Nitric Oxide Production and Reactions in the Lung

描述(申请人提供)：原发性肺动脉高压(PPH)是一种病因不明的致命性疾病，其特征是肺血流动力学和血管生长调节受损。我们的初步数据显示，PPH肺来源的原代肺动脉内皮细胞(PAEC)在体外具有促进增殖、迁移和异常管状形成的作用。信号转导和转录激活因子(STAT)3最近被认为是血管生成的关键调节因子，在PPH PAEC中持续激活，但在对照细胞中不被激活。推测下游靶基因精氨酸酶II和血管内皮生长因子(VEGF)的高水平表达仅在PPH PAEC中存在。先前，我们已经证明血管扩张剂一氧化氮(NO)的减少在PPH的病理生理学中起重要作用，但NO合成酶(NOS)的表达是完整的。在这里，我们提出了一种低NO的翻译后机制，即精氨酸酶II，一种竞争一氧化氮合酶底物精氨酸的酶，被增加。因此，我们假设PPH的发病机制源于异常的内皮细胞，这些内皮细胞持续激活STAT3，继而表达VEGF和精氨酸酶II，导致细胞增殖增加，血管生成紊乱，NO丢失。首先，我们将对PPH PAEC的增殖、迁移和管状形成进行量化，并与健康和疾病对照组进行比较。为了确定PPH细胞生物学改变和PPH中低NO的机制，我们使用过度表达、RNA沉默或在评估酶底物和产物效应的条件下药物抑制精氨酸酶以及NO合成的策略来研究精氨酸酶II。为了探讨精氨酸酶在PPH中高水平表达的机制，我们计划分析STAT介导的精氨酸酶II的转录激活。我们的研究还包括评估与PPH发病有关的II型骨形态发生蛋白受体(BMPR2)突变和人类疱疹病毒8型(HHV8)感染，并使用蛋白质组学方法发现对照组和PPH之间的新差异。我们先前的研究表明，NO与肺动脉压呈负相关。在这里，我们建议对PPH患者进行一项纵向研究，以检验我们的假设，即精氨酸酶和精氨酸合成的决定因素预测PPH的结果。综上所述，这些研究将最终揭示PPH内皮细胞的根本性和内在变化，确定其原因机制，并导致治疗PPH的新疗法。

DESCRIPTION (provided by applicant): Primary pulmonary hypertension (PPH) is a fatal disease of unknown etiology characterized by impaired regulation of both pulmonary hemodynamics and vascular growth. Our preliminary data show that primary pulmonary artery endothelial cells (PAEC) from PPH lung have enhanced proliferation, migration and abnormal tube formation in vitro. The signal transducer and activator of transcription (STAT) 3, recently identified as a critical regulator for angiogenesis, is persistently activated in PPH PAEC, but not in control cells. High-level expression of putative downstream target genes, arginase II and vascular endothelial growth factor (VEGF), are present only in PPH PAEC. Previously, we showed that diminished vasodilator nitric oxide (NO) is important in the pathophysiology of PPH, but NO synthases (NOS) expression are intact. Here, we propose a post-translational mechanism for low NO, i.e. arginase II, an enzyme that competes for the NOS substrate arginine, is increased. Thus, we hypothesize that the pathogenesis of PPH stems from abnormal endothelial cells, which have persistent STAT3 activation with consequent expression of VEGF and arginase II, that leads to increased proliferation, deregulated angiogenesis, and loss of NO. Initially, we will quantitate proliferation, migration and tube formation of PPH PAEC in comparison to healthy and disease controls. To identify mechanisms that account for the altered biology of PPH cells and low NO in PPH, we focus our investigations on arginase II using strategies of over-expression, RNA silencing or pharmacologic inhibition of arginase under conditions that assess enzyme substrate and product effects, and NO synthesis. To investigate mechanisms for high-level arginase expression in PPH, we plan to analyze STAT-mediated transcriptional activation of arginase II. Our studies also include evaluation for type II bone morphogenetic protein receptor (BMPR2) mutations and Human Herpesvirus 8 (HHV8) infection, both implicated in PPH pathogenesis, and use proteomic methods to discover novel differences between control and PPH. Our previous study showed an inverse correlation of NO to pulmonary artery pressure. Here, we propose a longitudinal study of PPH patients to test our hypothesis that determinants of NO synthesis, i.e. arginase and arginine, predict outcomes in PPH. Taken together, these studies will conclusively reveal fundamental and inherent alterations in PPH endothelial cells, identify the causal mechanisms, and lead to novel therapies for treatment of PPH.