NFATc3 in chronic hypoxic pulmonary hypertension

描述（由申请人提供）：尽管肺动脉高压（PAH）的起源，但由于肺血管收缩、动脉重塑和红细胞增多症导致右心衰和死亡，肺血管阻力升高。暴露于慢性低压缺氧（CH）的啮齿动物发展为多环芳烃。发展多环芳烃的复杂过程部分是由基因表达的变化驱动的。在PAH中，平滑肌细胞内Ca2+增加，内皮素1 （ET-1）表达上调。Ca2+调节肺动脉平滑肌（PASMC）收缩，并通过活化T细胞核因子（NFAT）与基因转录相关。NFATc3亚型与脉管系统的发育和平滑肌分化表型的维持有特殊的关系。本提案的总体目标是确定NFATc3在与CH-PAH相关的血管变化的分子机制中的作用。假设是CH激活PASMC中的NFATc3介导肥大并增强肺动脉（PA）的收缩性，从而导致PAH。特异性目的1：确定NFATc3在ch诱导的PASMC肥大和PAH中的作用。我们将评估PA压力，测量PA中肥厚标记α -肌动蛋白和肌球蛋白重链mRNA和蛋白，NFATc3结合1-肌动蛋白和肌球蛋白重链启动子，并确定野生型+/-钙调磷酸酶/NFAT抑制剂和NFATc3敲除小鼠暴露于常氧和低压CH下肺血管的结构变化。目的探讨NFATc3在ch诱导的Kv通道表达下调和肺血管收缩反应性升高中的作用。我们将使用在ai1中提出的相同的动物模型，并确定mRNA和蛋白Kv亚型；NFATc3与KV 1.5和2.1启动子的结合以及与其他转录调控子的关联；PASMC膜电位和激动剂诱导的离体加压PA血管收缩。具体目的3：确定CH在PASMC中增加NFATc3转录活性的机制。我们将使用NFAT-荧光素酶报告小鼠和NFAT-荧光素酶与NFATc3 KO小鼠杂交来测定ch - NFAT活性增加的介质（ET-1、Ca2+、钙调磷酸酶和rho激酶）。这些研究结果将为PAH基因转录变化的信号机制调控提供新的信息。更好地了解PAH的机制将导致新的治疗方法的发展，以预防和治疗这种使人衰弱的疾病。

DESCRIPTION (provided by applicant): Despite the origin of pulmonary arterial hypertension (PAH), pulmonary vascular resistance rises due to pulmonary vasoconstriction, arterial remodeling and polycythemia leading to right heart failure and death. Rodents exposed to chronic hypobaric hypoxia (CH) develop PAH. The complex process of developing PAH is driven, in part, by changes in gene expression. In PAH, smooth muscle intracellular Ca2+ is increased and endothelin 1 (ET-1) expression is up-regulated. Ca2+ regulates pulmonary arterial smooth muscle (PASMC) contraction and is linked to gene transcription through the nuclear factor of activated T cells (NFAT). NFATc3 isoform is specifically implicated in the development of the vasculature and maintenance of smooth muscle differentiate phenotype. The overall goal of this proposal is to determine the role of NFATc3 in the molecular mechanisms underlying the vascular changes associated with CH-PAH. The hypothesis is that CH activates NFATc3 in PASMC to mediate hypertrophy and enhance contractility of pulmonary arteries (PA) contributing to PAH. Specific Aim 1: To determine the role of NFATc3 in CH-induced PASMC hypertrophy and PAH. We will estimate PA pressure, measure mRNA and protein of the hypertrophic markers alpha-actin and myosin heavy chain in PA, NFATc3 binding to 1-actin and myosin heavy chain promoters, and determine structural changes of the pulmonary vasculature on wild type +/- calcineurin/NFAT inhibitor and NFATc3 knockout mice exposed to normoxia and hypobaric CH. Specific Aim 2: To establish the contribution of NFATc3 to CH-induced downregulation of Kv channel expression and increases in pulmonary vasoconstrictor reactivity. We will use the same animal models proposed in aim1 and determine mRNA and protein Kv isoforms; NFATc3 binding to KV 1.5 and 2.1 promoters and association to additional transcriptional regulators; PASMC membrane potential and agonist-induced vasoconstriction in isolated pressurized PA. Specific Aim 3: To determine the mechanisms by which CH increases NFATc3 transcriptional activity in PASMC. We will determine the mediators (ET-1, Ca2+, calcineurin and Rho-kinase) of CH-increased NFAT activity using NFAT-luciferase reporter mice and NFAT-luciferase crossed with NFATc3 KO mice. Findings from the proposed studies will provide novel information about the signaling mechanisms regulating changes in gene transcription in PAH. A better understanding of this mechanisms in PAH will lead to the development of novel therapeutic approaches to prevent and cure this debilitating disease.