Impact of Insufficient Vascular EC-SOD in Pulmonary Hypertension

描述(由申请人提供)：氧化应激和活性氧物种信号现在被认为在肺动脉高压(PH)的发病机制中起着关键作用。对超氧化物(O2.-)的唯一胞外酶防御是抗氧化剂胞外超氧化物歧化酶(EC-SOD或SOD3)。虽然EC-SOD在大多数组织中只占总SOD活性的一小部分，但它是动脉中含量最丰富的SOD亚型。在初步数据和迄今为数不多的人体研究中，EC-SOD的表达和活性在终末期PH中降低。在PH的动物模型中，EC-SOD活性的丧失会恶化预后，尽管其机制尚不清楚。我们推测，EC-SOD定位于肺动脉壁对于防止血管炎症和重塑至关重要，因为它阻止了细胞外。 基质中的氧化还原敏感事件可以激活PA细胞中的重要信号通路。我们将使用新的小鼠模型、原代PASMC和巨噬细胞、有效的分子工具和有针对性的EC-SOD替代来验证这一假说。我们的第一个目标是测试血管EC-SOD不足在PH发生发展中的作用；以及其突出的血管定位是否可以通过将EC-SOD替换到PA来转化为更有效的治疗策略。我们将测试EC-SOD全身性丢失和血管EC-SOD优先丢失的小鼠品系，测量氧化还原状态、炎症、肺血管重构和PH；并测试是否可以通过有针对性的EC-SOD替代来逆转这些影响。我们的第二个目标是测试由于细胞外O2清除受损而导致的血管内皮细胞超氧化物歧化酶(EC-SOD)不足是否激活了转化生长因子--；如果这与ERK1/2的激活、Egr-1的表达以及随后PASMC的生长、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--转化生长因子--的依赖激活，如果这与ERK1/2依赖的转录因子上调有关，则早期生长反应-1，导致PASMC在PH中的生长、炎症和合成特性的变化。在第三个目标中，我们将测试低血管EC-SOD是否会导致基质成分HA的氧化断裂，从而导致巨噬细胞中NLRP3炎性小体的激活。这一目标将集中在一种特殊的基质成分，透明质酸，容易受到超氧化物介导的碎裂，并与PH有关。目的是测试血管EC-SOD不足是否会导致透明质酸的氧化裂解及其对NLRP3炎症体的激活，NLRP3炎症体是巨噬细胞中处理促炎细胞因子IL-1？和IL-18的蛋白质平台。这一建议提供了直接的翻译相关性，为开发适当针对特定血管间隔的新型抗氧化剂疗法奠定了基础。这些发现将对广泛的肺部疾病具有重要意义，最终改善患有这些严重问题的患者的健康结局。

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.