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。项目的叙述。在美国，估计每年有300例新的原发性肺动脉高压病例被诊断出来，但真正的患病率和发病率是未知的（NHLBI, Facts About primary pulmonary hypertension）。继发性肺动脉高压更为常见，因为它是由各种阻塞性肺部疾病和生活在高海拔地区引起的，这两种情况与慢性缺氧有关。肺动脉对血流的持续高阻力导致右心室充盈压力升高，最终导致右心室肥大、缺血、衰竭和心源性猝死。肺动脉高压患者最常见的死亡原因与进行性右侧心力衰竭和心源性猝死事件有关。因此，有必要了解慢性缺氧对细胞、分子和基因组水平生理反应的影响，以便为大量患者制定适当的治疗策略。我们的长期研究目标是明确慢性缺氧导致肺动脉高压病理的机制，以便更合理地设计药理学方法来改善慢性缺氧相关肺部疾病患者的生活质量，并降低与此相关的发病率和死亡率。发展肺动脉高压的复杂过程部分是由基因表达的变化驱动的。钙调节转录因子NFATc3在肺动脉高压中的作用此前尚未得到解决，这突出了拟议研究的影响和必要性。本研究将首次明确肺动脉高压破坏性病理状态下肺动脉平滑肌细胞中NFATc3信号的调控因子和靶点。NFATc3先前与血管发育、血管平滑肌细胞分化、增殖和收缩的调节有关。因此，更好地了解肺动脉高压中血管重构和血管收缩增加的分子机制有望导致新的治疗方法的发展，以预防和治疗这种疾病。该建议最新颖的方面是它能够以真正综合的方式检查NFATc3对肺血管功能的调节。计划中的实验将利用我们在分子生物学、血管生物学和综合系统生理学方面的专业知识。

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. Project Narrative. In the United States it is estimated that 300 new cases of primary pulmonary hypertension are diagnosed each year but the true prevalence and incidence is unknown (NHLBI, Facts About Primary Pulmonary Hypertension). Secondary pulmonary hypertension is much more common because it is caused by a variety of obstructive pulmonary diseases and living at high-altitude, two conditions associated with chronic hypoxia. Sustained high pulmonary arterial resistance to blood flow causes an increase in the right ventricular (RV) filling pressure, which will eventually cause RV hypertrophy, ischemia, failure, and sudden cardiac death. The most common causes of death among patients with pulmonary arterial hypertension are related to progressive right-sided heart failure and sudden cardiac death events. Thus there is a need to understand the impact of chronic hypoxia on physiological responses at the cellular, molecular, and genomic levels in order to develop appropriate treatment strategies for the large group of patients. Our long-term research goal is to define the mechanisms whereby chronic hypoxia leads to the pathologies of pulmonary hypertension to allow more rational design of pharmacological approaches to improve the quality of life of patients suffering lung diseases associated with chronic hypoxia, and to decrease the rate of morbidity and mortality linked to this condition. The complex process of developing pulmonary hypertension is driven, in part, by changes in gene expression. The role of the calcium-regulated transcription factor NFATc3 in pulmonary arterial hypertension has not been previously addressed, highlighting the impact of and need for the proposed research. The proposed studies will define for the first time the regulators and targets of NFATc3 signaling in pulmonary arterial smooth muscle cells in the devastating pathological condition of pulmonary arterial hypertension. NFATc3 has been previously linked to vascular development, regulation of vascular smooth muscle cell differentiation, proliferation and contractility. Therefore, a better understanding of the molecular mechanisms that underlie the vascular remodeling and increased vasoconstriction in pulmonary hypertension is expected to lead to the development of novel therapeutic approaches to prevent and treat this disease. The most novel aspects of this proposal are its ability to examine NFATc3 regulation of pulmonary vascular function in a truly integrated fashion. The planned experiments will utilize our expertise in molecular biology, vascular biology and integrated systems physiology.