Vascular Extracellular Superoxide Dismutase Modulation

描述(申请人提供)：细胞外超氧化物歧化酶 在维管系统中表达的一种主要的超氧化物歧化酶(EcSOD)是一种“分泌型”。 含铜酶，在调节血液中起着重要作用 压力和内皮功能通过调节血管内氧水平 细胞外空间。尤其是在血管紧张素H诱导的高血压模型中， 在血管壁观察到过量的02，高血压是 通过膜靶向形式的超氧化物歧化酶治疗而得到改善。此外，我们有 发现血压和血管中的O2产生高度升高 在注射血管紧张素II的EcSOD缺陷小鼠中。因此，ecS0D是一种 氧化现象在糖尿病发病机制中的潜在重要调节因子 高血压。最近，有研究表明，铜伴侣(CCS)是 对于铜的运输和向含铜的酶的输送至关重要。我们的 初步数据有力地提示CCS与信号肽(CCS-SP) 靶向高尔基体在铜向ECSOD的运输中起着重要的作用， 这是EcSOD充分发挥活性所必需的。我们将提出以下建议 解决铜转运如何控制EcSOD活性的具体目标 CCS和铜转运蛋白等系统在酵母系统、血管细胞中的应用 和体内高血压模型。在目标1中，我们将描述一个角色 利用酵母菌构建完整表达EcSOD活性的铜转运系统 系统。首先，通过产生几个CCS-SP cDNA构建物，包括 截断形式，我们将确定哪个区域对铜至关重要 正在加载-到EcSOD。其次，我们将确定将铜装载到EcSOD是否需要 MNK，跨高尔基体网络中的铜转运蛋白，使用酵母菌株 缺乏MNK。在目标2中，我们将确定内源性铜伴侣蛋白 人主动脉平滑肌细胞(HASM)高表达EcSOD， 通过使用CCS的高度保守区域作为探针检测到新的 HASM中CCS样转录本和蛋白。接下来，我们将确定铜是否 通过使用小鼠，向EcSOD递送需要哺乳动物细胞中的MTNK MNK突变小鼠的成纤维细胞和主动脉。在目标3中，我们将研究 ECSOD铜转运系统在血压、血管氧分压中的作用 血管紧张素II诱导的高血压的产生和内皮功能 使用MNK突变小鼠。这些研究将提供对铜的新见解 ECSOD作为一种新型氧化应激调节剂的转运系统 高血压的发病机制和AS的抗氧化治疗必不可少。

DESCRIPTION (provided by the applicant): Extracellular superoxide dismutase (ecSOD), a major form of SOD expressed in the vasculature, is a "secretory" copper-containing enzyme and plays an important role in regulating blood pressure and endothelial function by modulating the levels of O2 in the extracellular space. Particularly, in angiotensin H-induced hypertension model, the excessive 02 is observed in the vessel wall and the hypertension is ameliorated by treatment with membrane-targeted forms of SOD. Moreover, we have found that blood pressure and 02 production in the vessel were highly elevated in ecSOD-deficient mice infused with angiotensin II. Thus, ecS0D is a potentially important modulator of oxidative phenomena in the pathogenesis of hypertension. Recently, it has been shown that copper chaperones (CCS) are critical for copper transport and delivery to copper containing enzymes. Our preliminary data strongly suggests that CCS with signal peptide (CCS-SP) which targets to Golgi plays an important role in the transport of copper to ecSOD, which is required for full activity of the ecSOD. We will propose the following specific aim to address how ecSOD activity is controlled by copper transport system such as CCS and copper transporter in the yeast system, vascular cells and in vivo model of hypertension. In aim 1, we will characterize a role of copper transport system for full expression of ecSOD activity using the yeast system. First, by generating several CCS-SP cDNA constructs including the truncated form, we will determine which region is critical for copper loading-to ecSOD. Second, we will determine if copper loading to ecSOD requires MNK, a copper transporter in the trans-Golgi network, using the yeast strain deficient in MNK. In aim 2, we will identify endogenous copper chaperone for ecSOD in human aortic smooth muscle cells (HASM) that highly expresses ecSOD, by using the highly conserved region of CCS as a probe that have detected novel CCS-like transcript and protein in HASM. Next, we will determine if copper delivery to ecSOD requires MTNK in mammalian cells, by using the murine MNK-mutant fibroblast and aorta from MNK-mutant mice. In aim 3, we will examine the role of copper transport system for ecSOD in blood pressure, vascular O2 production and endothelial function in angiotensin II induced hypertension by using MNK-mutant mice. These studies will provide new insight into a copper transport system for ecSOD as a novel modulator of oxidative stress linked to the pathogenesis of hypertension and as essential to anti-oxidant therapy.