Modulation of Vascular Extracellular Superoxide Dismutase

描述(申请人提供)：这项拟议的研究将解决铜(铜)转运ATPase(ATP7A)的新功能，它是细胞外超氧化物歧化酶(EcSOD)的铜转运体，在预防糖尿病患者内皮功能障碍和eNOS解偶联方面具有重要作用。氧化应激和内皮功能障碍参与了糖尿病(DM)的发病机制，其特征是胰岛素-Akt信号受损。尽管氧化应激在糖尿病血管功能障碍中的作用已被广泛研究，但对抗氧化酶在这些病理疾病中的作用知之甚少。ECSOD是一种由血管平滑肌细胞(VSMCs)合成和分泌的含铜酶，锚定于内皮细胞表面和细胞外基质上。由于EcSOD位于细胞外，可通过阻止O2-介导的NO失活和过氧亚硝酸盐(ONOO-)的形成来增加从内皮释放的一氧化氮(NO)的生物利用度。我们的实验室先前证明，血管EcSOD的完全激活需要铜转运蛋白ATP7A。然而，ECSOD和ATP7A在糖尿病血管功能障碍中的作用尚不完全清楚。基于我们的初步研究，我们将检验这一新的假设，即需要Akt介导的ATP7A磷酸化来增加ATP7A的稳定性，并将其移位到EcSOD获得铜以提高其活性的小窝/脂筏。因此，糖尿病患者胰岛素-Akt信号的受损降低了VSMCs的ATP7A功能和ECSOD活性，从而促进了细胞外间隙中O2“-和ONOO-的过度产生。这可能有助于“外向内信号”导致内皮细胞eNOS解偶联和内皮功能障碍。AIM1将确定ECSOD的铜转运ATP7A酶在培养的糖尿病VSMC中功能受损的分子机制。我们将通过抑制ATP7A的泛素化/降解以及ATP7A的转位到腔/脂筏来验证这一假设，即胰岛素-Akt介导的ATP7A的磷酸化是ATP7A稳定所必需的，在那里EcSOD获得铜以促进VSMC中的EcSOD活性。目的研究ECSOD的铜转运酶ATP7A在糖尿病小鼠和人血管eNOS解偶联和血管功能障碍中的保护作用。我们将检查糖尿病小鼠或糖尿病患者活检组织的血管是否显示ECSOD活性和ATP7A表达减少，从而增加O2“-水平、ONOO-水平、eNOS解偶联和内皮功能障碍。我们将使用1型和2型糖尿病小鼠与或不与EcSODKO、ATP7A突变体或ATP7A过度表达的转基因小鼠杂交。我们还将利用腺病毒基因转移模拟ATP7A突变的Akt磷酸化位点在DM血管中进行体外救援实验。将使用X射线荧光显微镜分析铜在DM血管和VSMCs中的分布。这些研究将为ECSOD作为治疗糖尿病等氧化应激依赖型心血管疾病的一种新的治疗策略提供新的见解。

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.