NFATc3 in chronic hypoxic pulmonary hypertension

描述(申请人提供)：尽管起源于肺动脉高压(PAH)，但由于肺血管收缩、动脉重构和红细胞增多症导致的肺血管阻力增加，导致右心衰竭和死亡。暴露于慢性低压缺氧(CH)的啮齿动物会发生PAH。发展PAH的复杂过程在一定程度上是由基因表达的变化驱动的。在PAH中，平滑肌细胞内钙升高，内皮素-1(ET-1)表达上调。CA2通过活化T细胞核因子(NFAT)调节肺动脉平滑肌(PASMC)收缩，并与基因转录有关。NFATc3亚型与血管系统的发育和平滑肌分化表型的维持有关。这项建议的总体目标是确定NFATc3在与CH-PAH相关的血管变化的分子机制中的作用。假说是CH激活PASMC中的NFATc3，从而介导肺动脉肥大并增强参与PAH的肺动脉(PA)的收缩能力。目的1：探讨NFATc3在CH诱导的PASMC肥大和PAH中的作用。我们将评估PA压力，检测PA中肥大标志物α-肌动蛋白和肌球蛋白重链、与1-肌动蛋白和肌球蛋白重链启动子结合的NFATc3的mRNA和蛋白，并测定野生型/-钙调神经磷酸酶/NFAT抑制剂和NFATc3基因敲除小鼠暴露于常氧和减压条件下的肺血管结构变化。特异性目的2：确定NFATc3在CH诱导的Kv通道表达下调和肺血管收缩反应性增加中的作用。我们将使用AIM1中提出的相同的动物模型，并测定Kv亚型的mRNA和蛋白；NFATc3与Kv 1.5和2.1启动子的结合以及与其他转录调节因子的关联；PASMC膜电位和激动剂诱导的血管收缩。具体目的3：确定CH增强PASMC中NFATc3转录活性的机制。我们将利用NFAT-荧光素酶报告小鼠和NFAT-荧光素酶与NFATc3 KO小鼠杂交来确定CH增加NFAT活性的介体(ET-1、钙调神经磷酸酶和Rho-激酶)。这项研究的结果将为调节PAH基因转录变化的信号机制提供新的信息。更好地了解PAH的这种机制将导致开发新的治疗方法来预防和治愈这种令人衰弱的疾病。项目叙事。据估计，在美国，每年新诊断出300例原发性肺动脉高压，但真正的患病率和发病率尚不清楚(NHLBI，关于原发性肺动脉高压的事实)。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。持续的高肺动脉血流阻力导致右室充盈压升高，最终导致右室肥大、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心衰竭和心源性猝死事件有关。因此，有必要在细胞、分子和基因组水平上了解慢性低氧对生理反应的影响，以便为广大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的病理机制，以便更合理地设计药物治疗方法，以改善与慢性缺氧相关的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。发生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以前没有被讨论过，这突显了拟议研究的影响和必要性。建议的研究将首次明确NFATc3信号在肺动脉高压的破坏性病理条件下在肺动脉平滑肌细胞中的调节和靶点。NFATc3以前被认为与血管发育、调节血管平滑肌细胞分化、增殖和收缩有关。因此，更好地了解肺动脉高压血管重构和血管收缩增加的分子机制有望导致开发新的治疗方法来预防和治疗这种疾病。这一建议最新颖的方面是它能够以一种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、血管生物学和集成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.