Impact of Insufficient Vascular EC-SOD in Pulmonary Hypertension

描述（由申请人提供）：目前认为氧化应激和活性氧物质信号传导在肺动脉高压（PH）的发病机制中具有关键作用。唯一的细胞外酶防御超氧化物（O2.-）是抗氧化剂胞外超氧化物歧化酶（EC-SOD或SOD 3）。虽然EC-SOD在大多数组织中占总SOD活性的一小部分，但它是动脉中最丰富的SOD同工型。在初步数据和迄今为止的少数人类研究中，EC-SOD表达和活性在终末期PH中降低。在PH动物模型中EC-SOD活性的丧失导致预后不良，尽管其机制尚不清楚。我们假设，EC-SOD定位于肺动脉（PA）壁是至关重要的，以防止血管炎症和重塑，因为它阻止细胞外 基质中的氧化还原敏感事件，可以激活PA细胞中的重要信号传导途径。我们将使用新的小鼠模型、原发性PASMC和巨噬细胞、有效的分子工具和靶向EC-SOD替代物来验证这一假设。我们的第一个目标是测试血管EC-SOD不足对PH发展的贡献;以及其突出的血管定位是否可以通过靶向EC-SOD替代PA转化为更有效的治疗策略。我们将测试具有EC-SOD的普遍损失与血管EC-SOD的优先损失的小鼠品系，测量氧化还原状态、炎症、肺血管重塑和PH;并测试这些效应是否可以通过靶向EC-SOD替代来逆转。我们的第二个目标是测试血管EC-SOD是否不足，这是由于细胞外O2-清除受损，激活TGF-β;以及这是否与ERK 1/2激活、Egr-1表达以及随后PASMC生长、炎症和合成特性的变化有关。这一目标将使用体内和体外模型来系统地测试低血管EC-SOD是否会增加O2。关键生长因子TGF-β 1的依赖性激活，并且如果这负责转录因子早期生长反应-1的ERK 1/2依赖性上调，则导致PH中PASMC的生长、炎症和合成特性的变化。在第三个目的中，我们将测试低血管EC-SOD是否导致基质组分HA的氧化断裂，导致巨噬细胞中的NLRP 3炎性体活化。这一目标将集中在一个特定的基质成分，透明质酸，对超氧化物介导的碎片敏感，并参与PH。该目标将测试如果血管EC-SOD不足导致透明质酸的氧化碎片和NLRP 3炎性体的激活，一个蛋白质平台，在巨噬细胞中处理促炎细胞因子IL-1 β和IL-18。该提案通过建立开发适当靶向特定血管室的新型抗氧化剂疗法的基础，提供了直接的翻译相关性。这些发现将对广泛的肺部疾病产生重要影响，最终改善患有这些严重问题的患者的健康状况。

DESCRIPTION (provided by applicant): Oxidative stress and reactive oxygen species signaling are now recognized to have a critical role in the pathogenesis of pulmonary hypertension (PH). The sole extracellular enzymatic defense against superoxide (O2.-) is the antioxidant extracellular superoxide dismutase (EC-SOD or SOD3). Though EC-SOD constitutes a small fraction of total SOD activity in most tissues, it is the most abundant SOD isoform in arteries. In preliminary data and the few human studies to date, EC-SOD expression and activity are decreased in end- stage PH. Loss of EC-SOD activity in animal models of PH worsens outcome, though the mechanisms are poorly understood. We hypothesize that EC-SOD localized to the pulmonary artery (PA) wall is critical to the protection against vascular inflammation and remodeling because it prevents extracellular redox-sensitive events in the matrix that can activate important signaling pathways in PA cells. We will test this hypothesis using novel mouse models, primary PASMC and macrophages, potent molecular tools and a targeted EC-SOD replacement. Our first Aim will test the contribution of insufficient vascular EC-SOD to the development of PH; and whether its prominent vascular localization can be translated into a more effective therapeutic strategy by targeting EC-SOD replacement to the PA. We will test mouse strains with a generalized loss of EC-SOD versus a preferential loss of vascular EC-SOD, measure redox state, inflammation, pulmonary vascular remodeling and PH; and test if the effects can be reversed by targeted EC-SOD replacement. Our second Aim will test if insufficient vascular EC-SOD, as a result of impaired scavenging of extracellular O2.-, activates TGF-ß; and if this is responsible for ERK1/2 activation, Egr-1 expression and the subsequent changes in growth, inflammation and synthetic properties of PASMC. This aim will use in vivo and in vitro models to systematically test if low vascular EC-SOD, increases O2.---dependent activation of the key growth factor, TGF-ß, and if this is responsible for ERK1/2-dependent up-regulation of the transcription factor, early growth response-1, leading to changes in the growth, inflammatory, and synthetic properties of PASMC in PH. In the third Aim, we will test if low vascular EC-SOD results in oxidative fragmentation of matrix component, HA, leading to NLRP3 inflammasome activation in macrophages. This aim will focus on a specific matrix component, hyaluronan, susceptible to superoxide-mediated fragmentation and implicated in PH. The aim will test if insufficient vascular EC-SOD leads to oxidative fragmentation of hyaluronan and its activation of the NLRP3 inflammasome, a protein platform that processes pro-inflammatory cytokines IL-1ß and IL-18 in macrophages. This proposal provides direct translational relevance by establishing the foundation to develop novel antioxidant therapies appropriately targeted to a specific vascular compartment. These findings will have important implications in a wide range of lung diseases to ultimately improve health outcome for patients with these serious problems.