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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：
9389671
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 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9389671
关键词：
ATP phosphohydrolase Address Alpha Cell 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 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 Injection of therapeutic agent Ischemia Knockout Mice Lead Leukocytes Limb structure Measures Micronutrients Molecular Morbidity - disease rate Mus Patients Perfusion Peripheral Peripheral Vascular Diseases Permeability Play Protein-Lysine 6-Oxidase Recruitment Activity 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 Wound Healing angiogenesis ataxia telangiectasia mutated protein base cadherin 5 copper-transporting ATPase 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 repaired small hairpin RNA therapeutic target tissue repair transcription factor

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.

这项研究将阐明“铜转运ATPase(ATP7A)”的新的保护作用。糖尿病缺血性血管病变中受损的修复性新生血管。糖尿病并发症血管生成受损导致缺血性外周血管疾病新生血管缺陷内皮细胞(EC)屏障功能障碍，机制不明。铜是一种必需的微量营养素，与血管生成有关，而过量的铜会导致糖尿病等炎症性疾病。由于超额铜是有毒的，细胞内铜的生物有效性受到ATP7A的严格控制，ATP7A将铜输送到分泌型铜酶，或将铜输出到细胞外空间。我们的实验室发现VSMC中的ATP7A可以预防高血压。我们还鉴定了IQGAP1是一个与血管内皮生长因子受体2(VEGFR2)结合的支架蛋白，促进血管内皮生长因子信号与缺血后血管生成。然而，ATP7A在缺血后缺陷内皮细胞中的作用糖尿病的血运重建是完全未知的。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-Cad-herin)维持基本的EC屏障功能 CAD)。ATP7A在糖尿病小鼠内皮细胞或2型糖尿病微血管中的表达显著降低病人。铜转运功能降低的ATP7A突变(ATP7Amut)小鼠或糖尿病小鼠表现为受损组织铜过量和血管通透性/组织损伤的缺血诱导的修复性血管生成，它们是通过ATP7A的过度表达而拯救的。因此我们假设ATP7A的功能是促进和整合关键的血管修复计划，如血管生成和维持内皮屏障以铜依赖的方式发挥作用，这是糖尿病患者恢复新生血管所必需的缺血性血管疾病。目标1将确定ATP7A对：i)血管内皮生长因子诱导的损伤的保护作用血管生成通过稳定VEGFR2和II)内皮细胞屏障功能障碍通过维持铜的动态平衡从糖尿病小鼠和2型糖尿病患者的人微血管中分离出内皮细胞。目标2将决定糖尿病患者ATP7A下调损伤VEGFR2信号和血管内皮细胞的分子机制屏障完整性，重点是：i)ATP7A与IQGAP1结合，防止VEGFR2泛素化/降解以及ii)ATP7A在调节miR-125b中的作用，该miR-125b通过铜抑制VE-Cad。依赖转录因子Atox1。目标3将确定ATP7A对糖尿病诱导的保护作用损伤的缺血后新生血管和体内组织修复，并解决潜在的机制严重肢体缺血的动物模型。我们将使用ATP7Amut和ATP7A转基因小鼠；可诱导的EC特异性 ATP7A-/-或铜进口CTR1-/-小鼠或1型和2型糖尿病小鼠；创新的电感耦合等离子体质谱，X射线荧光显微镜分析细胞和组织中的[铜]i；FRET或基于BIFC的蛋白质-蛋白质相互作用；和活体显微镜。我们的研究将发现铜转运蛋白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。