Modulation of Vascular Extracellular Superoxide Dismutase

血管细胞外超氧化物歧化酶的调节

• 批准号: 8631664
• 负责人: TOHRU FUKAI
• 金额: $ 36.77万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631664
• 关键词: ATP phosphohydrolase; ATP7A protein; Address; Antioxidants; Atherosclerosis; Biological Assay; Biological Availability; Biopsy; Blood Vessels; Cardiovascular Diseases; Carrier Proteins; Caveolae; Cell Fractionation; Complication; Copper; Coupled; Data; Diabetes Mellitus; Diabetic Angiopathies; Diet; Disease; Endothelium; Enzymes; Extracellular Matrix; Extracellular Space; Fatty acid glycerol esters; Fluorescence Microscopy; Gene Transfer; Goals; Health; Human; Hyperglycemia; In Vitro; Insulin; Insulin Resistance; Insulin-Dependent Diabetes Mellitus; Laboratories; Location; Mass Spectrum Analysis; Mediating; Membrane Microdomains; Molecular; Morbidity - disease rate; Mus; Mutagenesis; Nitric Oxide; Non-Insulin-Dependent Diabetes Mellitus; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Peroxonitrite; Phosphorylation; Phosphorylation Site; Phosphotransferases; Plasma; Play; Production; Risk Factors; Roentgen Rays; Role; Side; Signal Transduction; Smooth Muscle Myocytes; Streptozocin; Superoxide Dismutase; Superoxides; Surface; Synchrotrons; System; Testing; Total Internal Reflection Fluorescent; Transgenic Mice; Ubiquitination; Vascular Diseases; Visceral; base; cofactor; diabetic; endothelial dysfunction; extracellular; insight; mimetics; mortality; mouse model; mutant; novel; novel therapeutics; overexpression; oxidant stress; prevent; protein expression; public health relevance; research study; therapeutic target; treatment strategy

DESCRIPTION (provided by applicant): The proposed study will address a novel function of Copper (Cu)-transporting ATPase (ATP7A), a Cu transporter for extracellular SOD (ecSOD), in preventing endothelial dysfunction and eNOS uncoupling in diabetes. Oxidative stress and endothelial dysfunction contribute to pathogenesis of diabetes melitus (DM), characterized by impaired insulin-Akt signaling. Although role of oxidative stress in vascular dysfunction in DM has been extensively studied, little is known about the function of antioxidant enzymes in these pathological diseases. ecSOD is a Cu-containing enzyme synthesized and secreted by vascular smooth muscle cells (VSMCs), and anchored to endothelial surfaces and extracellular matrix. Because of its extracellular location, ecSOD can increase nitric oxide (NO) bioavailability released from the endothelium by preventing O2¿-- mediated inactivation of NO and formation of peroxynitrite (ONOO-). Our laboratory previously demonstrated that full activation of vascular ecSOD requires Cu transporter ATP7A. However, the role of ecSOD and ATP7A in vascular dysfunction in DM is entirely unknown. Based on our preliminary studies, we will test the novel hypothesis that Akt-mediated phosphorylation of ATP7A is required for increasing ATP7A stability and translocation to the caveolae/lipid rafts where ecSOD obtains Cu to increase its activity. Thus, impaired insulin-Akt signaling in DM decreases ATP7A function and ecSOD activity in VSMCs, thereby promoting overproduction of O2"- and ONOO- in the extracellular space. This may facilitate the "out- side in signaling" leading to eNOS uncoupling and endothelial dysfunction in ECs. Aim1 will determine the molecular mechanism by which function of ATP7A, a Cu-transporting ATPase for ecSOD, is impaired in cultured diabetic VSMC. We will test the hypothesis that insulin-Akt-mediated phosphorylation of ATP7A is required for ATP7A stabilization by inhibiting its ubiquitination/degradation as well as ATP7A translocation to caveolae/lipid rafts where ecSOD obtains Cu to promote ecSOD activity in VSMCs. Aim 2 will determine the protective role of ATP7A, a Cu-transporting ATPase for ecSOD, in eNOS uncoupling and vascular dysfunction in murine and human diabetic vessels. We will examine if vessels from DM mice or those from biopsies obtained from patients with DM show decrease in ecSOD activity and ATP7A expression, thereby increasing O2"- level, ONOO- level, eNOS uncoupling, and endothelial dysfunction. We will use type1 and type2 DM mice models crossed with or without ecSOD KO, ATP7A mutant, or ATP7A overexpressing transgenic mice. We will also perform rescue experiments using adenoviral gene transfer of Akt phosphorylation sites mimetic ATP7A mutant in DM vessels ex vivo. X-ray fluorescence microscopy will be used to analyze Cu distribution in DM vessels and VSMCs. These studies should provide new insight into Cu transport system for ecSOD as a novel therapeutic strategy for treatment of oxidative stress-dependent cardiovascular diseases such as diabetes.

描述（由适用提供）：拟议的研究将介绍铜（CU）传输ATPase（ATP7A）的新功能，ATPase（ATP7A），一种用于防止内皮功能障碍和eNOS在糖尿病中偶联的CU转运蛋白（ECSOD）。氧化应激和内皮功能障碍有助于糖尿病（DM）的发病机理，其特征是胰岛素-AKT信号受损。尽管氧化应激在DM中血管功能障碍中的作用已广泛研究，但对这些病理疾病中抗氧化酶的功能知之甚少。 ECSOD是一种由血管平滑肌细胞（VSMC）合成并分泌的含Cu的酶，并锚定在内皮表面和细胞外基质上。由于其细胞外位置，ECSOD可以通过防止O2 wary从内皮中释放出一氧化氮（NO）生物利用度 - 介导的NO和过氧亚硝酸盐的形成和形成（ONOO-）。我们的实验室先前证明，血管ECSOD的完全激活需要Cu转运蛋白ATP7A。但是，ECSOD和ATP7A在DM中血管功能障碍中的作用是完全未知的。基于我们的初步研究，我们将检验以下新的假设：Akt介导的ATP7A磷酸化是提高ATP7A稳定性并转移到Caveolae/脂质筏的磷酸化所必需的，而Ecsod获得了Cu获得CU以增加其活性。 DM中的胰岛素-AKT信号受损会降低VSMC中的ATP7A功能和Ecsod活性，从而促进细胞外空间中的O2”的生产过多，并且在细胞外空间中 - 这可能会促进“信号中的“侧面”，从而导致ENOS UNOS和ENOSERIAL at ecs at ecs in ecs in ecs in ecs ains ains aint at ecs in ecs a in ecs ain aint at ecs。培养的糖尿病vSMC中，CU转发ATPase的ATPase受损。 vsmcs。 eNOS解偶联和内皮功能障碍。我们将使用带有或没有Ecsod KO，ATP7A突变体或ATP7A过表达转基因小鼠的类型1和2型DM小鼠模型。我们还将使用Akt磷酸化位点的腺病毒基因转移模拟于DM血管中的Akt磷酸化位点模拟ATP7A突变体。 X射线荧光显微镜将用于分析DM血管和VSMC中的CU分布。这些研究应为Ecsod的CU运输系统提供新的见解，作为治疗氧化应激依赖性心血管疾病（如糖尿病）的一种新型治疗策略。