，以及 ii) 通过维持 Cu 稳态来调节内皮屏障功能障碍从糖尿病小鼠和 2 型糖尿病患者的人体微血管中分离的 EC 将确定目标 2。糖尿病中 ATP7A 下调损害 VEGFR2 信号传导和内皮细胞的分子机制通过关注屏障完整性；i) ATP7A 与 IQGAP1 结合，防止 VEGFR2 泛素化/降解 ii) ATP7A 在调节 miR-125b 中的作用，miR-125b 通过 Cu- 抑制 VE-Cad 依赖转录因子 Atox1。目标 3 将定义 ATP7A 对糖尿病诱导的保护作用。受损的缺血后新生血管形成和体内组织修复，并使用以下方法解决潜在机制我们将使用 ATP7Amut 和 ATP7A 转基因小鼠； ATP7A-/- 或 Cu 输入 CTR1-/- 小鼠或 1 型和 2 型糖尿病小鼠；创新 ICP 质谱、X 射线荧光显微镜分析细胞和组织中的 [Cu]i；基于 FRET 或 BiFC 的蛋白质-蛋白质相互作用；我们的研究将揭示铜转运蛋白 ATP7A 作为一个新的潜在治疗靶点。加强糖尿病血管并发症患者的综合血管修复计划。